UNIVERSIDADE FEDERAL DO CEARÁ REDE NORDESTE DE ... · Todavia eu me alegrarei no Senhor, exultarei...

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UNIVERSIDADE FEDERAL DO CEARÁ

REDE NORDESTE DE BIOTECNOLOGIA - RENORBIO

PROGRAMA DE PÓS-GRADUAÇÃO EM BIOTECNOLOGIA

JOSÉ JACKSON DO NASCIMENTO COSTA

EXPRESSÃO DE MARCADORES DE CÉLULAS GERMINATIVAS E DE OÓCITOS

EM FIBROBLASTOS BOVINOS TRATADOS COM 5-AZA-CITIDINA E EM

CÉLULAS-TRONCO ADULTAS CULTIVADAS IN VITRO NA PRESENÇA DE BMP-2,

BMP-4 OU FLUIDO FOLICULAR

SOBRAL - CE

2016

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JOSÉ JACKSON DO NASCIMENTO COSTA

EXPRESSÃO DE MARCADORES DE CÉLULAS GERMINATIVAS E DE OÓCITOS

EM FIBROBLASTOS BOVINOS TRATADOS COM 5-AZA-CITIDINA E EM

CÉLULAS-TRONCO ADULTAS CULTIVADAS IN VITRO NA PRESENÇA DE BMP-2,

BMP-4 OU FLUIDO FOLICULAR

Tese apresentada ao Curso de Doutorado em

Biotecnologia da Rede Nordeste de Biotecnologia

– RENORBIO da Universidade Federal do Ceará,

como parte dos requisitos para obtenção do título

de Doutor em Biotecnologia. Área de

concentração: Biotecnologia em Agropecuária.

Orientador: Prof. Dr. José Roberto Viana Silva

Co-orientadora: Profa. Dra. Márcia Viviane Alves

Saraiva

SOBRAL - CE

2016

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A Deus, pela minha existência, pela força,

coragem e determinação que me foi dada para

alcançar mais esse objetivo, porque nada nos é

possível se não for de Sua vontade,

Dedico

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AGRADECIMENTOS

A Deus, pelo seu amor e pela sua infinita misericórdia manifestados a cada dia em

minha vida. Pela proteção, força e coragem para enfrentar todas as dificuldades da vida pessoal e

profissional. Senhor, a minha confiança descansa em tuas mãos. Sempre espero e confio em ti.

Obrigado por mais essa vitória.

À minha esposa Amélia Araújo, agradeço pelo amor, amizade, companheirismo,

apoio nos momentos mais difíceis e pela compreensão que me deram suporte na trajetória final

deste trabalho. “Entre as coisas mais lindas que eu conheci, só reconheci suas cores belas quando

eu te vi. Entre as coisas bem-vindas que já recebi, eu reconheci minhas cores nela então eu me

vi.” Sou muito mais feliz com você e com nossa filha Val. Que o Senhor continue nos

abençoando cada dia mais.

Aos meus pais José Ponte Costa e Maria de Fátima do Nascimento, por terem me

dado a vida e por todo amor e dedicação fundamentais em todos os momentos da minha vida.

Aos meus irmãos Janilson Costa e sua esposa Cristiane Santos Costa, Jailson Costa e Jamile

Costa, com os quais dividi momentos de alegrias e tristezas, e que sempre estarão me

incentivando e torcendo pelo meu sucesso. De forma especial, agradeço à minha sobrinha, Ana

Clara Costa, uma das maiores alegrias da minha vida, você é muito especial.

Aos meus avós Pedro do Nascimento e Maria da Conceição do Nascimento, por todo

o amor e carinho dedicados a mim, vocês são essenciais para a minha vida. E ainda agradeço a

todos os meus tios(as) e primos(as), que longe ou perto sempre me ajudaram, torcem por mim e

sempre estarão no meu coração. Em especial ao meu tio José Maria Costa e José Astélio Costa,

por cuidarem de mim desde a infância, educando e sempre orientando os caminhos retos a seguir.

À família Araújo, em especial à dona Amélia Araújo, sr. João Elmiro Soares,

Marcigleide Araújo, Elenilton Roratto, Júlia Roratto, Ana Sofia Roratto, Olga Araújo e Samira

Araújo. Por fazerem me sentir parte da família, dividindo comigo momentos de tristezas, mas

principalmente momentos de muitas alegrias. Vocês moram no meu coração.

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Ao orientador Prof. Dr. José Roberto Viana Silva, pela orientação deste trabalho,

paciência, pela dedicação dispensada, confiança e profissionalismo demonstrados no decorrer de

nossa convivência. Agradeço pela contribuição decisiva na minha formação e pelo muito que

aprendi durante os anos de mestrado e doutorado.

À co-orientadora e amiga Profa. Dra. Márcia Viviane Alves Saraiva pela dedicação

constante e por sempre estar disposta a ajudar em todos os momentos.

Aos queridos amigos integrantes do grupo de pesquisa de Reprodução e Cultura de

Células: Ellen de Vasconcelos da Cunha, Francisco Taiã Gomes Bezerra, Laís Feitosa, Ana Kelry

Carneiro Lopes, Éverton Pimentel, Adriel Pereira, Edilcarlos Max, Hozana Braga, Pedro Alves,

Miguel Fernandes, Laryssa Barrozo, Bruno Matos Brito e Bianca Silva.

À querida amiga Juliane Passos e a toda família Passos, Cleuton Monteiro, tia Mazé e

Neyla, agradeço pela amizade, companheirismo, apoio e incentivo desde o início e por me

tratarem sempre com muito carinho, como se eu fosse um irmão. Obrigado pelo apoio e pelas

palavras de carinho nos momentos mais difíceis.

Aos amigos e vizinhos Jordânia Marques, Clayrtiano Freire, João Arthur Freire e

Maria das Graças Oliveira, agradeço de forma especial, pelo convívio, confiança, amizade e

companheirismo durante todos esses anos. Que Deus continue abençoando nossa amizade.

Aos amigos Anderson Weiny Silva, Regislane Pinto Ribeiro, José Renato de Souza

Passos e Glaucinete Borges, agradeço pela amizade, pelos conselhos, pelos momentos de trabalho

e pelos momentos de descontração. Admiro a competência de vocês.

À amiga Joyla Maria Pires Bernardo agradeço por ter tornado o dia a dia muito mais

divertido e agradável, sempre trazendo alegria e descontração durante a execução de todos os

experimentos.

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Aos amigos Tânia de Azevedo Lopes, Antônia Moemia Lucia Rodrigues Portela,

Gisvani Lopes de Vasconcelos, Katianne dos Santos Freitas e Danielle Val, obrigado por me

aguentarem todos esses anos, obrigado pela amizade, carinho e momentos de diversão e de

trabalho também. Sem as risadas de vocês tudo teria sido mais difícil. É bom ter o privilégio da

presença de vocês na minha vida. Sou grato a Deus por nossa amizade, vocês são especiais.

Ao prof. Dr. Rodrigo Maranguape pelo incentivo dado aos meus primeiros passos na

pesquisa durante minha iniciação científica e a toda equipe do Laboratório de Genética Molecular

do NUBIS, de forma especial a Auxiliadora Oliveira, João Garcia, Tatiana Farias, Nayane Hardy,

Aurilene Cajado, Jedson Aragão, pela convivência durante todos esses anos.

Aos integrantes da banca examinadora, Profa. Dra. Ana Paula Ribeiro Rodrigues;

Profa. Dra. Maria Helena Tavares de Matos; Profa. Dra. Valdevane Rocha Araújo; Prof. Dr. Igor

Iuco Castro da Silva; Prof. Dr. Antonio Silvio do Egito; Profa. Dra. Juliana Jales De Hollanda

Celestino; por terem gentilmente aceito o convite para participar da banca de defesa desta tese e

pela solicitude em contribuir no engrandecimento deste trabalho.

Aos funcionários da Faculdade de Medicina de Sobral pela convivência, atenção e

disponibilidade durante todos esses 10 anos de convívio.

A todos que de alguma forma me deram força e incentivo na realização do meu

doutoramento, seja profissionalmente ou sentimentalmente e por participarem da minha vida.

Por fim, agradeço a todos que contribuíram, de alguma forma, ou torceram para que

eu chegasse até aqui, compartilhando comigo um momento tão importante. A todos vocês, de

coração, o meu MUITO OBRIGADO!!!

8

“Porquanto, ainda que a figueira não floresça,

nem haja fruto na vide; o produto da oliveira

minta, e os campos não produzam mantimento;

Todavia eu me alegrarei no Senhor, exultarei no

Deus da minha salvação. O Senhor é minha força

e me fará andar sobre as minhas alturas.”

(HABACUQUE, 3:17-19)

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RESUMO

Este estudo teve como objetivo investigar o efeito da 5-Aza-citidina durante a indução da

pluripotência, e avaliar os efeitos do cultivo em meio contendo BMP-2, BMP-4 ou fluido

folicular na diferenciação de fibroblastos em células germinativas primordias (CGP) e oócitos

(fase 1). Além disso, isolar células-tronco do epitélio ovariano de bovinos e avaliar os efeitos da

BMP-2, BMP-4 ou fluido folicular sobre a diferenciação destas células-tronco em estruturas

semelhantes a oócitos (fase 2). Na fase 1, os fibroblastos foram tratados com 0.5, 1,0 ou 2.0 μM

de 5-Aza por 18, 36 ou 72 h. Foi avaliada a morfologia, viabilidade celular e a expressão gênica

(OCT-4, NANOG, REX e SOX2), para a seleção da concentração/tempo mais eficientes. Os

fibroblastos foram então cultivados em meio suplementado com 10 ng/mL de BMP-2, ou 10

ng/mL de BMP-4 ou 5% de fluido folicular bovino, por 7 ou 14 dias. Posteriormente, foi feita a

avaliação da morfologia e viabilidade celular, e expressão gênica (VASA, DAZL, C-KIT, SCP3,

ZPA e GDF-9). Para a fase 2, as células-tronco da superfície ovariana foram isoladas,

expandidas, cultivadas em meio de diferenciação contendo as 50 ng/mL de BMP-2, ou 50 ng/mL

de BMP-4, ou BMP-2+BMP-4 ou 5% de fluido folicular bovino por 14 dias. Foram avaliadas as

características morfológicas, viabilidade celular, e expressão da fosfatase alcalina e expressão

gênica (VASA, DAZL, C-KIT, SCP3, ZPA e GDF-9). Os resultados de expressão gênica foram

analisados usando ANOVA seguido pelo Teste de Kruskal Wallis (P<0,05). Na fase 1, o cultivo

com 2.0 μM de 5-Aza por 72 h provocou mudanças na morfologia e na taxa de proliferação

celular, e aumentou significativamente a expressão de fatores de pluripotência. Já o cultivo em

meio contendo BMP-2, BMP-4 ou fluido folicular, durante 7 ou 14 dias, alterou a morfologia

celular, e a expressão de genes específicos para células germinativas e oócitos. Na fase 2, as

células-tronco do ovário expressaram genes de pluripotência e após o cultivo, as células

apresentaram características morfológicas que se assemelhavam a CGP e a células semelhantes a

oócitos, incluindo a expressão da fosfatase alcalina e a expressão de genes específicos de células

germinativas e oócitos. Em conclusão, o presente estudo descreve a possibilidade de converter os

fibroblastos da pele de bovinos e células-tronco da superfície ovariana, em células semelhantes a

oócitos, através do processo de reprogramação celular, associado à suplementação do meio com

BMP-2, BMP-4 e fluido folicular.

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Palavras-chave: Expressão gênica. Fibroblastos bovinos. Células-tronco adultas. Cultivo in

vitro.

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ABSTRACT

This study aimed to investigate the effect of 5-Aza-cytidine during induction of pluripotency, and

evaluate the effects of culture in medium containing BMP-2, BMP-4 or follicular fluid in the

differentiation of fibroblasts in primordias germ cells (CGP) and oocytes (stage 1). Furthermore,

isolation of stem cells from the bovine ovarian epithelium and evaluate the effects of BMP-2,

BMP-4 or follicular fluid on the differentiation of these stem cells into structures similar to

oocytes (stage 2). In phase 1, fibroblasts were treated with 0.5, 1.0 or 2.0 µM of 5-Aza for 18, 36

or 72 h. Morphology cell viability and gene expression (OCT-4, NANOG, SOX2 and REX), were

assessed, for the selection of the concentration/time more efficient. The fibroblasts were then

cultured in medium supplemented with 10 ng/mL BMP-2 or 10 ng/mL BMP-4 or 5% bovine

follicular fluid by 7 or 14 days. Subsequently, evaluation of the morphology and cell viability

was taken, and gene expression (VASA, DAZL, c-Kit, SCP3, ZPA and GDF-9). For phase 2, the

stem cells of ovarian surface were isolated, expanded, grown in differentiation medium

containing 50 ng/mL BMP-2 or 50 ng/mL BMP-4, or BMP-2+BMP-4 or 5% bovine follicular

fluid for 14 days. Morphological characteristics, cell viability and expression of alkaline

phosphatase and gene expression (VASA, DAZL, C-KIT, SCP3, ZPA and GDF-9), were evaluated.

The gene expression results were analyzed using ANOVA followed by Kruskal-Wallis test (P

<0.05). In stage 1, the culture with 2.0 µM of 5-Aza for 72 h caused changes in morphology and

cell proliferation rate, and significantly increased the expression of pluripotency factors. The

culture in medium containing BMP-2, BMP-4 or follicular fluid for 7 or 14 days, altered cellular

morphology, and expression of specific genes for stem cells and oocytes. In stage 2, the ovarian

stem cells expressed pluripotent genes, and after culture, this cells showed morphologic

characteristics similar to PGC and oocytes, including the expression of alkaline phosphatase, and

the expression of specific genes for PCG and oocytes. In conclusion, the present study describes

the possibility of conversion of the skin fibroblasts and stem cells from ovarian surface of the

bovine, in oocyte-like cells, similar oocyte cells, through the cell reprogramming process

associated with the supplementation of BMP-2, BMP- 4 and follicular fluid.

Keywords: Gene expression. Bovine fibroblasts. Adult stem cells. In vitro culture.

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LISTA DE ILUSTRAÇÕES

Figura 1 - Desenho esquemático do ovário de mamíferos com suas diversas

estruturas........................................................................................................................

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Figura 2 - Representação esquemática do desenvolvimento das CGP e dos sinais

envolvidos na regulação da migração e colonização CGP em camundongos............... 31

Figura 3 - Especificação da linhagem germinativa e eventos moleculares.................. 34

Figura 4 – Representação esquemática do papel da sinalização das BMPs e WNT

durante a formação das células germinativas primordiais (CGPs) em embriões..........

36

Figura 5 – Regulação da transcrição da especificação das CGPs................................. 37

Figura 6 – Isolamento e diferenciação das células-tronco embrionárias...................... 43

Figura 7 – Isolamento e diferenciação de células-tronco mesenquimais..................... 45

Figura 8 – Reprogramação de células adultas e formação das células iPS.................. 50

CAPÍTULO 1

Figure 1 - Schematic presentation of the development of stem cells into oocytes-like

cells, and the stimulating substances and markers for each stage of differentiation…. 92

Figure 2 - Procedure for restoring fertility by differentiating iPS into oocytes............ 93

CAPÍTULO 2

Figure 1 - Representative pictures of the morphological changes in bovine skin

fibroblasts exposed to 5-Aza. (A) The fibroblast cells isolated from bovine fetal ear

skin (untreated cells). (B) Fibroblasts exposed to 2.0 µM of 5-Aza for 72 h. (C)

Fluorescence staining of viable cells for Calcein AM. (D) Fluorescence staining of

apoptotic cells for ethidium homodimer-1. Scale bar = 100

µm………………………………………….………………………...……………….. 133

Figure 2 - Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in

fibroblasts cultured for 18 h in with different concentrations of 5-Aza (0.5, 1.0 or 2.0

µM)………………………………………………………………………………….. 134

Figure 3 - Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in


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µM)…………………………………………………………………………………... 135

Figure 4. Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in


µM)…………………………………………………………………………………… 136

Figure 5. Levels of mRNA for of pluripotency genes, (A) SOX2, (B) NANOG, (C)

OCT4 and (D) REX, in fibroblasts cultured for 18 h, 36 h or 72 h in with different

concentrations of 5-Aza (0.5, 1.0 or 2.0 µM)………………………………………… 137

Figure 6. Representative pictures of the morphological characterization in bovine

skin fibroblasts exposed to 5-Aza and cultured in differentiation medium for 14

days. Fibroblast cultured for 14 days in differentiation medium supplemented with

10 ng/mL of BMP-2 (line 1), 10 ng/mL of BMP-4 (line 2), 5% follicular fluid (line

3), (A, D, G) cell analyzed by light microscopy, (B, E, H) Fluorescence staining of

viable cells for Calcein AM; (C, F, I) Fluorescence staining of apoptotic cells for

ethidium homodimer-1. Scale bar = 100 µm…………………………………………. 138

Figure 7. Levels of mRNA for markers of germ cells [VASA (A, B), DAZL (C, D),

C-KIT (E, F)] in cells cultured for 7 (A, C, E) or 14 (B, D, F) days in control

medium or supplemented with BMP-2 (10 ng/mL), BMP-4 (10 ng/mL) and 5%

follicular fluid………………………………………………………………………… 139

Figure 8. Levels of mRNA for markers of oocytes [ZPA (A, B), GDF-9 (C, D),

SCP3 (E, F)] in cells cultured for 7 (A, C, E) or 14 (B, D, F) days in control medium

or supplemented with BMP-2 (10 ng/mL), BMP-4 (10 ng/mL) and 5% follicular

fluid…………………………………………………………………………………… 140

Figure 9. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-

KIT (C)] and oocytes [ZPA (D), GDF-9(E), C-KIT (F)] after culture cells for 0 h (5-

Aza), 7 or 14 days in control medium……………………………………………....... 141


KIT (C)] and oocytes [ZPA (D), GDF-9(E), C-KIT(F)] after culture cells for 0 h (5-

Aza), 7 or 14 days in medium supplemented with 10 ng/mL of BMP-2……………... 142


KIT (C)] and oocytes [ZPA (D), GDF-9(E), C-KIT(F)] after culture cells for 0 h (5-

Aza), 7 or 14 days in medium supplemented with 10 ng/mL of BMP-4…………… 143

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KIT (C)] and oocytes [ZPA (D), GDF-9(E), C-KIT(F)] after culture cells for 0 h, 7 or

14 days in medium supplemented with 5% follicular fluid…………………………... 144

CAPÍTULO 3

Figure 1. Fluorescence staining of ovarian stem cells before (A-B) and after 14 days

culture in minimum essential medium (α-MEM) (C-D) or supplemented with BMP2

(E-F), BMP4 (G-H), both BMP2 and BMP4 (I-J) and follicular fluid (K-L). Scale

bar = 100 µm. Green (calcein) staining cells are viable and red (ethidium

homodimer) staining cells are not…………………………………………………….. 167

Figure 2. Alkaline phosphatase activity in ovarian stem cells at day 0. Scale bar =

100 µm………………………………………………………………………………... 168

Figure 3. Cycle threshold (CT) after amplification of mRNA for pluripotency stem

cell markers (OCT4 and SOX2) by real time qRT-PCR……………………………… 168

Figure 4. Levels of mRNA for specific germline cell [VASA (A), DAZL (B)] and

oocyte [SCP3 (C), GDF9 (D) and ZPA (E)] markers in ovarian stem cells cultured

for 14 days in MEM supplemented with BMP2, BMP4, BMP2 and BMP4 and

follicular fluid. Significant differences at P<0.05……………………………………. 169

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LISTA DE TABELAS

CAPÍTULO 1

Table 1. Molecular markers for stem cells, PCGs, oocytes and their functions............ 89

CAPÍTULO 2

Table 1. Primer pairs used in real-time PCR for quantification of markers of

pluripotency, germ cells and oocytes genes expressed in cells cultured……………...

132

CAPÍTULO 3

Table 1. Primer pairs used in real-time PCR…………………………………………. 166

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LISTA DE ABREVIATURAS E SIGLAS

Português Inglês

Alk2 Receptor de ativina semelhante à

quinase tipo 2

Activin receptor-like kinase-2

AP Fosfatase alcalina Alkaline phosphatase

AP2g Proteína de ligação e ativação do

regulador - 2 gama

Activating enhancer binding protein 2

gamma

ASC Células-tronco adultas Adult stem cells

AVE Endoderma visceral anterior Anterior visceral endoderm

Blimp1 Proteína de maturação induzida por

linfócitos B - 1

B lymphocyte-induced maturation

protein-1

BMP Proteína morfogenética óssea Bone morphogenetic protein

BMP-15 Proteína morfogenética óssea - 15 Bone morphogenetic protein – 15



BMP-8b Proteína morfogenética óssea - 8b Bone morphogenetic protein - 8b

BOULE Proteína Boule Boule protein

BRG1 Brahma-Related Gene - 1

BSA Albumina sérica bovina Bovine serum albumin

CD133 Antígeno também conhecido por

prominina-1

Antigen also known as prominin-1

CDH1 Caderina-1 Cadherin-1

CDX2 Caudal-related homeobox

CGP Célula Germinativa Primordial Primordial germ cell

c-Kit Receptor para kit ligante Kit ligand receptor

c-MYC Myc proto-oncogene protein

COLA4 Colagenase microbiana Microbial collagenase

Cx43 Conexina 43 Connexin 43

DA Aorta dorsal Dorsal aorta

DAZL Suprimido na azoospermia Deleted in azoospermia like

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DDX-4 Polipeptídeo caixa DEAD 41 DEAD box polypeptide 4

Dmc1 Proteina de recombinação meiótica 1 DNA meiotic recombinase 1

DMEM/F12 Dulbecco's Modified Eagle Medium:

Nutrient Mixture F-12

DNA Ácido desoxirribonucleico Deoxyribonucleic acid

DNMT DNA metiltransferase Deoxyribonucleic acid

methyltransferase

DNMT1 DNA metiltransferase 1 Deoxyribonucleic acid

methyltransferase 1

Dpc Dias pós-concepção Days post coitum

Dppa3 Gene de pluripotência associada ao

desenvolvimento – 3

Developmental pluripotency

associated 3 pseudogene 2

DVE Endoderma visceral distal Distal visceral endoderm

EBs Corpos embrióides Embryoid bodies

EM Mesoderma embrionária Embryonic mesoderm

Epi Epiblasto Epiblast

ESC Células-tronco embrionárias Embryonic stem cells

Evx1 Even-Skipped Homeobox 1

ExE Ectoderma extra-embrionário Extra-embryonic ectoderm

ExM Mesoderma extra-embrionário Extra-embryonic mesoderm

FGSCs Células-tronco de linha germinativa

feminina

Female germline stem cell

FIGα Factor in the germline alpha

FOP Falha ovariana precoce Premature ovarian failure

FOXH1 Forkhead box protein H1

Fragilis Mouse interferon-induced protein like

gene-1

FSH Hormônio Folículo Estimulante Follicle-stimulating hormone

FSHR Receptor do Hormônio Folículo

Estimulante

Follicle-stimulating hormone receptor

GATA3 Proteína 3 de ligação a GATA GATA binding protein 3

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GDF3 Fator de diferenciação de crescimento

tipo 3

Growth differentiation factor type 3

GDF-9 Fator de diferenciação de crescimento

tipo 9

Growth differentiation factor type 9

GFP Proteína fluorescente verde Green fluorescent protein

giPS Células-tronco de pluripotência

induzidas caprinas

Goats induced pluripotent stem cells

GV Vesícula germinativa Germinal vesicle

H2AX Histona membro da família X H2A histone family member X

HDAC Histona deacetilase Histone deacetylase

HDM Histona demetilase Histone demethylase

hESC Células-tronco embrionárias de

humanos

Human embryonic stem cells

Hg Intestino primitivo Hindgut

HGF Fator de crescimento de hepatócitos Hepatocyte Growth Factor

HMT Histona metiltransferase Histone methyltransferase

Hoxa1 Homeobox protein Hox-A1

Hoxb1 Homeobox protein Hox-B1

ICM Massa celular interna Inner cell mass

Ifitm3 Proteína transmembranar induzida 3 Interferon-induced transmembrane

protein 3

IGF2 Fator de crescimento semelhante à

insulina-2

Insulin like growth factor-2

IM Mesoderma intermediário Intermediate mesoderm

iPS Células-tronco de pluripotência

induzidas

Induced pluripotent stem cells

IVF Fertilização in vitro In vitro fertilization

KLF4 Fator semelhante a Kruppel – 4 Kruppel-like factor 4

KRT7 Queratina 7 Keratin 7

KRT8 Queratina 8 Keratin 8

LIF Fator inibidor de leucemia Leukemia inhibitory fator

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LIN28 Lin-28 homolog A

MAPK Proteína cinase ativada por mitógenos Mitogen-activated protein kinase

MEM Meio essencial mínimo Minimal essential medium

mESCs Células-tronco embrionárias de ratos Mouse embryonic stem cells

mRNA Ácido Ribonucléico mensageiro Messenger Ribonucleic Acid

MSC Células-tronco mesenquimais Mesenchymal stem cells

mTS Células-tronco do trofoblasto murinho Stem cells from murine trophoblast

Mvh Proteína de rato homóloga a proteína

VASA

Mouse vasa homolog

NaB Butirato de sódio Sodium butyrate

Nanog Nanog Homeobox

NANOS3 Nanos Homolog 3

NeC Cordão nefrogênico Nephrogenic cord

NoC Notocorda Notochord

NODAL Fator de diferenciação de crescimento

nodal

Nodal growth differentiation factor

NT Tubo neural Neural tube

Oct4 Fator de transcrição ligado ao

octâmero 4

Octamer-binding transcription factor 4

OCT-4A Fator de transcrição ligado ao

octâmero 4A

Octamer-binding transcription factor

4A

OGSCs Células-tronco germinativas do ovário Ovarian germ stem cells

OLC Células semelhantes a oócitos Oocyte-like cells

OMS Organização Mundial de Saúde World Health Organization

ORF Quadro aberto de leitura Open reading frame

OSC Células-tronco ovarianas Ovarian stem cells

OSE Epitélio de superficie do ovário Ovarian surface epithelium

OSKM OCT4, SOX2, KLF4 e MYC OCT4, SOX2, KLF4 and MYC

PGC Célula germinativa primordial Primordial germ cell

Pgc7 Proteína 7 em células germinativas

primordiais

Primordial germ cell protein 7

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PGCLC Células semelhantes a células

germinativas primordiais

Primordial germ cell-like cells

pH Potencial hidrogeniônico Potential hydrogen

PMSG Gonadotrofina de égua prenhe Pregnant mare serum gonadotropin

Pou5f1 Domínio POU, classe 5, fator de

transcrição 1

POU domain, class 5, transcription

factor 1

Prdm1 Proteína dedo de zinco de domínio

PR1

PR domain zinc finger protein 1

Prdm14 Proteína dedo de zinco de domínio PR

14

PR domain zinc finger protein 14

REC8 Proteína de recombinação meiótica 8 Meiotic recombination protein 8

RG108 N - Ftalil – L – Triptofano N-Phthalyl-L-Tryptophan

RNA Ácido Ribonucléico Ribonucleic acid

RNAm Ácido Ribonucléico mensageiro Messenger ribonucleic acid

R-Smads Receptor regulado dos mensageiros

intracelulares

Receptor-regulated mothers against

decapentaplegic homolog

SCP1 Proteína 1 do complexosinaptonêmico Synaptonemal complex protein 1



Smad1 Mensageiro intracelular do tipo 1 Mothers against decapentaplegic

homolog 1

SMAD2 Mensageiro intracelular do tipo 2 Mothers against decapentaplegic

homolog 2


homolog 4


homolog 5

Sox2 Região determinante do sexo no

cromossomo Y - Caixa 2

Sex determining region Y-box 2

SSEA Antígeno embrionárioestágio-

específico

Stage specific embryonic antigen

21

SSEA-1 Antígeno embrionárioestágio-

específico 1

Stage specific embryonic antigen 1


específico – 3

Stage specific embryonic antigen – 3


específico – 4

Stage specific embryonic antigen – 4

STAT3 Transdutor de sinal e ativador da

transcrição - 3

Signal transducer and activator of

transcription 3

Stella Gene de pluripotência associado ao

desenvolvimento - 3

Developmental pluripotency-associated

3

STELLAR Pseudogene de pluripotência 2

associado ao desenvolvimento - 3

Developmental pluripotency

associated 3 pseudogene 2

Stra8 Gene 8 estimulado pelo ácido

retinóico

Stimulated by retinoic acid gene 8

SYCP3 Proteína 3 do complexosinaptonêmico Synaptonemal complex protein 3

Tcfap2c Fator de transcrição AP-2 gama Transcription factor AP-2, gamma

TEKT1 Tektina-1 Tektin-1

TERT Transcriptase inversa da telomerase Telomerase reverse transcriptase

TGF-β Superfamília de fatores de

crescimento transformante beta

Transforming growth factor beta

Tnap Fosfatase alcalina não especifica a

tecidos

Tissue non-specific alkaline

phosphatase

TRA1-60 Gene semelhante a podocalixina 60 Podocalyxin like 60

TRA1-81 Gene semelhante a podocalixina 81 Podocalyxin like gene 80

TS Células-tronco do trofoblasto Trophoblast stem cells

TSA Tricostatina A Trichostatin A

UCB Sangue de cordão umbilical Umbilical cord blood

Vasa Proteína Vasa Vasa protein

VE Endoderma visceral Visceral endoderm

VEGF Fator de crescimento endotelial

vascular

Vascular endothelial growth fator

22

VPA Ácido valpróico Valproic acid

VSEL Células-tronco semelhante à células

embrionárias pequenas

Small embryonic-like stem cells

WNT3 Wingless-Type MMTV Integration

Site Family, Member 3

XEN Células-tronco do endoderma extra-

embrionário

Extraembryonic endoderm stem cells

ZP2 Glicoproteína da zona pelúcida 2 Zona pellucida glycoprotein 2

ZPA Glicoproteína da zona pelúcida A Zona pellucida glycoprotein A

ZPC Glicoproteína da zona pelúcida C Zona pellucida glycoprotein C

β-hCG Gonadotrofina coriônica humana beta Beta human chorionic gonadotropin

23

LISTA DE SÍMBOLOS

Português Inglês

% Percentagem Percentage

~ Aproximadamente Aproximately

± SEM Erro padrão da média Standard error of the mean

°C Graus Celsius Degrees Celsius

µg Micrograma Microgram

µL Microlitro Microliter

µm Micrômetro Micrometer

µM Micromolar Micromolar

CO2 Dióxido de carbono Carbon dioxide

h Hora Hour

IU/mL Unidades internacionais por mL International units per mL

min Minuto Minute

mg Miligrama Milligram

mL Mililitro Milliliter

mM Milimolar Millimolar

mm Milímetro Millimeter

ng Nanograma Nanogram

nm Nanômetro Nanometer

P < 0,05 Probabilidade de erro menor do que

5%

Error probabilities is less than 5%

P > 0,05 Probabilidade de erro maior do que

5%

Error probabilities is more than 5%

24

SUMÁRIO

1 INTRODUÇÃO............................................................................................. 25

2 REVISÃO DE LITERATURA..................................................................... 28

2.1 Ovário mamífero........................................................................................... 28

2.2 Formação e desenvolvimento das células germinativas e de oócitos........ 29

2.2.1 Mecanismos de especificação das CGPs........................................................ 32

2.2.1.1 Sinalização da especificação das CGPs.......................................................... 35

2.2.2 Migração de células germinativas primordiais............................................. 37

2.3 Células-tronco: definição, classificação e aplicações.................................. 39

2.3.1 Células-tronco embrionárias.......................................................................... 42

2.3.2 Células-tronco adultas.................................................................................... 44

2.3.2.1 Células-tronco ovarianas................................................................................ 47

2.3.3 Células-tronco de pluripotência e reprogramação celular.......................... 49

2.4 Proteínas morfogenéticas ósseas e seu papel durante a formação das

CGPs............................................................................................................... 53

2.5 Fluido folicular e seu papel durante a formação das CGPs e

oócitos............................................................................................................. 56

3 CAPÍTULO 1

ARTIGO I: In vitro differentiation of primordial germ cells and oocytes-

likes cells from stem cells................................................................................

58

4 PROBLEMA.................................................................................................. 94

5 JUSTIFICATIVA.......................................................................................... 95

6 HIPÓTESES CIENTÍFICAS....................................................................... 97

7 OBJETIVOS.................................................................................................. 98

7.1 Objetivos gerais............................................................................................. 98

25

7.2 Objetivos específicos...................................................................................... 98

8 CAPÍTULO 2

ARTIGO II: Expression of markers for germ cells and oocytes in cow

dermal fibroblast treated with 5-aza-cytidine and cultured in presence of

BMP-2, BMP-4 and follicular fluid.................................................................

99

9 CAPÍTULO 3

ARTIGO III: Bovine ovarian stem cells differentiate into germ cells and

oocyte-like structures after culture in vitro.....................................................

145

10 CONCLUSÕES............................................................................................. 170

11 PERSPECTIVAS........................................................................................... 171

REFERÊNCIAS............................................................................................ 172

25

1 INTRODUÇÃO

A infertilidade tem sido reconhecida pela Organização Mundial de Saúde (OMS),

como um problema de saúde pública em todo o mundo, emergindo como um dos principais

desafios do novo milênio para aqueles envolvidos no tratamento de infertilidade e da reprodução

assistida (VAYENA et al., 2001). No mundo, estima-se que a infertilidade afete

aproximadamente 14% dos casais (BOIVIN et al., 2007). Assim, nos últimos anos, novas

tecnologias reprodutivas tem sido desenvolvidas na tentativa de ajudar a reduzir este problema.

Entretanto, nos países em desenvolvimento, as tecnologias reprodutivas mais recentes não estão

disponíveis ou tem elevado custo financeiro (OMBELET et al., 2008). Além disso, a elucidação

de mecanismos e fatores envolvidos na biologia reprodutiva possibilitará o desenvolvimento de

protocolos de preservação de gametas de animais de elevado patrimônio genético ou em risco de

extinção. As biotécnicas reprodutivas devem garantir a manutenção da morfologia folicular e a

obtenção de oócitos viáveis a serem utilizados em programas de criopreservação, cultivo e

fecundação in vitro, melhorando a produtividade de animais de alto valor zootécnico ou em via

de extinção.

A infertilidade feminina, com a exaustão prematura do pool de oócitos, pode ocorrer

em decorrência de diferentes patologias e devido a uma série de perturbações como doenças auto-

imunes, doenças genéticas ligadas ao cromossomo X, ooforectomia por neoplasias ovarianas

benignas ou malignas, endometriose e cistos ovarianos, por exemplo. Além disso, os tratamentos

de quimioterapia convencionais são as causas mais comuns de infertilidade em pacientes com

câncer. A quimioterapia exerce efeito anti-mitótico inibindo a divisão celular e a replicação do

DNA, e a exposição do ovário a estas drogas induz danos irreversíveis aos folículos e aos oócitos,

resultando em infertilidade (SILVESTRIS et al., 2015). Há várias técnicas de reestabelecimento

da fertilidade, como a criopreservação de tecido ovariano seguido por transplante oocitário

autólogo, congelamento de embriões, cultivo in vitro do tecido ovariano, de folículos e de

oócitos, os quais previnem danos ao conteúdo nuclear (SILVESTRIS et al., 2015). No entanto,

estas técnicas não são totalmente seguras, pois, com o efeito da estimulação hormonal para a

maturação do pool de oócitos, tumores sensíveis ao estrogênio como os cânceres de mama e de

endométrio podem crescer rapidamente antes do tratamento de quimioterapia. Por outro lado, as

técnicas baseadas na criopreservação de tecido ovariano, após a estimulação hormonal e

subsequente transplante autólogo, implica no risco de reintroduzir as células malignas,

26

especialmente em pacientes com leucemia que podem abrigar células malignas na corrente

sanguínea.

Além dos tratamentos de reprodução assistida estabelecidos acima, os cientistas estão

desenvolvendo novas abordagens baseadas em células-tronco para o tratamento da infertilidade

(CHIRPUTKAR; VAIDYA, 2015), visto que as células-tronco têm a capacidade de reconstituir

os diversos tecidos corporais. As células-tronco embrionárias (ESC) são isoladas a partir da

massa celular interna, permanecendo em estado indiferenciado e capazes de diferenciar-se em

células das três camadas germinativas in vitro e in vivo. Já as células-tronco adultas (ASC) são

conhecidas por estar presentes em vários órgãos do corpo, como a pele, medula óssea, cérebro,

coração, tecido adiposo, etc. Estas células embora presentes na fase quiescente, podem ser

estimuladas pela secreção de fatores solúveis para a reconstituição tecidual (CHIRPUTKAR;

VAIDYA, 2015). As células de pluripotência induzida (iPS) foram mencionadas pela primeira

vez em 2006, por Takahashi e Yamanaka, sendo então intensamente estudadas. Através desta

técnica, células somáticas adultas, são modificadas e adquirem comportamento muito semelhante

ao das células-tronco embrionárias, inclusive molecularmente. O uso das iPS reduz questões

éticas relacionadas ao uso de células-tronco de origem embrionária. No entanto, os mecanismos

biológicos relacionados à formação e diferenciação das iPS bovinas ainda não foram

completamente elucidados (TAKAHASHI; YAMANAKA, 2006; YAMANAKA, 2008). Além

disso, durante muitos anos, acreditou-se que não haveria a possibilidade de renovação da a

reserva oocitária. Porém, com o avanço dos estudos da oogênese, a teoria se tornou alvo de

grande controvérsia (TILLY; JOHNSON, 2007). Diante do exposto, as pesquisas que visam

solucionar os problemas de infertilidade em humanos, bem como aumentar a eficiência

reprodutiva de animais de alto valor genético ou em via de extinção, são de grande importância.

Assim, a possibilidade de formação de novos oócitos após o nascimento, utilizando-se células-

tronco de diferentes origens, pode ter uma importante aplicação terapêutica reprodutiva, mas

ainda não se sabe quais os mecanismos ideiais para a produção de gametas.

Para um melhor entendimento da importância deste trabalho, a seguir serão

abordados aspectos relacionados ao ovário mamífero, formação das células germinativas e

diferenciação em oogônias, formação e diferenciação de células-tronco embrionárias, células-

tronco adultas, células-tronco de pluripotência induzida, as funções das proteínas morfogenéticas

27

ósseas (BMP) e do fluido folicular no processo de formação de células germinativas e

diferenciação em oócitos.

28

2 REVISÃO DE LITERATURA

2.1 Ovário mamífero

A palavra ovário é derivada do latim ovum que significa ovo. Este órgão, além de ser

a gônada feminina que contém o suprimento de células germinativas para a produção da próxima

geração, funciona também como uma glândula reprodutiva que controla diversos aspectos

fisiológicos e do desenvolvimento das fêmeas (EDSON; NAGARAJA; MATZUK, 2009). Dessa

forma, o ovário possui dois papéis primários: (i) a liberação de um oócito inteiramente

competente para fertilização e desenvolvimento embrionário, caracterizando a função

gametogênica e (ii) a preparação de órgãos reprodutivos acessórios para a gestação e o

nascimento através da produção de hormônios esteroides, que retrata a função endócrina deste

órgão (GOUGEON, 2004).

Figura 1 - Desenho esquemático do ovário de mamíferos com suas diversas estruturas.

O ovário mamífero apresenta folículos em diferentes estágios de desenvolvimento (primordiais, primários,

secundários, terciários e pré-ovulatórios), corpo lúteo e oogônias, na superfície ovariana (TILLY; JOHNSON, 2007).

Fonte: Adaptado de http://academic.rcc.edu/moore/docs/Bio30/Lecture%208%20-%20Oogenesis.pdf.

http://academic.rcc.edu/moore/docs/Bio30/Lecture%208%20-%20Oogenesis.pdf

29

Nos mamíferos, o tamanho e o formato do ovário variam de acordo com a espécie e

com a fase do ciclo estral/menstrual (HAFEZ; HAFEZ, 2004). Em geral, os ovários dos

mamíferos estão organizados em córtex e medula (Figura 1) (ARAKI, 2003; GARTNER;

HIATT, 2007). O epitélio superficial, pode variar o formato de pavimento à cuboide (mais

comum), que cobre o ovário é denominado epitélio germinativo. Imediatamente abaixo deste

epitélio, fica a túnica albugínea, uma cápsula de tecido conjuntivo denso não modelado, que é

pouco vascularizada (GARTNER; HIATT, 2007). O córtex do ovário, com uma alta densidade

celular, é constituído por uma estrutura de tecido conjuntivo, o estroma, que contém células

semelhantes a fibroblastos (GARTNER; HIATT, 2007), assim como folículos ovarianos e/ou

corpos lúteos em vários estágios de desenvolvimento e regressão (HAFEZ; HAFEZ, 2004). A

medula, por sua vez, é constituída principalmente por tecido conjuntivo frouxo, altamente

vascularizado (vasos sanguíneos e linfáticos), e fibras nervosas (GARTNER; HIATT, 2007).

Histologicamente, não há um limite bem definido entre essas duas regiões (GARTNER; HIATT,

2007).

Os eventos que marcam a morfogênese do ovário fetal incluem a colonização por

células germinativas primordiais (CGP), interação das células germinativas primordiais com

células somáticas, formação dos cordões ovígeros e, finalmente, o desaparecimento dos cordões

ovígeros com concomitante estabelecimento de uma população de folículos primordiais e uma

complexa rede vascular (JUENGEL et al., 2002). A cronologia no desenvolvimento desses

eventos que culminam na formação dos folículos primordiais parece ser semelhante em todos os

mamíferos (SAWYER et al., 2002).

Durante muitos anos, acreditou-se que a reserva folicular ovariana fosse formada na

embriogênese e que não haveria a possibilidade de renovação. Porém, com o avanço da ciência e

consequente aumento de metodologias para o estudo da foliculogênese, a teoria se tornou alvo de

grande controvérsia, pois demonstrou-se, então, a possibilidade de renovação, todavia, ainda não

se sabe se ela realmente ocorre naturalmente e em quais condições poderia ocorrer (TILLY;

JOHNSON, 2007).

2.2 Formação e desenvolvimento das células germinativas e de oócitos

30

As CGP foram identificadas pela primeira vez em mamíferos por Chiquoine em

1954, que encontrou uma população de células capaz de gerar oócitos e espermatozoides

(CHIQUOINE, 1954). As CGP, que se originam do epiblasto proximal, são os precursores

embrionários dos gametas e a especificação destas células é uma das primeiras decisões do

destino celular feita pelo embrião (YOUNG; DIAS; LOVELAND, 2010). Em camundongos,

após cerca de 4,0-4,5 dias pós-concepção (dpc), o blastocisto é composto por três tipos de células,

o trofectoderma, o ectoderma primitivo e a endoderme primitivo (ROSSANT; TAM, 2009). Após

a implantação, em torno de 6,0 dpc, um pequeno grupo de células do epiblasto proximal,

originado do ectoderma primitivo, é direcionado para entrar na linhagem germinativa e formar as

CGPs (DE SOUSA LOPES et al., 2004; 2007) (Figura 2).

As CGP tornam-se identificáveis durante o início da gastrulação pela a atividade

positiva da fosfatase alcalina (AP). Além disso, formam um grupo de ~40 células na base do

alantóide incipiente no mesoderma extra-embrionário (ExM), por volta do 7,25 dia embrionário

(GINSBURG; SNOW; MCLAREN, 1990; LAWSON; HAGE, 1994). Posteriormente, e

concomitante com um aumento em seu número, elas começam a migrar em direção ao

endoderma do intestino primitivo em desenvolvimento e se movem através dele. Através de

movimentos ameboides, as CGP, então, saem do endoderma e seguem para o mesentério, em

torno do dia E10,5 para colonizar gônadas embrionárias, onde elas proliferam (elas estarão

sofrendo mitoses, e o número dessas células aumenta significativamente) (SOTO-SUAZO;

ZORN, 2005), e iniciam mais uma diferenciação em oócitos ou espermatozóides dependendo do

sexo (BOWLES; KOOPMAN, 2007). Nem todas as CGP irão obter sucesso nesta migração, e

aquelas que não alcançarem a crista genital irão se degenerar (ADAMS et al., 2008). Um evento

chave que ocorre nas CGP durante esta fase proliferativa em ambos os sexos (machos e fêmeas) é

uma reprogramação epigenética, mais notavelmente, uma desmetilação do DNA de todo o

genoma, que inclui a inativação do imprinting genômico (SAITOU; KAGIWADA; KURIMOTO,

2012). No embrião fêmea (XX), as CGP continuam a proliferar até ~E13,5 (quando atingem

cerca de 25.000 células) e, posteriormente, entram na prófase I das divisões meióticas

(HILSCHER et al.,1974; SPEED, 1982). Em ovinos e bovinos, a diferenciação das CGP em

oogônias ocorre no 31º e 42º dia de gestação, respectivamente (RÜSSE, 1983). A fase que

antecede este processo meiótico é marcada pela replicação do DNA (GORDON, 1994). Nesta

fase, as oogônias se diferenciam e passam então a ser denominadas de oócitos (FAIR, 2003).

31

Figura 2 - Representação esquemática do desenvolvimento das CGP e dos sinais envolvidos na

regulação da migração e colonização CGP em camundongos.

Breve resumo da especificação e migração das CGP, bem como colonização das gônadas em camundongos. Epi:

epiblasto; (AVE: endoderma visceral anterior; ExE: ectoderma extra-embrionário; CGP: Células germinativas

primordiais; EM: mesoderma embrionário; ExM: mesoderma extra-embrionário; Hg: intestino primitivo; DA: aorta

dorsal; NT: tubo neural; NoC: notocorda; IM: mesoderma intermediário; NeC: cordão nefrogênico. Fonte: Adaptado

de SAITOU; YAMAJI, (2010); NIKOLIC et al., (2016).

32

As oogônias são células-tronco germinativas que expandem sua população por meio

de uma alta frequência de divisões mitóticas (PICTON; BRIGGS; GOSDEN, 1998). Devido às

rápidas e consecutivas divisões mitóticas, as oogônias são muitas vezes unidas em aglomerados

por pontes citoplasmáticas intercelulares, formando um sincício ou ninhos de oogônias,

organizados em cordões ovígeros (PICTON, 2001; TINGEN; KIM; WOODRUFF, 2009).

Durante a replicação do DNA, o ácido retinoico atua ativando proteínas responsáveis por

desencadear o início da meiose, como por exemplo, STRA-8, REC-8, DMC-1 e SCP-3

(MARQUES-MARI et al., 2009; BOWLES et al., 2006; KOUBOVA et al., 2006). As oogônias

apresentam um maior número de organelas intracelulares, antes de se diferenciarem em oócitos

primários (SUH; SONNTAG; ERICKSON, 2002; ABIR et al., 2006). Os oócitos primários

continuam a divisão meiótica passando pelos estágios de leptóteno, zigóteno e paquíteno da

prófase I, até atingirem o estágio de diplóteno (ARAKI, 2003). Os oócitos permanecem nesse

estágio até pouco antes da ovulação, em que após estimulação hormonal os oócitos completam a

primeira divisão meiótica com a extrusão concomitante do primeiro corpo polar e param

novamente na segunda divisão meiótica (oócito secundário), a qual será somente é concluída

após a fertilização (BOWLES; KOOPMAN, 2007). Durante a fertilização pelo espermatozoide, o

oócito completa a segunda divisão meiótica e extrusa o segundo corpo polar.

2.2.1 Mecanismos de especificação das CGPs

A origem exata da linhagem de células germinativas e o mecanismo de especificação

das CGP são ainda indefinidos, especialmente devido à ausência de marcadores específicos que

retratem os primeiros processos de especificação destas células (SAITOU; YAMAJI, 2010).

Entretanto, sabe-se que as CGP precursoras são formadas sob o controle de sinais oriundos das

células vizinhas, tais como as proteínas morfogenéticas ósseas (BMPs), -2, -4, e -8 (Figura 3)

(HUMMITZSCH et al., 2015). A análise da expressão gênica da população de CGP fundadoras

com E7,25 identificou dois genes, Fragilis e Stella, que são altamente e especificamente

expressos em CGP, respectivamente (SAITOU; BARTON; SURANI, 2002). Fragilis (Mouse

interferon-induced protein like gene-1, mil-1 ou ainda conhecido como interferon-induced

transmembrane protein 3, Ifitm3) (TANAKA; MATSUI, 2002) é um membro das proteínas

transmembranares induzíveis pelo interferon. Enquanto que Stella (também conhecido como

33

primordial germ cell 7, Pgc7, ou ainda conhecido como Developmental pluripotency-associated

3, Dppa3) (SATO et al., 2002) é uma pequena proteína que se transloca do citoplasma ao núcleo

e ainda na direção oposta. Fragilis começa a ser expresso nas células no epiblasto proximal, com

~E6,25-E6,5, e sua expressão se intensifica no mesoderma extra-embrionário posterior. Já a

expressão de Stella começa especificamente nas células que já estão expressando Fragilis no

mesoderma extra-embrionário (~E7,0-E7,25), e continua a ser expressa na migração das CGP.

Em aproximadamente E7, pequenos grupos de CGP, que são estabilizados por E-caderina, foram

localizados posterior à linha primitiva no mesoderma extra-embrionário (HUMMITZSCH et al.,

2015).

As células Stella-positivas mostram uma elevada expressão de fosfatase alcalina

tecido não específica (Tnap), um gene para a atividade AP em CGP (MACGREGOR;

ZAMBROWICZ; SORIANO,1995). As células com expressão positiva para Stella e altos níveis

de RNAm para Fragilis reprimem a expressão de genes Homeobox tais como Hoxb1, Hoxa1,

Evx1 e VL1, enquanto que as células Fragilis-positivas, mas Stella-negativas mantém a expressão

de genes Hox (SAITOU; BARTON; SURANI, 2002). Saitou et al. (2002) propuseram que as

células Stella-positivas e Homeobox-negativas são importantes para o estabelecimento das CGP.

Além disso, estudos revelaram que nem Fragilis e nem Stella são genes essenciais para a

especificação das CGP (PAYER et al., 2003; LANGE et al., 2008).

Essa variação na expressão dos genes Homeobox é importante, uma vez que o papel

desses genes é especificar a identidade das células ao longo do eixo do corpo ou induzir a

diferenciação das células para linhagens celulares somáticas específicas. Isto sugere que as

células germinativas fundadoras adquirem a capacidade de evitar a especificação somática,

prevenindo ou suprimindo a expressão dos genes Homeobox. Esta poderia ser uma das principais

características que as células germinativas de mamíferos possuem e lhes permite manter ou

recuperar a totipotência (NIKOLIC et al., 2016). Este conceito é apoiado pela expressão

continuada de Oct4 (Octamer-binding transcription factor 4, também conhecido por Pou5f1), e

outros genes de pluripotência em células germinativas (YEOM et al., 1996). O gene Oct4 é

considerado um gene chave para pluripotência (NIWA, 2007), além de ser usado como um

marcador específico para a linhagem de células germinativas, apenas após ~E7,75 (YEOM et al.,

1996).

34

A análise do transcriptoma das CGP levou à identificação de dois genes reguladores

chave para a especificação das CGP, Blimp1 (B lymphocyte-induced maturation protein-1,

também conhecido como PR domain zinc finger protein 1 ou ainda PR domain containing 1,

[Prdm1]) e Prdm14 (PR domain zinc finger protein 14 ou ainda PR domain containing 14)

(OHINATA et al., 2005; VINCENT et al., 2005; YABUTA et al., 2006; KURIMOTO et al.,

2008; YAMAJI et al., 2008). Ainda para a especificação de CGP em mamíferos, a expressão do

gene Tcfap2c (também conhecido como AP2g) é necessária (WEBER et al., 2010). Todos os

precursores de CGP Blimp1-positivas inicialmente expressam os genes Hox e reprimem Sox2

(YABUTA et al., 2006; KURIMOTO et al., 2008). No entanto, a partir E6.75, as células Blimp1-

positivas começam a reprimir os genes Hox e expressam Sox2, Stella, e Nanog (YAMAGUCHI

et al., 2005; KURIMOTO et al., 2008). As células Blimp1-positivas continuam a expressar Oct4,

que regula o aumento da expressão de cerca de 500 genes de “especificação de células

germinativas” e reduz a expressão de cerca de 330 genes “somáticos” (KURIMOTO et al., 2008).

Figura 3 - Especificação da linhagem germinativa e eventos moleculares.

Durante o desenvolvimento embrionário in vivo, como resultado da estimulação por BMP-4, BMP-2 e BMP-8b, um

subconjunto de células do epiblasto começa a expressar os genes Fragilis e Blimp1 e tornam-se CGP que migram em

35

direção ao cume gonadal, onde proliferam. Durante a migração destas células, há a expressão de genes como Stella e

c-Kit e iniciam a colonização do cume gonadal. Neste momento, as CGP expressam os marcadores pré-meióticos

Dazl e Vasa. As proteínas SCP1, SCP2 e SCP3 são específicas de células germinativas em meiose, assim como o

gene Dmc1. Nas fêmeas, as células germinativas entram em meiose e param na prófase I. As células germinativas

masculinas não entram em meiose, apenas após o nascimento. Os marcadores pós-meióticos GDF-9 e TEKT1 são

específicos de gametas haplóides maduros. Fonte: adaptado de Marques-Mari (2009).

2.2.1.1 Sinalização da especificação das CGPs

Por volta de 5,5 dias de desenvolvimento embrionário em camundongos, as células

do epiblasto adquirem competência para responder a BMP-4 pelas atividades de NODAL e

WNT3. Por outro lado, a sinalização NODAL mediada por SMAD2/FOXH1 específica de

células da endoderme visceral distal, começam a fornecer sinais agonistas. Sinais do ectoderma

extraembrionário, incluindo BMP8b, aparentemente previne o endoderma visceral proximal de

diferenciar-se em endoderme visceral distal, restringindo assim, a atividade anti-agonista. Por

volta dos dias 6,0-6,25, a endoderme visceral distal move-se para formar endoderme visceral

anterior, e um subconjunto de células do epiblasto recebem altos níveis de sinais da BMP-4 a

partir do ectoderma extraembrionário, os quais são especificados e iniciam a síntese de PRDM1 e

PRDM14, que são marcadores positivos de CGPs (Figura 4). Desta forma, estes dois fatores têm

sido utilizados para induzir a especificação de CGPs a partir de células-tronco embrionárias.

As BMP-4 e BMP-8b induzem a formação de CGPs na região do epiblasto do disco

embrionário (Figura 4, YING; ZHAO, 2001; GÜNESDOGAN; MAGNÚSDÓTTIR; SURANI,

2014). As CGP parecem requerer as proteínas BMP4 ou BMP8b sozinhas ou em combinação. A

proteína BMP-4 secretada a partir do ectoderma extra-embrionário (ExE) ativa a a expressão de

Blimp1 e Prdm14 de maneira dose-dependente. BMP-2 expresso no endoderma visceral proximal

aumenta a via de sinalização, assegurando níveis mais elevados de sinalização de BMP no

epiblasto mais proximal (LAWSON et al., 1999; OHINATA et al., 2005; VINCENT et al.,

2005). Nesse contexto, foi observado que individuos mutantes para os genes Bmp4, Bmp8b,

Bmp2, Smad1, Smad4, Smad5, e Alk2 apresentaram uma redução ou ausência de CGPs AP-

positivas (ZHAO, 2003; SAITOU; YAMAJI, 2010). Ainda não se sabe se as Smads ativam a

transcrição de Blimp1 e Prdm14 direta ou indiretamente (OHINATA et al., 2009). Baseados

nesses achados foi proposta uma via de sinalização para a especificação das CGPs, com base na

influência da expressão de Blimp1 e Prdm14 no epiblasto (Figura 5) (OHINATA et al., 2009).

36

Figura 4 – Representação esquemática do papel da sinalização das BMPs e WNT durante a

formação das células germinativas primordiais (CGPs) em embriões.

A BMP-4 produzida no ectoderma extraembrionário e BMP-2 produzida a partir do endoderma visceral induz a

fosforilação de SMAD1 e SMAD5, que formam um complexo com SMAD4. Este complexo transloca-se para o

núcleo e liga-se, presumivelmente, a reguladores e promotores de genes que são necessários para estabelecimento do

destino das CGP. Além disso, a sinalização das BMPs resulta na ativação direta ou indireta da WNT3, que também é

necessária para induzir o destino das CGP. DVE, endoderme visceral distal; ectoderma extraembrionário (ExE),

endoderme visceral (VE); endoderme visceral distal (DVE); endoderme visceral anterior (AVE). Fonte: adaptado de

YING; ZHAO, 2001; GÜNESDOGAN; MAGNÚSDÓTTIR; SURANI, 2014.

37

Figura 5 – Regulação da transcrição da especificação das CGPs.

Esquema das vias genéticas para a especificação das CGPs. Setas e linhas com barras de terminais as indicam vias de

ativação e vias de repressão, respectivamente, conforme demonstrado por experimentos in vivo (KURIMOTO et al.,

2008; YAMAJI et al., 2008); Setas e linhas pontilhadas com barras de terminais indicam vias de ativação e

repressão, respectivamente, como proposto com base em experimentos in vitro (COVELLO et al., 2006; WEST et

al., 2009; WEBER et al., 2010). Fonte: Adaptado de SAITOU; YAMAJI, (2012).

A sinalização WNT também é essencial para o destino das CGP (Figura 4),

possívelmente através de interações pós-transcricionais, sugerindo que a expressão dos membros

do sistema WNT podem atuar na sinalização das BMPs de maneira pós-transcricional

(OHINATA et al., 2009). Em ~E9,5, as CGP migram para o intestino primitivo, e mais tarde

através do mesentério dorsal, para os cumes genitais em desenvolvimento. Durante o processo de

migração, CGPs ainda expressam Tnap, mas também Oct3/4, o proto-oncogene c-Kit, e SSEA

(antígeno específico da fase embrionária) 1 e 3.

2.2.2 Migração de células germinativas primordiais

A migração através da linha primitiva para o endoderma embrionário posterior

adjacente, endoderma extraembrionário e alantóide inicia a especificação das CGP, as células

38

começam a apresentar morfologia polarizada e extensões citoplasmáticas (ANDERSON et al.,

2000). Em roedores, o primeiro passo na migração das CGP é o movimento das células da linha

primitiva para o endoderma posterior em E7,5. Entre E8,5 e E13,5, as CGP Tnap-positivas

proliferam e migram para as cristas genitais, entrando em meiose nas fêmeas ou em mitose nos

machos, e ainda iniciam a diferenciação em oócitos ou espermatozoides (MOLYNEAUX et al.,

2001; TILGNER et al., 2008). Embriões de camundongos E13,5 devem conter cerca de 24.000

CGPs em suas cristas genitais (TAM; SNOW, 1981). Ainda na fase migratória, as CGPs passam

por uma extensa reprogramação do genoma e alterações epigenéticas, tais como metilação do

DNA e modificações das histonas, além disso, o imprinting genômico pode ser necessário para

restaurar a totipotência das linhagens de células germinativas (TILGNER et al., 2008).

Atualmente, não há evidências sobre as diferenças específicas do sexo durante a

migração das CGP em qualquer espécie. Na gônada, um subconjunto de células germinativas

adquire a capacidade de funcionar como células-tronco de linhagem germinativa, que se

submetem a meiose para produzir oócitos ou espermatozoides e promover a geração do

desenvolvimento embrionário (NIKOLIC et al., 2016).

A proteína Vasa é um componente essencial de germoplasma e representa um

complexo pool de RNA e proteínas que são necessários para a determinação de células

germinativas. Mutações no gene para VASA leva à esterilidade em ratas resultante de defeitos

graves na oogênese (SAFFMAN; LASKO, 1999). Nos seres humanos, a expressão de VASA

começa no fim da fase migratória das CGP (CASTRILLON et al., 2000). Tilgner et al. (2010)

demonstraram que a expressão específica de Vasa nas células-tronco de linhagens germinativas

durante a colonização da crista gonadal sugerem que Vasa é requerido para manter as CGP

funcionais. Por exemplo, ratos machos homozigotos para Mvh (Mouse vasa homologue) são

estéreis e exibem defeitos graves na espermatogênese, enquanto as fêmeas homozigotas são

férteis (MENKE; KOUBOVA; PAGE, 2003; LASKO; ASHBURNER, 1990). Outros sinais

envolvidos na regulação da migração e colonização das CGPs são a molécula de adesão E-

caderina (BENDEL-STENZEL et al., 2000) e a moléculas da matriz extracelular integrina β1

(ANDERSON et al., 1999; CHEN et al., 2013). Infelizmente, a função exata desses fatores e as

vias de sinalização ainda não foram completamente elucidadas.

A migração das CGP mantém um programa genômico associada à pluripotência, em

que elas expressam genes de pluripotência (Oct4, Nanog e Sox2), sendo capazes de formar

39

teratomas após a injeção em ratos (SAITOU; YAMAJI, 2012; CHUMA et al., 2005; HAYASHI;

DE SOUSA LOPES; SURANI, 2007). Além disso, CGPs migratórias expressam antígeno

embrionário específico da fase 1 (stage-specific embryonic antigen 1 - SSEA1) (TILGNER et al.,

2008). Após a chegada na gônada, a proteína de ligação ao RNA específico de células

germinativas Dazl (Deleted in azoospermia-like) é essencial para o desenvolvimento das CGP

(LIN; PAGE, 2005).

Muitos estudos revelaram que o Dazl pode se ligar a proteínas específicas chamadas

de fatores de transcrição e potencializar a transcrição gênica (REYNOLDS et al., 2005; 2007;

ZENG et al., 2009). Diversos trabalhos indicam que a proteína DAZL pode ter papéis adicionais

nas CGP, especialmente estando envolvido com a apoptose, regulando a expressão das enzimas

caspases, agindo como um mecanismo que impede a formação de teratomas, eliminando as CGP

aberrantes (COOKE et al., 1996; RUGGIU et al., 1997; TSUI et al., 2000; CHEN et al., 2014).

Gill et al. (2011) indicam que na ausência de DAZL, as CGP não se desenvolvem para além do

estágio de CGP, mostrado pela expressão contínua de marcadores de pluripotência.

A pós-migração das CGP é marcada pela expressão de várias proteínas de ligação ao

RNA, tais como MVH, DAZL e NANOS3 (REYNOLDS et al., 2005; ZENG et al., 2009;

RUGGIU et al., 1997; TSUI et al., 2000; COOKE et al., 1996; CHEN et al., 2014; GILL et al.,

2011; MCLAREN, 2003). Em camundongos, as CGP femininas rapidamente iniciam a meiose e

param no estágio diplóteno da prófase I da meiose I, enquanto em machos as células se dividem

mitoticamente e entram em repouso quando passam a ser chamados de gonócitos (NIKOLIC et

al., 2016). Especificamente, as CGP aumentam a expressão de genes que lhes permitam sofrer

diferenciação sexual e a gametogênese, como Stra8 (gene requerido para a iniciação meiótica)

junto com Rec8 e Dmc1, enquanto suprimem a pluripotência (LIN et al., 2008; BALTUS et al.,

2006; KOUBOVA et al., 2014; HU et al., 2015).

2.3 Células-tronco: definição, classificação e aplicações

O conceito de células-tronco surgiu a partir de experimentos pioneiros realizados no

início dos anos 1960 por Ernest A. McCulloch e James E. Till que observaram a presença de

colônias hematopoiéticas no baço de camundongos irradiados e que haviam recebido transplante

40

de medula. Essas colônias eram derivadas de uma única célula, a célula-tronco (TILL;

MCCULLOCH; SIMINOVITCH, 1964).

As células-tronco são uma população de células indiferenciadas, caracterizadas pela

capacidade de sofrer auto-renovação e diferenciação em vários tipos de tecido (KOŹLIK;

WÓJCICKI, 2014). Com base no seu potencial de diferenciação, as células-tronco são

classificadas como totipotentes, pluripotentes, multipotentes e unipotentes. As células-tronco

totipotentes estão presentes nas primeiras fases da ontogênese e podem diferenciar-se em todos os

tecidos embrionários e na placenta. As células pluripotentes podem ser coletadas a partir da

camada interna do blastocisto, e podem originar células das 3 camadas germinativas (ectoderma,

endoderma e mesoderma). As células-tronco multipotentes podem ser encontradas em quase

todos os tecido. Durante muito tempo acreditou-se que elas poderiam se diferenciar em células de

uma camada germinativa (por exemplo, as células-tronco hepáticas poderiam diferenciar-se

apenas em hepatócitos ou células dos ductos biliares), mas estudos recentes têm revelado que

algumas células multipotentes têm o mesmo potencial das células-tronco pluripotentes. Já as

células-tronco unipotentes, com menor potencial de diferenciação, são capazes de originar apenas

um tipo de célula (por exemplo, as células-tronco da epiderme podem diferenciar-se apenas em

células epiteliais escamosas queratinizadas superficiais) (BECK; BLANPAIN, 2012; KOŹLIK;

WÓJCICKI, 2014).

As células-tronco também podem ser classificadas em 4 grupos correspondentes às

suas diferentes origens: células-tronco embrionárias (ESC), células-tronco fetais, células-tronco

adultas (ASC) e células-tronco de pluripotência induzidas (iPS) (KOŹLIK; WÓJCICKI, 2014). O

zigoto, resultante da fecundação, é caracterizado ser totipotente. Após ~4 dias de crescimento, o

zigoto se transforma em blastocisto. A camada interna é composta por células-tronco

embrionárias, que são pluripotentes. As células-tronco embrionárias podem proliferar-se de

maneira quase ilimitada, além disso, são identificados pela expressão de fatores de transcrição

específicos, como Nanog, Oct4 e Sox2 (WANG et al., 2012). Com a utilização de meios de

cultivo e fatores de crescimento específicos, as células-tronco embrionárias foram cultivadas in

vitro e diferenciadas em outros tipos celulares, incluindo músculos esqueléticos, células

endoteliais, condrócitos, miocardiócitos, entre outros (ROHWEDEL; MALTSEV; BOBER,

1994; MALTSEV et al., 1993; ZHU et al., 2013; LEYDON et al., 2013). Há uma certa restrição

com os trabalhos envolvendo células-tronco embrionárias e fetais, levando em consideração os

41

dilemas éticos e sociais envolvidos na coleta, cultivo e experimentação dessas células-tronco,

além disso, o uso deste tipo celular é restrito em muitos países (ZARZECZNY; CAULFIELD,

2009). Wakitani et al. (2003) indicam que o uso de células-tronco embrionárias pode levar a

formação de células cancerígenas e de teratomas. Isto provavelmente está relacionada a métodos

falhos e a limitações tecnológicas (WAKITANI et al., 2003).

As ASC são específicas para cada órgão, e estão presentes em pequenas quantidades

em todos os tecidos. Por exemplo, as células-tronco hematopoiéticas compõem cerca de 0,001-

0,01% das células sanguíneas (DA SILVA MEIRELLES; CAPLAN; NARDI, 2008). A

morfologia e os marcadores proteicos permitem que cada células-tronco adulta possa ser

classificada de acordo com o tecido que a origina. As ASC foram previamente classificadas como

multipotentes, mas atualmente são conhecidos por serem pluripotentes. Devido ao fenômeno

conhecido como “plasticidade” ou “transdiferenciação”, algumas destas células-tronco adultas

podem produzir um novo tipo célular, quando transferida para outros tecidos (KOŹLIK;

WÓJCICKI, 2014). Em 2006, Takahashi e Yamanaka, demonstraram que, mesmo células

somáticas maduras, quando expostas a genes associados a pluripotência específica, podem

“recuperar” a pluripotência de maneira semelhante às células-tronco embrionárias, com

capacidade de diferenciação em células de qualquer camada germinativa. Estes experimentos

foram realizados primeiro com fibroblastos de camundongos, e posteriormente com fibroblastos

humanos, sendo estas células denominadas de células-tronco de pluripotência induzida

(TAKAHASHI; YAMANAKA, 2006; TAKAHASHI et al., 2007).

As células-tronco mesenquimais multipotentes têm uma ampla aplicação na prática

clínica. As células-tronco mesenquimais da medula óssea são obtidas rapidamente e tem seu uso

amplamente conhecido, no entanto, a sua obtenção envolve um procedimento invasivo e

ineficiente (KOŹLIK; WÓJCICKI, 2014). Novos procedimentos foram desenvolvidos para a

coleta de vários tipos de células-tronco mesenquimais a partir de outras estruturas do corpo

humano, incluindo o tecido adiposo (células-tronco derivadas do tecido adiposo), o sangue de

cordão umbilical (UCB), o periósteo, os tendões, os músculos (células-tronco derivadas dos

músculos), membranas mucosas e pele (DA SILVA MEIRELLES; CAPLAN; NARDI, 2008).

Uma análise comparativa de todos os tipos de células-tronco mesenquimais não revelou grandes

diferenças entre as suas habilidades de se diferenciar em tecidos específicos (KERN et al., 2006).

Para a diferenciação celular, as células-tronco mesenquimais desenvolvem sinalização parácrina,

42

sendo capazes de induzir neovascularização do tecido a partir da liberação de fatores de

crescimento, tais como fator de crescimento do endotélio vascular (VEGF), fator de crescimento

transformante (TGF) e fator de crescimento de hepatócitos (HGF) (CAI et al., 2009).

2.3.1 Células-tronco embrionárias

As células-tronco embrionárias (ESC) são linhagens de células pluripotentes,

geralmente derivadas a partir da massa celular interna do blastocisto. Estas células são

consideradas como um modelo para estudar a embriogênese (Figura 6, DE PAEPE et al., 2014).

Podem ser propagadas indefinidamente em cultura, permanecendo em estado indiferenciado.

Essa pluripotência refere-se à capacidade de uma célula em diferenciar-se em células das três

camadas germinativas in vitro e in vivo. Os fatores de transcrição OCT4 (POU5F1), SOX2 e

NANOG desempenham um papel importante na manutenção deste estado indiferenciado

(BOYER et al., 2005; 2006).

Em ratos, células-tronco embrionárias, as células-tronco do trofoblasto (TS) e células-

tronco do endoderma extra-embrionário (XEN) foram derivadas do blastocisto (YAMANAKA et

al., 2006). As linhagens de ESC e mTS (células-tronco do trofoblasto murinos) têm sido

derivadas de blastômeros no estágio de 1 a 8 células (CHUNG et al., 2006).

Em humanos, as linhagens de células-tronco embrionárias pluripotentes (hESC)

(THOMSON et al., 1998; REUBINOFF et al., 2000) foram derivadas de blastocistos pré-

implantados, sendo caracterizadas por marcadores de superfície celular, capacidade de

diferenciação, transcriptômica e (epi)-genômica. As hESC são capazes de se diferenciar em

células do endoderma extra-embrionário in vitro (THOMSON et al., 1998; LEE et al., 2013), e

em células trofoblásticas (THOMSON et al., 1998; XU et al., 2002; GERAMI-NAINI et al.,

2004; HARUN et al., 2006). A diferenciação de linhagens de hESC em células trofoblásticas

induz a expressão de fatores de transcrição específicos (Cdx2 e Gata3), genes associados com o

citoesqueleto (Krt7 e Krt8) e a matriz extracelular (Cola4), genes envolvidos na invasão (Igf2,

Cdh1 ou E-caderina) e hormônios (β-hCG) (MARCHAND et al., 2011).

43

Figura 6 – Isolamento e diferenciação das células-tronco embrionárias.

As células-tronco embrionárias são removidas do blastocisto cerca de 4-5 dias após a fertilização. O processo ocorre

quando o embrião em desenvolvimento apresenta aproximadamente 150 células. Essas células, quando estimuladas,

apresentam o potencial de diferenciação de linhagens celulares. As células coletadas são cultivadas sob condições

especiais de laboratório, podendo ser usadas para o tratamento de diferentes tipos de doenças, bem como para o

transplante em órgãos vitais. Fonte: Adaptado de

<http://sgugenetics.pbworks.com/w/page/38198357/Embryonic%20Stem%20Cells>.

Inicialmente, as hESC foram obtidas a partir da massa celular interna (THOMSON et

al., 1998; REUBINOFF et al., 2000) com uma alta taxa de derivação a partir do 6º dia de

blastocisto (CHEN et al., 2009). As hESC também têm sido derivadas de blastômeros no estágio

de 4 a 8 células (KLIMANSKAYA et al., 2006, 2007; FEKI et al., 2008; GEENS et al., 2009;

ILIC et al., 2009). As hESC derivados da massa celular interna e derivados de blastômeros têm

perfis semelhantes de transcrição (GIRITHARAN et al., 2011; GALAN et al., 2013). As hESC

apresentam expressão de marcadores como Dazl e Stella, que são característicos de células

germinativas iniciais (ZWAKA; THOMSON, 2005). No entanto, a diferenciação de hESC em

44

células germinativas ainda é um processo falho (GEIJSEN et al., 2004; NAYERNIA et al., 2006;

AFLATOONIAN et al., 2009).

2.3.2 Células-tronco adultas

Em organismos multicelulares, grupos de células semelhantes especializadas são

agrupadas em tecidos e órgãos para executar funções específicas. No decorrer da vida adulta,

estas células podem perder de maneira progressiva as suas funções. Para compensar essa perda

contínua de células diferenciadas, novas células funcionais devem ser geradas de modo que

tecidos permaneçam em homeostase. A manutenção e o reparo deste ciclo nos tecidos adultos,

geralmente dependem de uma pequena população de células, as chamadas células-tronco adultas,

que possuem a capacidade de se auto-renovar, dando origem a células diferenciadas, mantendo o

seu número de células constante nos tecidos (WATT; HOGAN, 2000; FUCHS; CHEN, 2013).

A capacidade de auto-renovação tem sido considerada a definição característica de

ASC (CLERMONT; LEBLOND, 1952, 1953; LEBLOND; STEVENS, 1948). Para a

manutenção da homeostase, a proliferação e diferenciação de ASC deve ser perfeitamente

equilibrada, de modo que, a divisão seguinte, uma das células- filha permanece como célula-

tronco, ao passo que as outras se diferenciam diretamente, ou ainda através de uma série de

divisões (KRIEGER; SIMONS, 2015).

O processo pelo qual as células-tronco dão origem a diferentes tipos celulares não é

completamente compreendido e é denominado de diferenciação celular. Acredita-se que entre os

fatores responsáveis por este fenômeno, estejam substâncias secretadas por células vizinhas, além

de componentes do microambiente, presentes de forma solúvel ou ligados a matriz-extracelular

(CARMO; SANTOS, 2009). As ASC estão presentes em praticamente todos os tipos de tecidos,

no entanto, sua proporção em relação aos outros tipos celulares é baixa, o que torna estas células,

difíceis de identificar, isolar e purificar (CARMO; SANTOS, 2009).

As ASC podem ser divididas em dois tipos principais: as células-tronco

hematopoiéticas (responsáveis pela formação dos diferentes tipos celulares sanguíneos), e as

células-tronco mesenquimais (MSC) (pode formar tecido cartilaginoso, ósseo, adiposo e

muscular). As MSC são um tipo de células-tronco multipotentes que podem ser isoladas de vários

tecidos e membranas fetais ou adultas (BIANCHI et al., 2001; FILIOLI URANIO et al., 2011)

45

incluindo tecido adiposo, medula óssea, sangue do cordão umbilical (KERN et al., 2006;

TAKEMITSU et al., 2012; STRIOGA et al., 2012), polpa dentária (XIAO; TSUTSUI, 2013),

placenta e músculos (KISIEL et al., 2012). Ambas podem ser encontradas na medula óssea, mas

a última também pode ser encontrada em outros tecidos (ZAGO; COVAS, 2006). A figura 7

ilustra um exemplo de diferenciação de células-tronco mesenquimais.

Devido a sua função de manutenção de tecidos adultos e de resposta a lesões do

organismo, atribui-se as ASC um grande potencial terapêutico, no tratamento das mais variadas

injúrias, como diabetes, doenças auto-imunes, na hematologia, na oftalmologia e na regeneração

de lesões provocadas por acidentes. Devido a este provável potencial, tais células têm se tornado

alvo de inúmeras pesquisas nos últimos anos, muitas das quais obtiveram resultados favoráveis

(CARMO; SANTOS JÚNIOR, 2009).

Figura 7 – Isolamente e diferenciação de células-tronco mesenquimais.

As MSC podem ser isoladas de diferentes tecidos como adiposo, medula óssea, sangue do cordão umbilical, polpa

dentária, placenta e músculos. Essas células, quando estimuladas podem se diferenciar tipos celulares, como

adipócitos, condrócitos e osteócitos. Fonte: Disponível em: <http://www.eurostemcell.org/factsheet/mesenchymal-

stem-cells-other-bone-marrow-stem-cells>.

Além de classificadas como multipotentes, as MSC expressam um nível

relativamente elevado de marcadores de pluripotência semelhantes às ESC, como Oct4, Nanog e

Sox2 (SACHS et al., 2012; TAKEMITSU et al., 2012; KISIEL et al., 2012). Estes fatores de

transcrição estão envolvidos na regulação da multipotência, auto-renovação e proliferação das

MSC (TAKEMITSU et al., 2012; KISIEL et al., 2012). Oct4 está envolvido no desenvolvimento

inicial dos mamíferos e é essencial para a formação de massa celular interna de embriões e para a

46

manutenção de ESC (NICHOLS et al., 1998). Sox2 regula a expressão de Oct4, e mantém o

estado pluripotente de ESC, enquanto Nanog é necessário para a manutenção do estado

indiferenciado e para a auto-renovação das células-tronco (TAKEMITSU et al., 2012; ZOMER et

al., 2015).

In vivo, as MSC proporcionam um suporte estrutural em diferentes órgãos e regulam

o fluxo de algumas substâncias. Além disso, elas apresentam uma alta e rápida taxa de

proliferação em meio de cultura simples, e podem ser mantidas in vitro, sem alterações do

cariótipo por várias passagens (WEBSTER et al., 2012). As MSC têm a capacidade de se

diferenciar em vários tipos de células, tais como adipócitos, osteócitos e condrócitos, a partir da

camada germinativa mesodérmica (SACHS et al., 2012; DU et al., 2010). Esta plasticidade

depende do ambiente da matriz extracelular e da presença de fatores de crescimento solúveis

(VIDANE et al., 2013). Alguns autores induziram a diferenciação de MSC em células de outras

camadas germinativas, como neurônios (KRAMPERA et al., 2007), que são originados do

ectoderma, e hepatócitos que são derivados a partir do endoderma (AURICH et al., 2009). No

entanto, a diferenciação em tecidos não-mesodérmicos ainda é controversa, devido a falta de

resultados in vivo (STRIOGA et al., 2012).

Devido à sua plasticidade, as MSC são consideradas o tipo de célula mais importante

para medicina regenerativa, e são as mais extensamente estudadas em ensaios pré-clínicos e

clínicos. Suas vantagens para aplicação clínica incluem o fácil isolamento e alto rendimento, alta

plasticidade, bem como a capacidade para mediar a inflamação e promover o crescimento e

diferenciação celular, reparação de tecidos por imunomodulação e imunossupressão, e ainda,

estão isentas de implicações éticas (LAGE-CONSIGLIO et al., 2013; PLOCK et al., 2013;

INSAUSTI et al., 2014). Além disso, as MSC não formam teratomas após o transplante,

garantindo a segurança para o receptor (ZOMER et al., 2015).

As MSC derivadas a partir da medula óssea têm sido as mais intensivamente

estudadas. No entanto, procedimentos invasivos são necessários para o seu isolamento, e a

quantidade e a qualidade das células isoladas variam de acordo com a idade do doador (ZOMER

et al., 2015). Um número pequeno de MSC está presente em aspirados de medula óssea em

comparação com as células totais da composição do estroma da medula óssea (BYDLOWSKI et

al., 2009). Devido à heterogeneidade da população de células, as suas propriedades imunogênicas

dependem de várias configurações, tais como métodos de isolamento, de superfície e meio de

47

cultura e suplementação química (WEBSTER et al., 2012). Portanto, a identificação de fontes

alternativas de MSC tem sido o ponto focal de pesquisas recentes. Entre diferentes fontes de

MSC, o tecido adiposo destaca-se pela sua acessibilidade e pela abundância de células isoladas

(LAM; LONGAKER, 2012; DU et al., 2010; HOUSMAN et al., 2002; CHERUBINO et al.,

2011; KIM et al., 2011). Cada isolamento resulta em aproximadamente 100 vezes mais células do

que o isolamento da medula óssea (DEY; EVANS, 2011), e o processo é muito menos invasivo

(SACHS et al., 2012).

2.3.2.1 Células-tronco ovarianas

Diversos trabalhos indicam a existência das células-tronco ovarianas (OSC) nos

ovários das fêmeas adultas (ZOU et al., 2009; WHITE et al., 2012; ZHANG et al., 2012;

BHARTIYA et al., 2013; PARTE et al., 2014). Esta tese refuta as observações de Zuckerman et

al. (1951), que postularam o dogma de que nos ovários de mamíferos na vida pós-natal não há

OSC germinativas renováveis, assim, apoiando a hipótese de que durante a vida há um pool

numericamente fixo de oócitos que serão direcionados para a fertilidade. Este pool contaria com

cerca de 106 oócitos na puberdade. No entanto, esse número diminui com o envelhecimento até a

completa exaustão na menopausa (ZUCKERMAN et al., 1951; SILVESTRIS et al., 2015). Esta

hipótese foi posteriormente refutada por Johnson et al. (2004), que identificaram a presença de

OSC mitoticamente ativas em ovários murinos jovens e adultos, capazes de garantir a

disponibilidade de oócitos após o nascimento.

Em seus experimentos, Johnson et al. (2004) observaram uma discordância entre a

taxa de depleção folicular e a vida útil reprodutiva, e ainda, encontraram aspectos histológicos

normais nos ovários de ratos que foram esterelizados quimicamente por busulfan (quimioterápico

usado para o tratamento de leucemia), mostrando folículos saudáveis em maturação e corpo lúteo

após o receberem fragmentos de ovários de animais saudáveis. No entanto, Bristol-Gould et al.

(2006) simularam a dinâmica da progressão folicular murina durante a vida útil de camundongos

através de dois modelos matemáticos, chamados de “células-tronco” e “modelo de pool fixo”,

que argumentam que o declínio fisiológico da população folicular apoia a teoria da “população

fixa de oócitos” como reserva ovariana. Na verdade, foi descrito a expressão de marcadores de

linhagens de células germinativas incluindo Oct4, MVH, Dazl, Stella e Fragilis na medula óssea

48

de camundongas fêmeas adultas, e depois transplantadas para a medula óssea de fêmeas adultas

pré-esterilizadas com ciclofosfamida e bussulfan, detectando-se assim a geração significativa de

novos folículos contendo oócitos e também a formação de corpo lúteo (JOHNSON et al., 2005).

Assim, essas observações preliminares deram origem a novos estudos que visam o isolamento

das OSC e sua transferência para animais estéreis com o intuito de recuperar a sua fertilidade

(SILVESTRIS et al., 2015).

A primeira tentativa de isolar e cultivar as OSCs germinativas em mamíferos foi

realizada por Zou et al. (2009) que purificaram OSCs de camundongas fêmeas neonatais e

adultos, que foram infectadas com vírus GFP (proteína fluorescente verde) e transplantadas para

ovários de camundongas inférteis. As células transplantadas participaram da oogênese e geraram

descendentes (ZOU et al., 2009). White et al. (2012) isolaram e purificaram OSC humanas

baseados na detecção imunológica de um marcador de superfície celular, o Dead box polypeptide

4 (Ddx-4). Essas células mostraram um padrão de expressão de genes de linhagens germinativas

e, portanto, foram estabelecidas em cultura, injetadas em biópsias no tecido cortical de ovários

humanos adultos. Foi observada a formação de novos folículos contendo oócitos do tecido

transplantado (WHITE et al., 2012). Em contrapartida, Zhang et al. (2012) através de imagens de

células vivas e de experimentos de neofoliculogênese, mostram que as células de ovários de ratas

pós-natais, que expressam Ddx4, não entraram em mitose, e nem contribuem para renovação de

oócitos durante a neofoliculogênese.

Virant-Klun et al. (2008; 2009) isolaram OSC a partir do epitélio de superfície do

ovário (OSE) de mulheres normais, de mulheres na pós-menopausa e em mulheres com falha

ovariana precoce (FOP). As células obtidas expressaram Ssea-4, Oct-4, Nanog, Sox-2 e c-Kit.

Durante o cultivo, foi observada a diferenciação em células semelhantes à oócitos, que atingiram

o diâmetro de até 95 µm e expressaram Oct-4, c-Kit, Vasa e Zp2. Os resultados indicam que as

células expressaram marcadores de células-tronco embrionárias, e esses dados podem ser usados

para estudos que visem o tratamento autólogo da infertilidade ovariana e a cura doenças

degenerativas como FOP (VIRANT-KLUN et al., 2008; 2009).

Bhartiya et al. (2013) descreveram a presença de pequenas células-tronco

embrionárias (small embryonic-like stem cells - VSEL) e OSC germinativas no OSE de

mamíferos adultos. As OSC foram coletadas e analisados quanto à expressão de Oct-4a, Ssea-4,

Fragilis, Cd133, dentre outros. Estas células foram cultivadas por 21 dias e sofreram

49

diferenciação espontânea em estruturas semelhantes à oócitos (BHARTIYA et al., 2013). Com

base nestas observações, Parte et al. (2014) isolaram células-tronco do OSE de ovários de

ovelhas usando SSEA-4 como marcador de superfície. Essas células foram analisadas por

imunocitoquímica, imunofluorescência e por RT-PCR para detectar os marcadores específicos de

linhagens de células-tronco e linhagens germinativas. Além disso, durante o cultivo, as células

sofreram mudanças em suas características e diferenciaram-se espontâneamente em estruturas

semelhantes à oócitos (PARTE et al., 2014). Hayashi e colaboradores (2012) induziram células-

tronco embrionárias de camundongas para se diferenciarem em células semelhantes a células

germinativas primordiais (PGCLC) que foram utilizadas para reconstituir o córtex ovariano.

Após o transplante na bursa, os ovários foram reconstituídos, e em seguida, as PGCLCs foram

isoladas e histologicamente analisadas, indicando que as PGCLCs contribuiram para a formação

de oócitos. Os oócitos destes animais foram fertilizados, gerando filhotes que atingiram a idade

adulta (HAYASHI et al., 2012).

Stimpfel et al. (2013) isolaram OSC no OSE de mulheres que expressavam

marcadores de pluripotência como fosfatase alcalina, Ssea-4, Oct4, Ddx4. Essas células exibiram

um elevado grau de plasticidade, uma vez que, quando adequadamente estimuladas,

diferenciaram-se em várias células somáticas derivadas dos 3 folhetos germinativos (mesoderma,

ectoderma e endoderma), e ainda sem formar teratomas em camundongos imunodeficientes

(STIMPFEL et al., 2013).

2.3.3 Células-tronco de pluripotência induzidas e reprogramação celular

Em 2006, Yamanaka et al. identificaram quatro fatores de transcrição, Klf4, Sox2,

Oct4 E c-Myc, capazes de transformar fibroblastos de camundongos (TAKAHASHI;

YAMANAKA, 2006) e humanos (TAKAHASHI et al., 2007), em clones pluripotentes através de

transdução retroviral. As células iPS são geradas a partir da indução da expressão de fatores de

transcrição associados à pluripotência, permitindo que uma célula somática diferenciada possa

inverter a sua condição para o estágio embrionário (ZOMER et al., 2015), ver figura 8.

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Figura 8 – Reprogramação de células adultas e formação das células iPS.

Na formação das células iPS, genes de pluripotêmcia são introduzidos nas células adultas de maneira a induzir a

reprogramação. As células iPS resultantes se assemelham a células-tronco embrionárias e podem ser diferenciadas

em qualquer tipo de célula, que podem ser utilizadas para estudos de doenças, teste de medicamentos ou correção de

genes defeituosos e desenvolvimento de terapias com células. Fonte: Adaptado de

<http://www.eurostemcell.org/factsheet/ips-cells-and-reprogramming-turn-any-cell-body-stem-cell>.

A descoberta de tal tecnologia foi baseada na hipótese de que a reprogramação

nuclear é um processo conduzido por fatores que desempenham um papel crítico na manutenção

da pluripotência das células ESC (TAKAHASHI; YAMANAKA, 2006; YAMANAKA, 2008).

As iPS poderiam implicar na eliminação de problemas éticos e problemas de rejeição após

transplantes, uma vez que podem ser coletadas do próprio paciente (trasnplante autólogo),

ampliando as possibilidades de pesquisa (LAM; LONGAKER, 2012; TAKAHASHI;

YAMANAKA, 2006). Já é bem conhecido que um ou vários fatores de transcrição podem

converter uma célula em outra. Entretanto, os mecanismos pelos quais fatores exógenos alteram o

estado epigenético permanece desconhecida. Os fatores de Yamanaka são os mais utilizados,

51

outras combinações de fatores foram testadas com sucesso, tais como a substituição de c-Myc e

Klf4 por Nanog e Lin28 (YU et al., 2007), ou a exclusão de c-Myc (XU et al., 2013).

Nas células somáticas, os promotores de genes de pluripotência são altamente

metilados, refletindo um estado transcricional reprimido (ZOMER et al., 2015). Mikkelsen et al.,

(2008) mostraram que a geração de células iPS envolve a ativação destes genes, e a sua

desmetilação é utilizada para determinar o sucesso da reprogramação. Quando os genes de

pluripotência exógenos são introduzidos na célula, induzem a expressão de genes pluripotência

endógenos (JAENISCH; YOUNG, 2008). Por sua vez, o aumento da produção de fatores

endógenos induz o silenciamento de genes exógenos por metilação dos promotores (HOTTA;

ELLIS, 2008).

O valor terapêutico de células iPS é a presença de integrações provirais abrigando

oncogenes conhecidos, especialmente c-Myc, bem como Oct4 e Klf4. c-Myc foi dispensável para

a geração de iPS a partir de fibroblastos, e os camundongos quiméricos produzidos por células

iPS que receberam esses três fatores (Oct4, Sox2 e Klf4), não apresentaram formação do tumor,

enquanto as células derivadas dos quatro fatores (Klf4, Sox2, Oct4 e c-Myc) apresentaram

características tumorigênicas (NAKAGAWA et al., 2008; WERNIG et al., 2007). As técnicas de

reprogramação convencionais dependem da integração estável de transgenes, mas podem

introduzir o risco de mutagênese de inserção. Assim, diversas técnicas de reprogramação não-

integrativas foram desenvolvidas para melhorar a qualidade das células geradas (DIECKE et al.,

2014). Os sistemas integrativos consistem em vetores virais, tais como retrovírus (TAKAHASHI;

YAMANAKA, 2006) e lentivírus (PICANÇO-CASTRO et al., 2011). Já vetores não-integrativos

também tem sido utilizados, tais como adenovírus (STADTFELD et al., 2008) ou sistemas não

virais, como plasmídeos (OKITA; YAMANAKA, 2006), proteínas (ZHOU et al., 2009) e

RNAm, que não promovem a integração de DNA complementar (DNAc) dos fatores OSKM para

o genoma na célula (OKITA; YAMANAKA, 2006; WARREN et al., 2010; YU et al., 2009).

Recentemente, novas abordagens foram testadas para induzir a pluripotência, usando moléculas

químicas exógenas (HOU et al., 2013; PENNAROSSA et al., 2013) ou vetores epissomais (uso

de DNA plasmidial) (YU et al., 2009; DIECKE et al., 2014).

Diferentes razões tem levado os pesquisadores a desenvolver estratégias para gerar

células iPS sem a utilização de lentivírus. Em primeiro lugar, a introdução de DNA exógeno

capaz de se integrar em posições aleatórias do genoma representam um risco para a fisiologia da

52

célula, podendo dar origem a uma mutagênese de inserção. O DNA exógeno poderia destruir o

quadro aberto de leitura (ORF) de genes ou influenciar a regulação gênica. Além disso o

silenciamento de transgenes de lentivírus é incompleto, levando a reativação de vetores lentivirais

nas células iPS (WERNIG et al., 2007).

Há diversos estudos sobre a ação de moléculas que poderiam substituir os vetores

virais durante o processo de reprogramação. A maioria dessas moléculas podem melhorar a

eficiência da reprogramação (LIN; WU, 2015). Durante a reprogramação, as células sofrem

alterações a nível transcricional global e também passam por mudanças epigenéticas em seu

DNA (metilação e desmetilação), juntamente com modificações nas histonas, como acetilação e

desacetilação, ou ainda metilação e desmetilação (CHIN et al., 2009; MAHERALI et al., 2007;

HUANGFU et al., 2008; MIKKELSEN et al., 2008; NIE et al., 2012; HAWKINS et al., 2010;

SHU et al., 2013; ZHU et al., 2010; MALI et al., 2010). Huangfu et al. (2008) foram os

primeiros a estudar a aplicação de um composto na geração de iPS, em que avaliaram o efeito do

ácido valpróico (VPA), um inibidor de enzimas histona-desacetilase (HDAC), e descobriram que

a eficiência da reprogramação foi aumentada em 100 vezes, em relação ao método tradicional,

usando vetores de transcrição (HUANGFU et al., 2008). Um inibidor da DNA metiltransferase, o

5-aza-citidina (5-Aza), é um análogo da citosina que se converte em um nucleotídeo-trifosfato in

vivo, possibilitando a sua incorporação no DNA, o que influência a sua estrutura e estabilidade

(JUTTERMANN; LI; JAENISCH, 1994). Assim, o 5-aza-citidina é considerado um agente da

inibição da metilação do DNA, pois inibe a enzima DNA metiltransferase 1 (DNMT1), e é

amplamente utilizado no estudo da regulação epigenética da expressão gênica (JONES; TAILOR,

1980). Há ainda outros compostos que podem ser utilizados na reprogramação, como a

tricostatina A (TSA) que é um potente inibidor de HDAC, induz o acúmulo de histonas acetiladas

no núcleo e subsequente ativação de genes alvo (TADDEI et al., 2005; DOKMANOVIC;

CLARKE; MARKS, 2007; KIM et al., 2009). O butirato de sódio (NaB) aumenta a acetilação

das histonas e é um potente inibidor de HDAC provocando hiperacetilação de histonas H3 e H4

em células de mamíferos (CANDIDO; REEVES; DAVIE, 1978; NÖR et al., 2013).

Zomer et al. (2015) indicam que o estado de pluripotência nas células iPS pode ser

avaliado pela capacidade de formar teratomas in vivo e a formação de corpos embrionárias in

vitro. Além disso, as iPS podem apresentar morfologia semelhante as ESC, como formato

arredondo, grande nucléolo e escasso citoplasma (ZOMER et al., 2015). A capacidade de

53

diferenciação das iPS parece assemelhar-se a das células ESC. Vários estudos descrevem a

diferenciação de uma variedade de tipos de células a partir de células iPS murinas e humanas,

entre eles, cardiomiócitos (CHRISTOFOROU et al., 2013), células de músculo liso (WANG et

al., 2014), células hepáticas (TAKEBE et al., 2013) e neurônios (HEILKER et al., 2014). Além

disso, essas células apresentam comportamento semelhante, quando da diferencição de iPS e

células ESC.

Diversos trabalhos indicam que as células iPS são muito semelhantes, mas não

idênticas a ESC (TAKAHASHI; YAMANAKA, 2006; CHIN et al., 2010; PFANNKUCHE et

al., 2010). Chin et al. (2010) ao compararem o padrão de expressão de ESC e de células iPS em

humanos, observaram modificações das histonas e a expressão de miRNA não codificantes em

ambos os tipos celulares, e construíram um padrão de expressão, que distingue as iPS das ESC

(PFANNKUCHE et al., 2010). Pfannkuche et al. (2010) identificaram 318 genes

diferencialmente expressos entre ESC e iPS humanas e de células em qualquer fase. Além disso,

foi demonstrado que os genes que foram mais expressos em iPS também tiveram uma expressão

aumentada em fibroblastos, quando comparados à expressão em ESC. A mesma conclusão foi

observada quando foram avaliados os genes menos expressos, sendo eles menos expressos em

células iPS do que em ESC, também tendo uma baixa expressão em fibroblastos

(PFANNKUCHE et al., 2010). Dentre os genes expressos em ambas as células, tanto em iPS

como em ESC, há a expressão de inúmeros marcadores de pluripotência como, Oct4, Nanog,

Sox2, Ssea-1, Ssea-3, Ssea-4, Tra1-60, Tra1-81, bem como a atividade da fosfatase alcalina

(LEWITZKY; YAMANAKA, 2007; WANG et al., 2010; SCHEPER; COPRAY, 2009).

2.4 Proteínas morfogenéticas ósseas e seu papel durante a formação das CGPs

Os membros da superfamília Fator de Crescimento Transformante-β (TGF-β),

especialmente as BMPs, regulam uma grande variedade de funções celulares nas células-tronco

pluripotentes (MISHRA; DERYNCK; MISHRA, 2005; ZHANG; LI, 2005). Os membros destas

famílias se ligam especificamente aos seus respectivos complexos receptores, consistindo em

subunidades do tipo I e tipo II. Após a ligação do ligante ao receptor do tipo II, este induz a

fosforilação do receptor do tipo I, o qual por sua vez, transmite o sinal para moléculas de

transdução de sinal intracelular, chamadas receptores ativados ou R-Smads. As BMPs

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tipicamente utilizam como transdutores de sinal as R-Smads 1/5/8. A fosforilação mediada por

receptores desencadeia a associação dessas R-Smads com um mediador comum ou co-Smad-4, e

a subsequente translocação deste complexo fator de transcrição para o núcleo. Adicionalmente,

os receptores ativados podem influenciar vias Smad-independente, tais como a via MAPK

(Proteína cinase ativada por mitógenos) (TERADA et al., 1999). Para confirmar se as BMPs

atuam no processo de diferenciação, Park; Woods; Tilly, (2013) demonstraram que a BMP-4

regula diretamente a diferenciação de células-tronco oogoniais de mamíferos através da via de

sinalização das SMADs 1/5/8.

Em camundongos, a formação e a proliferação dos precursores das CGP são

dependentes de BMP-2, BMP-4, BMP-8b (LAWSON et al., 1999; DE SOUSA LOPES et al.,

2004; YING et al., 2000; YING; ZHAO, 2001). Camundongos mutantes para BMP-4

apresentaram defeitos graves no desenvolvimento das células germinativas, com uma quase

completa ausência de CGP, enquanto camundongos knockouts para BMP-7 e BMP-8b

demonstraram uma grave redução no número de células germinativas (ZHAO et al., 1996; 2001;

YING et al., 2000; ZHAO, 2003). Lawson et al. (1999) utilizando camundongos mutantes para

BMP-4 demonstraram que esses animais eram desprovidos de CGP e alantóide. Já em mutantes

para Bmp8b foi observada uma redução acentuada no número de células germinativas. Estudos in

vitro estabeleceram que as vias de sinalização da BMP-4 e BMP8b atuam sinergicamente (YING;

QI; ZHAO, 2001).

A proteína morfogenética óssea 2 (BMP-2) é um membro da superfamília BMP, que

desempenha um papel crucial na produção de CGPs (PERA; TROUNSON, 2004). Em ratos, esta

proteína é produzida pelo endoderma visceral e exerce um importante papel na formação de

células germinativas primoridiais positivas para fosfatase alcalina (SAITOU; YAMAJI, 2010). A

BMP-2 também é responsável por estimular a expressão de Prdm1 e Prdm14 no epiblasto

durante a formação das CGPs (SAITOU; YAMAJI, 2010).

Já foi demonstrado que a BMP-4 aumenta o número de CGP migratórias em culturas

de embrião (DUDLEY et al., 2007). Em um trabalho com células-tronco embrionárias, McLaren

(1999) mostrou que a BMP-4 foi requerida no ectoderma extraembrionário, mais do que nas

células epiblásticas (MCLAREN, 2003). O papel da sinalização das BMP na regulação do

comportamento pós-migratório das CGP é menos claro, pois o cultivo de ovários fetais de

camundongos com BMP-2 ou BMP-4 reduziu o número de células germinativas em meiose

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(ROSS et al., 2003). Além disso, a BMP-4 pode promover a proliferação de CGP pós-migratórias

isoladas de camadas alimentadoras cultivadas in vitro (PESCE et al., 2002). Childs et al. (2010)

identificaram que a sinalização das BMPs está associada ao desenvolvimento do ovário fetal

humano, além disso, a BMP-4 apresenta um papel pró-apoptótico na regulação das CGP pós-

migratórias em humanos. A combinação de LIF e BMP-4 ou LIF e BMP-2 induzem um aumento

na auto-renovação das células-tronco embrionárias, resultando em populações altamente puras e

não diferenciadas após 2 ou 3 passagens (YING et al., 2003).

A BMP-4 atua pela via de sinalização de mensageiros intracelulares (SMADs 1/5)

que se ligam a domínios específicos nas células, chamados de Prdm14 e Prdm1. Este mecanismo

de ação mostra os caminhos genéticos para a ativação e inibição gênica. Prdm1 é essencial para

inibir todos os genes somáticos, podendo também ser importante para a criação de um estado

epigenético para a expressão de genes específicos de CGPs. O Pdm14 é também um regulador

crítico para a especificação CGPs (YAMAJI et al., 2008). É importante ressaltar que a expressão

inicial de Prdm14 em CGPs é independente de Prdm1, mas a sua manutenção e/ou regulação

positiva subsequente é estritamente dependente Prdm1. Assim, Prdm1 e Prdm14 são os dois

principais reguladores transcricionais para o desenvolvimento da linhagem de células

germinativas em ratos.

As BMPs promovem uma melhoria na derivação das CGPs a partir de células-tronco

embrionárias de humanos (KEE et al., 2006) e camundongos (YOUNG; DIAS; LOVELAND,

2010). Childs et al. (2010) relataram que a BMP-4 aumenta a expressão de marcadores pré-

migratórios (Oct4, Nanog e c-Kit) e pós-migratórios (Dazl e Vasa) em CGPs na diferenciação de

células-tronco embrionárias. Por outro lado, o tratamento com BMP-4 diminuiu a expressão Vasa

em células-tronco embrionárias humanas após um cultivo longo (21 dias) em culturas em

monocamada (TILGNER et al., 2008). VASA é expresso apenas por células germinativas em

diferenciação, assim a BMP-4 tem um efeito negativo sobre a derivação de células germinativas

derivadas a partir de células-tronco embrionárias humanas, resultando em perda de CGP pós-

migratórias em ovários tratados com BMP. A exposição contínua a BMPs pode ser prejudicial

para a sobrevivência de células germinativas derivadas de células-tronco embrionárias (CHILDS

et al., 2010).

Estudos em murinos e humanos demonstram que as BMPs promovem a diferenciação

de células germinativas a partir de células-tronco embrionárias humanas (hESCs) ou células-

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tronco pluripotentes induzidas (iPS) in vitro (KEE et al., 2009; PANULA et al., 2011; WEI et al.,

2008). Para determinar se proteínas morfogenéticas ósseas recombinantes humanas (rhBMPs)

podem induzir a diferenciação de células germinativas a partir de células-tronco embrionárias,

Kee et al. (2006) ao utilizarem a de BMP-4 recombinante humana verificaram que ela aumentou

a expressão de marcadores específicos de células germinativas tais como: Vasa, Sycp3. Além

disso, BMP-7 e BMP8b associada à BMP-4 demonstraram efeitos sinérgicos na indução de

células germinativas. Deste modo, a adição de BMPs na diferenciação de células-tronco

embrionárias humanas tem aumentado o percentual de células marcadas positivamente para

VASA, bem como a diferenciação de células germinativas humanas (KEE et al., 2006). Shah et

al. (2015a,b) demonstraram que a BMP-4 induz a diferenciação de células-tronco embrionárias

de búfalos em células germinativas.

O papel das BMPs na especificação de células germinativas foi demonstrado in vitro,

Ying et al. (2001) adicionaram BMP-4 e BMP-8b em culturas de epiblastos e foi observada a

indução da formação das CGP. Toyooka et al. (2003) ao promoverem o co-cultivo de células

produtoras de BMP-4 com células-tronco embrionárias de camundongos, aumentou o número de

CGP formadas. Nesse contexto, foi sugerido que as BMPs recombinantes podem induzir

diferenciação de células germinativas in vitro, especialmente células-tronco embrionárias de

murinos. Xu et al. (2002) indicaram que quando as células-tronco embrionárias humanas foram

incubadas com BMP4 recombinante, houve a indução na transcrição de diversos genes

relacionados com o desenvolvimento do trofoblasto.

2.5 Fluido folicular e seu papel durante a formação das CGPs e oócitos

O fluido folicular serve como uma importante fonte de substâncias regulatórias ou

moduladoras derivadas do sangue ou das secreções de células foliculares (BARNETT et al.,

2006). Atualmente, a principal hipótese sobre o mecanismo de formação do antro folicular,

sugere que as células da granulosa geram um gradiente osmótico ao produzir substâncias de alto

peso molecular, como os glicosaminoglicanos e os proteoglicanos, acumulando líquido entre as

células (SCHOENFELDER; EINSPANIER, 2003; CLARKE et al., 2006). O gradiente formado

atrai fluido derivado dos vasos presentes na teca e induz um relativo movimento das células da

granulosa para permitir que o fluido se acumule. Este movimento envolve a remodelação das

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junções gap entre as células, ou mesmo a morte de algumas células da granulosa (RODGERS;

IRVING-RODGERS, 2010). Em adição, estudos têm demonstrado que a expressão de proteínas,

como as aquaporinas, nas células da granulosa facilitam o transporte de água através das células

(McCONNELL et al., 2002; SKOWRONSKI; KWON; NIELSEN, 2009).

O fluido folicular humano contém uma variedade de substâncias bioquímicas

transferidas a partir do plasma sanguíneo e segregadas a partir de células da teca e da granulosa

(FORTUNE, 1994). Como os oócitos secretam fatores de crescimento parácrinos que regulam o

desenvolvimento de células da granulosa, as células da granulosa, por sua vez regulam o

crescimento de oócitos durante a formação folicular (GILCHRIST et al., 2004). Desta forma, é

evidente que as substâncias bioquímicas das células da granulosa podem desempenhar um papel-

chave na formação de células germinativas e desenvolvimento do oócito. Estudos mostram que o

fluido folicular contém diversos fatores biativos tais como: GDF-9 (PROCHAZKA et al., 2004;

WANG; ROY, 2004), BMP-15 (BERTOLDO et al., 2013) e gonadotrofinas (WANG; ROY,

2004). Vários estudos têm relatado que o fluido folicular suíno promove efetivamente a formação

de células germinativas a partir de células-tronco (DYCE; WEN; LI, 2006; CHENG et al., 2012;

DYCE et al., 2011). Vários trabalhos (DYCE et al., 2004; DYCE; WEN; LI, 2006; LINHER;

DYCE; LI, 2009) mostraram que células-tronco isoladas a partir da pele de fetos suínos tiveram a

capacidade de se diferenciar em células semelhantes a oócitos após cultivo em meio contendo

fluido folicular. Estas células expressaram marcadores de células germinativas (Dazl e Vasa), e

marcadores de oócitos e meiose (Gdf-9 e Oct4) (DYCE; WEN, 2006).

Em suínos, para a indução da diferenciação, as células-tronco isoladas a partir da pele

de fetos suínos foram cultivadas por até 42 dias na presença de fluido folicular e, em seguida,

observou-se a presença de marcadores específicos de células germinativas, tais como Oct4, Bmp-

15, Dazl e Vasa, comprovando a formação de células germinativas. Logo, estas células formaram

estruturas semelhantes a folículos ovarianos secretando estradiol e progesterona, e respondiam ao

estímulo de gonadotrofinas (DYCE et al., 2011), e expressavam marcadores de oócitos, como por

exemplo, proteínas da zona pelúcida e Scp3 (DYCE et al., 2011).

Um estudo mais detalhado sobre os mecanismos de indução da diferenciação de

células germinativas primordiais e estruturas semelhantes a oócitos a partir de células-tronco deu

origem ao artigo de revisão que será mostrado a seguir.

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3 ARTIGO I

In vitro differentiation of primordial germ cells and oocytes-likes cells from stem cells

(Diferenciação in vitro de celulas germinativas primordiais e células semelhantes à oócitos a

partir de células-tronco)

Artigo submetido ao periódico Histology and Histopathology

(Qualis B1 – Biotecnologia)

59

In vitro differentiation of primordial germ cells and oocytes-likes cells from stem cells

José J.N.Costa1; Glaucinete B. Souza1; Maria A.A Soares1; Regislane P. Ribeiro1; Robert van den

Hurk2, José R.V. Silva1

1Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042-280,

Sobral, CE, Brazil. 2Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht

University, Utrecht, The Netherlands.

*Corresponding address (J. R. V. Silva): Biotechnology Nucleus of Sobral - NUBIS, Federal

University of Ceara, Av. Comandante Maurocélio Rocha Ponte 100, CEP: 62.041-040, Sobral,

CE, Brazil. [[email protected]]

Abstract

Infertility is the result of failure due to an organic disorder of the reproductive organs, especially

their gametes. Recently, much progress has been made on generating germ cells, including

oocytes, from various types of stem cells. This review focuses on advances in female germ cell

differentiation from different kinds of stem cells, with emphasis on embryonic stem cells, adult

stem cells, and induced pluripotent stem cells. The advantages and disadvantages of the

derivation of female germ cells from several types of stem cells are also highlighted, as well as

the ability of stem cells to generate mature and functional female gametes. This review shows

that stem cell therapies have opened new frontiers in medicine, especially in the reproductive

area, with the possibility of regenerating fertility.

Keywords: Adult stem cells. Embryonic stem cells. Induced pluripotent stem cells. Primordial

germ cells. Oocytes-likes cells

1. Introduction

It has been estimated that human infertility affects approximately 14% of couples (Boivin

et al., 2007) and thus, in recent years, new reproductive medicine technologies have been

developed to help to reduce this problem. Women are born with a finite complement of eggs and

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an ovulated oocyte in a woman has the same age as she. It is known that cellular DNA is not

completely invulnerable to the passage of years, the impact of age on oocytes being consistent

with its effect on the risk of congenital abnormalities (Balen, 2011). Recently, it was

demonstrated that the ovaries contain cells which can be isolated and propagated, and have the

characteristics of oogonial stem cells. After reintroduction into the ovary, or in reaggregation

models, these primitive cells can form new oocytes and follicles, that can generate healthy

offspring (Johnson et al., 2004; White et al., 2012). Stem cell-based strategies for ovarian

regeneration and oocyte production have been proposed as future clinical therapies for treating

infertility in women (Volarevic et al., 2014). Stem cells are undifferentiated cells that are present

in embryonic, fetal, and adult stages of life and give rise to differentiated cells that make up

tissues and organs (Volarevic et al., 2014). From literature, it is known that oocyte-like cells

expressing different oocyte-specific genes can be developed in vitro from mouse embryonic stem

cells (mESCs) (Psathaki et al., 2011) or human embryonic stem cells (hESCs) (Richards et al.,

2010; Medrano et al., 2012). In addition, stem cells from human amniotic fluid (Cheng et al.,

2012) and from porcine fetal skin (Dyce et al., 2011) were also found to differentiate into oocyte-

like structures. Furthermore, human primordial germ cells have been differentiated from induced

pluripotent stem cells (iPS) (Panula et al., 2011; Eguizabal et al., 2011). Hayashi et al. (2012)

recently proposed that hiPS can potentially be utilized to give rise to de novo oocytes for use in

in-vitro fertilization (IVF) clinics, thus allowing sterile women to conceive a child.

In this review, we discuss the advances in our knowledge about the derivation of female

differentiated germ cells from several types of stem cells, including ES cells and iPS cells, as

well as, the mechanisms of reprogramming somatic cells into pluripotent stem cells.

2. Differentiation of primordial germ cells and oocytes-likes cells from embryonic stem cells

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ESCs are derived from totipotent cells of the early mammalian embryo from the inner cell

mass (ICM) of blastocysts and can give rise to all three germ layers (ectoderm, endoderm and

mesoderm) of the developing embryo (Evans and Kaufman, 1981; Martin, 1981; Singhal et al.,

2014). ESCs are capable of self-renewing, but also of exhibiting pluripotency, a feature

maintained by the core network of transcription factors comprising OCT4, SOX2 and NANOG

(Boiani and Schöler, 2005; Singhal et al. 2014). Dyce et al. (2014) found that expression of the

gap junction protein, CONNEXIN43 (Cx43), begins early during embryogenesis and is

maintained in many different cell types. Thus, CX43 can play an important role in somatic stem

cell proliferation and is required in at least some cases for maintaining a pluripotent,

undifferentiated stem cell population. Therefore, ESCs have been used in an in-vitro model of

early embryogenesis to investigate the detailed molecular mechanisms for developmental

processes (Itoh et al., 2014).

Female reproductive potential is limited in the majority of species due to oocyte depletion.

Functional human oocytes are restricted in number and accessibility. A robust system to

differentiate oocytes from stem cells would need a thorough investigation of the genetic,

epigenetic, and environmental factors affecting human oocyte development. The differentiation

of functional oocytes from stem cells may help to restore fertility in women. In mice, Hayashi et

al. [20] demonstrated the generation of primordial germ cell-like cells (PGCLCs) from ESCs with

high capacity for differentiation. Hayashi et al. (2011) have demonstrated that female ESCs were

induced into PGCLCs, which underwent proper development in reconstituted ovaries in vitro and

matured further into fully functional GV oocytes upon transplantation in vivo. Upon

transplantation under mouse ovarian bursa, PGCLCs in the reconstituted ovaries mature into

germinal vesicle-stage oocytes, which then contribute to fertile offspring after in-vitro maturation

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and fertilization. This system serves as a robust foundation to investigate and further reconstitute

female germline development in vitro in mammals (Hayashi et al., 2012), including the human.

Several studies in different species such as mouse (Nagano et al., 2002; Hübner et al.,

2003) and human (Clark et al., 2004; Park et al., 2009), indicate that ESCs can differentiate in

vitro into oocyte- or sperm-like cells. Nicholas et al. (2009) observed endogenous and ESC-

derived human oocyte development. Hübner et al. (2003) were the first to report that

differentiating mouse ESCs can spontaneously form oocyte-like cells in vitro, when cultivated

without LIF and feeder cells. Psathaki et al. (2011) indicated that follicle-like structures formed

by mouse ESCs in vitro consist of a single oocyte-like cell that can grow as large as 70 mm in

diameter, surrounded by one or more layers of tightly adherent somatic cells. Besides presenting

the expression of genes associated with steroidogenesis, these structures are connected via

intercellular bridges with their enclosed germ cells, which may serve to facilitate cell-to-cell

interaction required for normal follicle development (Hübner et al., 2003; Novak et al., 2006;

Albertini et al., 2001). Lacham-Kaplan et al. (2006) have cultured mouse ESC-derived embryoid

bodies (EBs), which formed ovarian-like structures containing oocyte-like cells, surrounded by

one or two layers of flattened cells, which expressed specific oocyte marker genes (FIGα and

ZP3). Kerkis et al. (2007) induced oocyte differentiation by stimulating EBs with retinoic acid.

Yu et al. (2009) reported that Deleted in Azoospermia-Like (DAZL) is a master gene controlling

germ cell differentiation and that ectopic expression of DAZL promotes the dynamic

differentiation of mouse ES cells into gametes in vitro. Furthermore, transient overexpression of

DAZL led to suppression of NANOG, but induced germ cell nuclear antigen in mESCs, whereas

DAZL knockdown resulted in reduction in the expression of germ cell markers, including

STELLA, MVH and PRDM1 (Yu et al., 2009).

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In humans, Clark et al. (2004a) demonstrated that differentiation of ESCs into EBs in

vitro results in formation of cells that express markers specific for gonocytes and thus are capable

to form germ cells in vitro. In undifferentiated cells, NANOG, STELLA, OCT4 and DAZL

appeared expressed, whereas after differentiation in EBs, mRNAs and proteins for VASA, SCP1,

SCP3, BOULE and GDF3 were found, which are germ cell specific markers (Clark et al., 2004b).

Kee et al. (2006) demonstrated that addition of recombinant human BMP4 increased the

expression of the germ cell-specific markers VASA and SCP3 during differentiation of hESCs to

embryoid bodies. In addition, BMP2 and BMP8b associated with BMP-4 demonstrated additive

effects on germ cell induction. Thus, several BMPs could induce differentiation of germ cells

from human hESCs. Medrano et al. (2012) examined whether intrinsic germ cell translational,

rather than transcriptional factors might drive germline formation and/or differentiation from

human pluripotent stem cells in vitro. They found that overexpression of VASA and/or DAZL

promoted differentiation of both hESCs into primordial germ cells. Additionally, maturation and

progression through meiosis was enhanced. Over the last decade, much progress has been made

in the differentiation of human germ cells from both hESCs (Ishii et al., 2013). Nicholas et al.

(2009) established fundamental parameters of oocyte development during ESC differentiation.

They demonstrated a timeline of definitive germ cell differentiation from ESCs in vitro that

initially parallels endogenous oocyte development in vivo by single-cell expression profiling and

analysis of functional milestones, including responsiveness to defined maturation media, shared

genetic requirement of DAZL, and entry into meiosis (Nicholas et al., 2009). Thus, ESCs can be

used in an in-vitro system to study oocyte development and could be of help in the treatment of

female infertility. Figure 1 shows the differentiation of stem cells in oocytes-like cells, as well as

the stimulating substances and markers for each stage of differentiation.

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3. Differentiation of primordial germ cells and oocytes from adult stem cells

After embryonic development, adult stem cells (ASCs) exist in almost all tissues of the

body. They are ready for emergencies after tissue injury, as well as for maintenance of tissue

homeostasis (Itoh et al., 2014). Recent literature (Johnson et al., 2004; 2005; Lee et al., 2007;

Bukovsky et al., 2008; Niikura et al., 2009; Selesniemi et al., 2009; Gong et al., 2010; Bukovsky,

2011; White et al., 2012; Tilly and Sinclair, 2013; Gheorghisan-Galateanu et al., 2014;

Hummitzsch et al., 2015) suggests the presence of germ line stem cells in mammalian ovary,

however, their ability to replenish the germ cell pool at an adult or postnatal stage during the

reproductive phase is not very clear. In this context, Zou et al. (2009) isolated female germline

stem cells (FGSCs) in postnatal mammalian ovaries from adult mice. After culture, these stem

cells underwent oogenesis, where after the recipient mice produced offspring. White et al. [4]

demonstrated that both adult mice and human ovaries possess mitotically active germ cells that

can differentiate into oocytes both in vitro and in vivo. Germ-line stem cells have been reported in

mice and human ovaries by several research groups and have been recently reviewed elaborately

(Virant-Klun et al., 2011; Telfer and Albertini, 2012; Woods et al., 2012; 2013; Dunlop et al.,

2014; Hanna and Hennebold, 2014; Hummitzsch et al., 2015); but, studies on human ovarian

stem cells are relatively few in number because of scarcity of ovarian tissue for research.

Bukovsky et al. (2005) demonstrated differentiation of surface epithelium of post-menopausal

human ovary and development into oocytes and blastocysts in vitro. Thus, those stem cells

generated functional oocytes in vitro irrespective of age and condition (menopausal and

premature ovarian failure) (2012). Already McLaughlin et al. (2015) developed a model of

estimated a woman’s ovarian reserve by non-invasive means, which is calculated a non-growing

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follicle density in the human ovarian cortex, using ovarian biopsies, and could be used in a

variety of experimental and pathological situations.

By using various techniques, Virant-Klun et al. (2008; 2009; 2013) and Virant-Klun and

Skutella (2010) identified putative stem cells in ovarian sections and in scraped ovarian surface

epithelium (OSE) from post-menopausal women and from those with premature ovarian failure

(POF), which could differentiate into oocyte-like structures and parthenotes in vitro. Parte et al.

(2011) reported two distinct stem cell populations namely, pluripotent very small embryonic-like

stem cells expressing nuclear OCT4 and slightly bigger progenitor stem cells, termed ovarian

germ stem cells (OGSCs), which express cytoplasmic OCT4 in peri-menopausal women and

other higher mammalian species. Similar results were observed in adult human (Bhartiya et al.,

2010) and mice (Bhartiya et al., 2012) ovaries. The putative stem cells differentiated

spontaneously in cultures to give rise to oocyte-like structures and parthenotes (Parte et al.,

2011), and expressed both pluripotent (OCT4, OCT4A, SSEA4, NANOG, SOX2, TERT, STAT3)

and germ cell (c-KIT, DAZL, GDF9, VASA) markers.

Bhartiya et al. (2012) reported that mouse ovarian stem cells lodged in the ovarian surface

epithelium (OSE) are modulated by pregnant mare serum gonadotropin (PMSG) resulting in neo-

oogenesis and postnatal follicular assembly. There is evidence that FSH modulates ovarian stem

cells through an alternatively spliced variant of FSH receptor. After in in-vitro cultured sheep

OSE, stem cells underwent potential self-renewal and clonal expansion as "germ cell cysts"

(Patel et al., 2013). In mice, PMSG treatment resulted in augmented stem cell activity, neo-

oogenesis and primordial follicle assembly (Bhartiya et al., 2012). Stem cells isolated from the

skin of porcine fetuses also have the ability to differentiate into oocyte-like cells (OLCs) after

stimulation with follicular fluid (Dyce et al., 2006). These cells i) express germ-cell markers,

(OCT4, GDF9B, DAZL and VASA), ii) form follicle-like aggregates that secrete oestradiol and

66

progesterone, iii) respond to gonadotropin stimulation, and iv) express oocyte markers, such as

zona pellucida (ZP), and the meiosis marker, synaptonemal complex protein 3 (SCP3) (Dyce et

al., 2006). In mice, Dyce et al. (2011) reported that newborn mice skin-derived stem cells are also

capable of differentiating into early OLCs that express oocyte markers. Linher et al. (2009) also

demonstrated that stem cells isolated from fetal porcine skin have the potential to form primordial

germ cells (PGCs) that express specifc markers (OCT4, FRAGILIS, STELLA, DAZL and VASA)

and originate putative oocytes after culture.

Stem cells were also isolated from adipose tissue and ovarian stroma from pigs and

cultured in presence of follicular fluid (Song et al., 2011). These cells expressed transcription

factors, such as OCT4, NANOG and SOX2 and exhibited their potential for in-vitro oogenesis,

since expressed markers like OCT4, GDF9B, C-MOS, VASA, DAZL, ZPC and FSHR after

culture. The described results indicate that stem cells from ovarian tissue may differentiate into

oocytes (Song et al., 2011). Figure 1 shows the differentiation of adults stem cells into oocytes-

like cells, as well as the stimulating substances and markers for each stage of differentiation. The

function of markers for stem cells, PGCs and oocytes are shown in Table 1.

4. Differentiation of primordial germ cells and oocytes from the iPS cells

The differentiation of induced pluripotent stem cells (iPS) has been observed in several

studies. Park et al. (2009), demonstrated the formation of PGCs from reprogrammed skin

fibroblasts of human. However, PGCs derived from iPS cells did not start imprint erasing

efficiently, suggesting that more studies are needed to elucidate the germ cell induction

mechanisms from iPS. In another study, Panula et al. (2011) compared the efficiency of human

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iPS derived from adult and fetal somatic cells to form primordial and meiotic germ cells. They

showed that around 5% of human iPS have the potential to differentiate into PGCs after induction

with BMPs. In addition, they had the ability to express specific markers of germ cells (DAZ,

DAZL and BOULE) and the iPS cells formed meiotic cells with extensive synaptonemal

complexes. In human species, Eguizabal et al. (2011) reported generation of postmeiotic cells

from human hiPS from different origin (keratinocytes and cord blood). These cells demonstrated

complete and robust meiotic competence, opening the way for the production of in-vitro gametes

in the human species.

Hayashi et al. (2011) demonstrated that mouse iPS cells differentiate into epiblast-like

cells, primordial germ cell-like cells (PGCLCs), and fertile oocytes, successively. After

reconstitution of ovaries with PGCLCs, these cells differentiated into germinal vesicle-stage

oocytes and contributed to fertile offspring after in-vitro maturation and fertilization (Hayashi et

al., 2012). Eguizabal et al. (2011) demonstrated the differentiation of female iPS into haploid

cells following the detection of SCP3 and H2AX proteins, which are indicators of meiotic

competence. Singhal et al. (2015) demonstrated the generation of germ cell-like cells and oocyte-

like cells from goat iPS (giPS) from fibroblast cells. These germ cell-like cells were characterized

by expression of germ cell specific markers (VASA, DAZL, STELLA and SCP3) at transcription

and protein level. These giPS differentiated into primordial germ cell-like cells in the presence of

retinoic acid and BMP4. Among the differentiated germ cell-like cells, oocyte-like structures

were observed. Figure 2 shows the strategy to restore fertility by differentiating iPS into oocytes.

4.1. Mechanisms of cellular reprogramming to produce iPS

Efficient reprogramming methods have been explored since the first report of the

generation of human induced pluripotent stem cells (Takahashi et al., 2007; Yu et al., 2007). In

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differentiated cells (adult cells), absence of expression of the pluripotency genes may be due to

changes in chromatin due to DNA methylation or histone acetylation in the promoter region of

these genes (Li, 2002; Li et al., 2007; Cedar and Bergman, 2009). Currently, new methods have

been applied in the cellular reprogramming process, especially in the production of iPS cells,

which include the use of episomal plasmids (Yu et al., 2009) or excisable expression systems

(Soldner et al., 2009), recombinant cell-penetrating reprogramming proteins (Kim et al., 2009;

Zhou et al., 2009), reprogramming mRNAs (Warren et al., 2010; Yakubov et al., 2010), or

microRNAs (Anokye-Danso et al., 2011; Miyoshi et al., 2011). Furthermore, a growing number

of compounds have been identified that can functionally replace reprogramming transcription

factors, enhance efficiency of iPS generation and accelerate the reprogramming process (Zhang et

al., 2012).

Takahashi and Yamanaka (2006) tested 24 different candidate factors that play important

roles in the maintenance of pluripotency. They found that four transcription factors (OCT4,

SOX2, C-MYC and KLF4) are involved in the generation of iPS, which are similar to ESCs in

morphology, proliferation, and teratoma formation. In regard to aging and rejuvenation, the

reprogramming process resets an aged, somatic cell to a more youthful state, elongating

telomeres, rearranging the mitochondrial network, reducing oxidative stress, restoring

pluripotency, and making numerous other alterations (Rohani et al., 2014). In goats, Singhal et al.

(2013) reprogrammed adult female goat fibroblast cells into induced pluripotent stem cells using

ectopic expression of OCT4, NANOG and SOX2 genes and the germ-cells-like cells, generated

from reprogrammed giPS, could be differentiated into goat oocytes-like structure. However, there

have been concerns over the use of integrating retroviruses to deliver the iPS factors, which could

potentially compromise the quality of or even cause tumorigenicity in the resultant iPS (Zhang et

al., 2012). The efficiency of iPS cell induction is quite low: less than 1% of human fibroblasts

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that received transcription factors OCT4/SOX2 and the oncogenes KLF4/C-MYC (together

abbreviated to OSKM) become iPS cells. However, Tanabe et al. (2013) showed that, after

receiving OSKM (Tanabe et al., 2013; 2014), the reprogramming process initiates in more than

20% of human fibroblasts.

There are several genes related to cell reprogramming, like OCT4. This gene is encoded

by the gene POU5F1 (Wu and Schöler, 2014) and is restricted in the blastomeres of the

developing mouse embryo, the ICM of blastocysts, the epiblast, germ cells and oocytes. It is also

expressed in pluripotent stem cells, including ESCs (Pesce et al., 1998; Yamanaka, 2007).

Singhal et al. (2014) demonstrated that Brahma-Related Gene 1 (BRG1) is essential for early

embryonic development, but also enhances the efficiency of reprogramming somatic cells in

murine species.

During the reprogramming process, remodeling of the epigenome and modulations of

the epigenetic processes may facilitate conversion of cell fate by making cells more permissive to

these epigenomic changes. Enzymes that stand out in this process are histone deacetylase

(HDAC), histone methyltransferase (HMT), histone demethylase (HDM) and DNA

methyltransferase (DNMT) (Zhang et al., 2012).

Among the compounds that can be used in reprogramming, 5-azacytidine, N-Phthalyl-L-

Tryptophan (RG108), Valproic acid (VPA), Trichostatin A (TSA), Sodium butyrate (NaB), and

other molecules (Federation et al., 2014) can be highlighted. Demethylation of one or more

(unknown) loci of DNA is a critical step in the late stages of direct reprogramming. Inhibition of

DNMT1 lowers this kinetic barrier, thereby facilitating the transition to pluripotency (Mikkelsen

et al., 2008).

70

DNA demethylation has been recently reported after use of 5-Azacytidine (5-Aza) or

RG108 during cellular reprogramming (Shi et al., 2008; Okita and Yamanaka, 2011; Pennarossa

et al., 2013; 2014; Federation et al., 2014). 5-Aza is a chemical derivative of the DNA nucleoside

cytidine, the only difference being the presence of a nitrogen atom at position 5 of the cytosine,

the same site at which DNA methylation occurs. Thus, 5-Aza causes DNA demethylation or

hemi-demethylation. DNA demethylation can regulate gene expression by relaxing chromatin

structure. This is detectable as an increase in nuclease sensitivity. This remodeling of chromatin

structure allows transcription factors to bind to the promoter regions, assembly of the

transcription complex, and gene expression, particularly genes associated with cell pluripotency.

As an analog of cytosine, DNA polymerase does not recognize the difference between 5-Aza and

cytosine and will incorporate 5-Aza during DNA replication (Christman, 2002; Federation et al.,

2014). Pennarosa et al. (2014) exposed pig dermal fibroblasts to 5-Aza for 18 h, followed by a

protocol for the induction of endocrine pancreatic differentiation. The results demonstrated

changes in cell morphology and expression of pluripotency genes (OCT4, NANOG, SOX2 and

REX1) (Pennarosa et al., 2014). The cells expressed insulin and were able to release it in response

to a physiological glucose challenge.

Valproic acid (VPA), 2-propyl-pentanoic acid, is a short-chain branched fatty acid

(Almutawaa et al., 2014). This VPA, an HDAC inhibitor, increases reprogramming efficiency of

human fibroblasts, enabling the efficiency of primary human fibroblasts infected with the

transcription factors OCT4, SOX2 and KLF4 (Huangfu et al., 2008). The molecular mechanisms

of VPA action involves several protein kinase pathways that have been suggested to be the

targets for this drug (Monti et al., 2009). VPA has been shown to inhibit the activity of histone

deacetylases (HDACs), resulting in chromatin remodelling and changes in gene expression (Phiel

et al., 2001; Gotfryd et al., 2011). Trichostatin A (TSA) is one of the most potent known histone

71

deacetylase inhibitor. This hydroxamic acid is in vitro active at nanomolar concentrations and

inhibits HDACs with zinc-containing catalytic sites, leading to accumulation of acetylated

histones in the nucleus and subsequent activation of target genes (Taddei et al., 2005;

Dokmanovic et al., 2007; Kim et al. 2009;). Sodium butyrate (NaB) increases histone acetylation

and is a potent HDAC inhibitor that causes hyperacetylation of histones H3 and H4 in

mammalian cells (Candido et al., 1978; Nör et al., 2013).

5. Clinical potential

Pluripotent stem cells hold great promise in the field of regenerative medicine, because

they can propagate indefinitely. In addition, these cells can lead to every other cell type (sperm,

oocytes, neurons, heart, pancreatic, and liver cells), they represent a single source of cells that

could be used to replace those lost to damage or disease.

Nearly 72.4 million people have fertility problems, caused by various underlying

pathologies, with exposure to toxicants or immune-suppressive treatments, in cases with gonadal

insufficiency due to POF or azoospermia (Boivin et al., 2007; Bhartiya et al., 2014; Volarevic et

al., 2014). Currently, several advancements have been made in assisted reproduction treatment,

especially the generation of gametes derived from pluripotent stem cells (Bhartiya et al., 2014).

Considering that the use of ESCs has led to deliberate ethical controversy (Klimanskaya et al.,

2006), ASCs or iPS can be considered the most suitable type of cells to produce patient-matched

oocytes that can be used to recover fertility.

6. Final considerations

72

This review emphasizes the potential of embryonic stem cells, adult stem cells and

induced pluripotent stem cells to produce germ cells and oocytes. Recent advances in cellular

therapies have led to the understanding how stem cells can give rise to gametes and how somatic

stem cells differentiate into germ cells and oocytes. This knowledge gives significant insight into

the regulation of developmental gametes and has important implications for female fertility and

regenerative medicine. More studies are needed in this area, to clarify what problems affect the

achievement of female germ cells. Various studies addressed the potential of stem cells to

differentiate into oocytes. However, more information is necessary for obtaining mature gametes

of good quality, which subsequently have to go through a process of fertilization to produce

viable offspring. It is expected that, in the near future, stem cells can be isolated that are able to

differentiate into viable oocytes in a reproducible manner. The findings will not only contribute

to enhancement of female germ cell induction but also to reduction of female infertility rates.

Acknowledgments

This work was financially supported by CNPq (Grant N° 478198/2013-2), and the authors thank

the members of the Laboratory of Animal Reproduction of the Biotechnology Nucleus of Sobral.

Compliance with Ethical Standards

Conflict of Interest

All authors have no conflicts of interest, including employment, financial ownership, and grants

or other funding.

References

73

Albertini D.F., Combelles C.M., Benecchi E. and Carabatsos M.J. (2001). Cellular basis for

paracrine regulation of ovarian follicle development. Reproduction 121(5), 647-653.

Almutawaa W., Kang N.H., Pan Y. and Niles L.P. (2014). Induction of Neurotrophic and

Differentiation Factors in Neural Stem Cells by Valproic Acid. Basic. Clin. Pharmacol. Toxicol.

115(2), 216-221.

Anokye-Danso F., Trivedi C.M., Juhr D., Gupta M., Cui Z., Tian Y., Zhang Y., Yang W., Gruber

P.J., Epstein J.A. and Morrisey E.E. (2011). Highly efficient miRNA-mediated reprogramming of

mouse and human somatic cells to pluripotency. Cell Stem Cell 8, 376-388.

Balen A.H. (2011). Infertility in practice. Fourth edition. London: CRC Press.

Bayne R.A.L., Kinnell H.L., Coutts S.M., He J., Childs A.J. and Anderson R.A. (2015). GDF9 is

Transiently Expressed in Oocytes before Follicle Formation in the Human Fetal Ovary and is

Regulated by a Novel NOBOX Transcript. PLoS One 10(3), e0119819.

Bhartiya D., Hinduja I., Patel H. and Bhilawadikar R. (2014). Making gametes from pluripotent

stem cells – a promising role for very small embryonic-like stem cells. Reprod. Biol. Endocrinol.

12, 114.

Bhartiya D., Kasiviswanathan S., Unni S.K., Pethe P., Dhabalia J.V., Patwardhan S. and

Tongaonkar H.B. (2010). Newer insights into pre-meiotic development of germ cells in adult

human testis using Oct-4 as a stem cell marker. J. Histochem. Cytochem. 58, 1093-1106.

Bhartiya D., Sriraman K., Gunjal P. and Modak H. (2012). Gonadotropin treatment augments

postnatal oogenesis and primordial follicle assembly in adult mouse ovaries? J. Ovarian Res.

5(1), 32.

Boiani M. and Schöler H.R. (2005). Regulatory networks in embryo derived pluripotent stem

cells. Nat. Rev. Mol. Cell. Bio. 6, 872-884.

74

Boivin J., Bunting L., Collins J.A. and Nygren K. G. (2007). International estimates of infertility

prevalence and treatment-seeking: potential need and demand for infertility medical care. Hum.

Reprod. 22(6), 1506-1512.

Bukovsky A. (2011). Ovarian stem cell niche and follicular renewal in mammals. Anat. Rec. 294,

1284-1306.

Bukovsky A. and Caudle M.R. (2012). Immunoregulation of follicular renewal, selection, POF,

and menopause in vivo, vs. neo-oogenesis in vitro, POF and ovarian infertility treatment, and a

clinical trial. Reprod. Biol. Endocrinol. 10, 97.

Bukovsky A., Caudle M.R., Svetlikova M., Wimalasena J., Ayala M.E. and Dominguez R.

(2005). Oogenesis in adult mammals, including humans: a review. Endocrine 26(3), 301-16.

Bukovsky A., Gupta S.K., Virant-Klun I., Upadhyaya N.B., Copas P., Van Meter S.E.,

Svetlikova M., Ayala M.E. and Dominguez R. (2008). Study origin of germ cells and formation

of new primary follicles in adult human and rat ovaries. Method. Mol. Biol. 450, 233-265.

Cai L., Rothbart S.B., Lu R., Xu B., Chen W-Y., Tripathy A., Rockowitz S., Zheng D., Patel

D.J., Allis C.D., Strahl B.D., Song J. and Wang G.G. (2013). An H3K36 methylation-engaging

Tudor motif of polycomb-like proteins mediates PRC2 complex targeting. Mol. Cell 49, 571-582.

Candido E.P.M., Reeves R. and Davie J.R. (1978). Sodium butyrate inhibits histone deacetylation

in cultured cells. Cell 14, 105-113.

Cedar H. and Bergman Y. (2009). Linking DNA methylation and histone modification: patterns

and paradigms. Nat. Rev. Genet. 10, 295-304.

Cheng X., Chen S., Yu X., Zheng P. and Wang H. (2012). BMP15 gene is activated during

human amniotic fluid stem cell differentiation into oocyte-like cells. DNA and Cell Biol. 31,

1198-1204.

75

Christman J. K. (2002). 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA

methylation: mechanistic studies and their implications for cancer therapy. Oncogene 21(35),

5483-5491.

Clark A.T., Bodnar M.S., Fox M., Rodriquez R.T., Abeyta M.J., Firpo M.T. and Pera R.A.

(2004). Spontaneous differentiation of germ cells from human embryonic stem cells in vitro.

Hum. Mol. Genet. 13(7), 727-739.

Cooper G.M. (1994). Expression and Function of c-MOS in Mammalian Germ Cells. Adv. Dev.

Biochem. 3, 127-148.

Dokmanovic M., Clarke C. and Marks P.A. (2007). Histone deacetylase inhibitors: overview and

perspectives. Mol. Cancer Res. 5, 981-989.

Dunlop C.E., Telfer E.E. and Anderson R.A. (2014). Ovarian germline stem cells. Stem Cell Res.

Ther. 5(4), 98.

Dyce P.W., Liu J., Tayade C., Kidder G.M., Betts D.H. and Li J. (2011). In Vitro and In Vivo

Germ Line Potential of Stem Cells Derived from Newborn Mouse Skin. PLoS ONE 6(5),

e20339.

Dyce P.W., Wen L., and Li J. (2006). In vitro germline potential of stem cells derived from fetal

porcine skin. Nat. Cell Biol. 8, 384-390.

Dyce P.W., Li D., Barr K.J. and Kidder G.M. (2014). Connexin43 is required for the maintenance

of multipotency in skin-derived stem cells. Stem Cells and Dev. 23(14), 1636-1646.

Eguizabal, C., Montserrat, N., Vassena, R., Barragan, M., Garreta, E., Garcia-Quevedo, L., Vidal

F., Giorgetti A., Veiga A. and Izpisua Belmonte J. C. (2011). Complete meiosis from human

induced pluripotent stem cells. Stem Cells 29, 1186-1195.

76

El-Mestrah M., Castle P.E., Borossa G. and Kan F.W. (2002). Subcellular distribution of ZP1,

ZP2 and ZP3 glycoproteins during folliculogenesis and demonstration of their topographical

disposition within the zona matrix of mouse ovarian oocytes. Biol. Reprod. 66, 866-876.

Evans M.J. and Kaufman M.H. (1981). Establishment in culture of pluripotential cells from

mouse embryos. Nature 292(5819), 154-156.

Federation A.J., Bradner J.E. and Meissner A. (2014). The use of small molecules in somatic-cell

reprogramming. Trends Cell Biol. 24(3), 179-187.

Gheorghisan-Galateanu A.A., Hinescu M.E. and Enciu A.M. (2014). Ovarian adult stem cells:

hope or pitfall? J. Ovarian Res. 7, 71.

Gong S.P., Lee S.T., Lee E.J., Kim D.Y., Lee G., Chi S.G., Ryu B.K., Lee C.H., Yum K.E., Lee

H.J., Han J.Y., Tilly J.L., Lim J.M. (2010). Embryonic stem cell-like cells established by culture

of adult ovarian cells in mice. Fertil. Steril. 93, 2594-2601.

Gotfryd K., Hansen M., Kawa A., Ellerbeck U., Nau H., Berezin V., Bock E. and Walmod P.S.

(2011). The teratogenic potencies of valproic acid derivatives and their effects on biological end-

points are related to changes in histone deacetylase and Erk1/2 activities. Basic Clin. Pharmacol.

Toxicol. 109(3), 164-174.

Hanna C.B. and Hennebold J.D. (2014). Ovarian germline stem cells: an unlimited source of

oocytes? Fertil. Steril. 101(1), 20-30.

Hayashi K., Ogushi S., Kurimoto K., Shimamoto S., Ohta H. and Saitou, M. (2012). Offspring

from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 338, 971-975.

Hayashi K., Ohta H., Kurimoto K., Aramaki S. and Saitou M. (2011). Reconstitution of the

mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532.

77

Huangfu D., Osafune K., Maehr R., Guo W., Eijkelenboom A., Chen, S., Muhlestein W. and

Melton D.A. (2008). Induction of pluripotent stem cells from primary human fibroblasts with

only Oct4 and Sox2. Nat. Biotechnol. 26, 1269-1275.

Hübner K., Fuhrmann G., Christenson L.K., Kehler J., Reinbold R., De La Fuente R., Wood J.,

Strauss J.F., Boiani M. and Schöler H.R. (2003). Derivation of oocytes from mouse embryonic

stem cells. Science 300(5623), 1251-1256.

Hummitzsch K., Anderson R.A., Wilhelm D., Wu J., Telfer E.E., Russell D.L., Robertson S.A.

and Rodgers R.J. (2015). Stem Cells, Progenitor Cells, and Lineage Decisions in the Ovary.

Endocrine Rev. 36(1), 65-91.

Hu W., Qiu B., Guan W., Wang Q., Wang M., Li W., Gao L., Shen L., Huang Y., Xie G., Zhao

H., Jin Y., Tang B., Yu Y., Zhao J. and Pei G. (2015). Direct Conversion of Normal and

Alzheimer's Disease Human Fibroblasts into Neuronal Cells by Small Molecules. Cell Stem Cell

17(2), 204-212.

Ishii T., Pera R.A. and Greely, H.T. (2013). Ethical and legal issues arising in research on

inducing human germ cells from pluripotent stem cells. Cell Stem Cell 13, 145-148.

Itoh F., Watabe T. and Miyazono, K. (2014). Roles of TGF-β family signals in the fate

determination of pluripotent stem cells. Semin. Cell. Dev. Biol. 32, 98-106.

Johnson J., Bagley J., Skaznik-Wikiel M., Lee H. J., Adams G. B., Niikura, Y., Tschudy K.S.,

Tilly J.C., Cortes M.L., Forkert R., Spitzer T., Iacomini J., Scadden D.T. and Tilly J.L. (2005).

Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and

peripheral blood. Cell 122, 303-315.

Johnson J., Canning J., Kaneko T., Pru J.K. and Tilly, J.L. (2004). Germline stem cells and

follicular renewal in the postnatal mammalian ovary. Nature 428, 145-150.

78

Kee K., Angeles V.T., Flores M., Nguyen H.N. and Reijo Pera R.A. (2009). Human DAZL, DAZ

and BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature 462,

222-225.

Kee K., Gonsalves J.M., Clark A.T. and Reijo Pera, R.A. (2006). Bone Morphogenetic Proteins

Induce Germ Cell Differentiation from Human Embryonic Stem Cells. Stem Cells Dev. 15, 831-

837.

Kerkis A., Fonseca S.A., Serafim R.C., Lavagnolli T.M., Abdelmassih S., Abdelmassih R.,

Kerkis I. (2007). In vitro differentiation of male mouse embryonic stem cells into both

presumptive sperm cells and oocytes. Cloning Stem Cells 9, 535-548.

Kerr C.L. and Cheng L. (2010). The dazzle in germ cell differentiation. J. Mol. Cell Biol. 2(1),

26-29.

Kim D., Kim C.H., Moon J.I., Chung Y.G., Chang M.Y., Han B.S., Ko S., Yang E., Cha K.Y.,

Lanza R. and Kim K.S. (2009). Generation of human induced pluripotent stem cells by direct

delivery of reprogramming proteins. Cell Stem Cell 4(6), 472–476.

Klimanskaya I., Chung Y., Becker S., Lu S-J. and Lanza R. (2006). Human embryonic stem cell

lines derived from single blastomeres. Nature 444, 481-485.

Lacham-Kaplan O., Chy H. and Trounson A. (2006). Testicular cell conditioned medium

supports differentiation of embryonic stem cells into ovarian structures containing oocytes. Stem

Cells 24, 266-273.

Lavial F., Acloque H., Bertocchini F., Macleod D.J., Boast S., Bachelard E., Montillet G., Thenot

S., Sang H.M., Stern C.D., Samarut J. and Pain B. (2007). The Oct4 homologue PouV and Nanog

regulate pluripotency in chicken embryonic stem cells. Development 134, 3549-3563.

Lee H.J., Selesniemi K., Niikura Y., Niikura T., Klein R., Dombkowski D.M. and Tilly J.L.

(2007). Bone marrow transplantation generates immature oocytes and rescues long-term fertility

79

in a preclinical mouse model of chemotherapy-induced premature ovarian failure. J. Clin. Oncol.

25, 3198-3204.

Levine A. and Brivanlou A. (2006). GDF3 at the crossroads of TGF-beta signaling. Cell Cycle

5(10), 1069–1073.

Liang L., Soyal S.M. and Dean J. (1997). FIGalpha, a germ cell specific transcription factor

involved in the coordinate expression of the zona pellucida genes. Development 124(24), 4939-

4947.

Li E. (2002). Chromatin modification and epigenetic reprogramming in mammalian

development. Nat. Rev. Genet. 3, 662-673.

Li J., Wang G., Wang C., Zhao Y., Zhang H., Tan Z., Song Z, Ding M, Deng H. (2007).

MEK/ERK signaling contributes to the maintenance of human embryonic stem cell self-renewal.

Differentiation 75, 299-307.

Linher K., Dyce P. and Li J. (2009). Primordial Germ Cell-Like Cells Differentiated In Vitro

from Skin-Derived Stem Cells. PLoS ONE 4(12), e8263.

Marqués-Marí A.I., Lacham-Kaplan O., Medrano J.V., Pellicer A. and Simón, C. (2009).

Differentiation of germ cells and gametes from stem cells. Hum. Reprod. Update 1(1), 1-12.

Martin G.R. (1981). Isolation of a pluripotent cell line from early mouse embryos cultured in

medium conditioned by teratocarcinoma stem cells. PNAS 78(12), 7634-7638.

McLaughlin M., Kelsey T.W., Wallace W.H.B., Anderson R.A. and Telfer, E.E. (2015). An

externally validated age-related model of mean follicle density in the cortex of the human ovary.

J. Assist. Reprod. Genet. 32, 1089-1095.

Medrano J.V., Ramathal C., Nguyen H.N., Simon C. and Reijo Pera R.A. (2012). Divergent

RNA-binding proteins, DAZL and VASA, induce meiotic progression in human germ cells

derived in vitro. Stem Cells 30, 441-451.

80

Medrano J.V, Marqués-Marí A.I., Aguilar C.E., Riboldi M., Garrido N., Martínez-Romero A.,

O'Connor E., Gil-Salom M. and Simón C. (2010). Comparative analysis of the germ cell markers

c-KIT, SSEA-1 and VASA in testicular biopsies from secretory and obstructive azoospermias.

Mol. Hum. Reprod. 16, 811-817.

Mikkelsen T.S., Hanna J., Zhang X., Ku M., Wernig M., Schorderet P., Bernstein B.E., Jaenisch

R., Lander E.S. and Meissner A. (2008). Dissecting direct reprogramming through integrative

genomic analysis. Nature 454, 49-55.

Miyoshi, N., Ishii, H., Nagano, H., Haraguchi, N., Dewi, D. L., Kano, Y., Nishikawa S.,

Tanemura M., Mimori K., Tanaka F., Saito T., Nishimura J., Takemasa I., Mizushima T., Ikeda

M., Yamamoto H., Sekimoto M., Doki Y. and Mori M. (2011). Reprogramming of mouse and

human cells to pluripotency using mature microRNAs. Cell Stem Cell 8(6), 633-638.

Monti B., Polazzi E. and Contestabile A. (2009). Biochemical, molecular and epigenetic

mechanisms of valproic acid neuroprotection. Curr. Mol. Pharmacol. 2, 95-109.

Nagano, M., Patrizio, P. and Brinster, R.L. (2002). Long-term survival of human spermatogonial

stem cells in mouse testes. Fertil. Sterility. 78, 1225-1233.

Nicholas C.R., Haston K.M., Grewall A.K., Longacre T.A. and Reijo Pera R.A. (2009).

Transplantation directs oocyte maturation from embryonic stem cells and provides a therapeutic

strategy for female infertility. Hum. Mol. Genet. 18, 4376–4389.

Niikura Y., Niikura T. and Tilly J.L. (2009). Aged mouse ovaries possess rare premeiotic germ

cells that can generate oocytes following transplantation into a young host environment. Aging 1,

971-978.

Niwa H., Ogawa K., Shimosato D. and Adachi, K. (2009). A parallel circuit of LIF signalling

pathways maintains pluripotency of mouse ES cells. Nature 460, 118-122.

81

Nör C., Sassi F.A., de Farias C.B., Schwartsmann G., Abujamra A.L., Lenz G., Brunetto A.L.

and Roesler R. (2013). The histone deacetylase inhibitor sodium butyrate promotes cell death and

differentiation and reduces neurosphere formation in human medulloblastoma cells. Mol.

Neurobiol. 48, 533-543.

Novak I., Lightfoot D.A., Wang H., Eriksson A., Mahdy E. and Höög C. (2006). Mouse

embryonic stem cells form follicle-like ovarian structures but do not progress through meiosis.

Stem Cells 24(8), 1931-1936.

Okita K. and Yamanaka S. (2011). Induced pluripotent stem cells: opportunities and challenges.

Philos. Trans. R. Soc. Lond. B Biol. Sci. 366(1575), 2198–2207.

Panula S., Medrano J.V., Kee K., Bergström R., Nguyen H.N., Byers B., Wilson K.D., Wu J.C.,

Simon C., Hovatta O. and Reijo Pera R.A. (2011). Human germ cell differentiation from fetal-

and adult-derived induced pluripotent stem cells. Hum. Mol. Genet. 20, 752-762.

Park T.S., Galic Z., Conway A.E., Lindgren A., van Handel B.J., Magnusson M., Richter L.,

Teitell M.A., Mikkola H.K., Lowry W.E., Plath K. and Clark A.T. (2009). Derivation of

primordial germ cells from human embryonic and induced pluripotent stem cells is significantly

improved by coculture with human fetal gonadal cells. Stem Cells 27, 783-795.

Parte S., Bhartiya D., Telang J., Daithankar V., Salvi V., Zaveri K., Hinduja, I. (2011). Detection,

characterization, and spontaneous differentiation in vitro of very small embryonic-like putative

stem cells in adult mammalian ovary. Stem Cells Dev. 20(8), 1451-1464.

Patel D.M., Shah J. and Srivastava A.S. (2013). Therapeutic Potential of Mesenchymal Stem

Cells in Regenerative Medicine. Stem Cells Int. 496218.

Pennarossa G., Maffei S., Campagnol M., Rahman M.M., Brevini T.A.L. and Gandolfi F. (2014).

Reprogramming of Pig Dermal Fibroblast into Insulin Secreting Cells by a Brief Exposure to 5-

aza-cytidine. Stem Cell Rev Rep. 10(1), 31-43.

82

Pennarossa G., Maffei S., Campagnol M., Tarantini L., Gandolfi F. and Brevini T.A.L. (2013).

Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-

secreting cells. PNAS 110(22), 8948-8953.

Pesce M., Gross M.K. and Scholer, H.R. (1998). In line with our ancestors: Oct-4 and the

mammalian germ. Bioessays 20, 722-732.

Phiel C.J., Zhang F., Huang E.Y., Guenther M.G., Lazar M.A. and Klein P.S. (2001). Histone

deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and

teratogen. J. Biol. Chem. 276(39), 36734-36741.

Psathaki O.E., Hübner K., Sabour D., Sebastiano V., Wu G., Sugawa, F., Wieacker P.,

Pennekamp P. and Schöler H.R. (2011). Ultrastructural Characterization of Mouse Embryonic

Stem Cell-Derived Oocytes and Granulosa Cells. Stem Cells Dev. 20(12), 2205-2215.

Richards M., Fong C.Y. and Bongso A. (2010). Comparative evaluation of different in vitro

systems that stimulate germ cell differentiation in human embryonic stem cells. Fertil. Steril.

93(3), 986-994.

Rohani L., Johnson A.A., Arnold A. and Stolzing A. (2014). The aging signature: a hallmark of

induced pluripotent stem cells? Aging Cell 13(1), 2-7.

Salvador L.M., Silva C.P., Kostetskii I., Radice G.L. and Strauss J.F. (2008). The promoter of the

oocyte-specific gene, Gdf9, is active in population of cultured mouse embryonic stem cells with

an oocyte-like phenotype. Methods 45, 172-181.

Selesniemi K., Lee H-J., Niikura T. and Tilly J.L. (2009). Young adult donor bone marrow

infusions into female mice postpone age-related reproductive failure and improve offspring

survival. Aging 1, 49-57.

83

Shi Y., Desponts C., Do J.T., Hahm H.S., Scholer H.R. and Ding S. (2008). Induction of

pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule

compounds. Cell Stem Cell 3, 568-574.

Singhal D.K., Malik H.N., Singhal R. Saugandhika S., Dubey A., Boateng, S., Kumar S.,

Kaushik J.K., Mohanty A.K. and Malakar D. (2013). 199 germ-cell-like cells generation from

goat induced pluripotent stem cells. Reprod. Fertil. Dev. 26(1), 214-214.

Singhal D.K., Singhal R., Malik H.N., Singh S., Kumar S., Kaushik J.K., Mohanty A.K. and

Dhruba, M. (2015). Generation of Germ Cell-Like Cells and Oocyte-Like Cells from Goat

Induced Pluripotent Stem Cells. J. Stem Cell Res. Ther. 5(5).

Singhal N., Esch D., Stehling M. and Schöler H.R. (2014). BRG1 Is Required to Maintain

Pluripotency of Murine Embryonic Stem Cells. BioRes. Open Access 3(1), 1-8.

Soldner F., Hockemeyer D., Beard C., Gao Q., Bell G.W., Cook E.G., Hargus G., Blak A.,

Cooper O., Mitalipova M., Isacson O. and Jaenisc R. (2009). Parkinson's disease patient-derived

induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964-977.

Song S-H., Kumar B.M., Kang E-J., Lee Y-M., Kim T-H., Ock S-A., Lee S.L., Jeon B.G. and

Rho G.J. (2011). Characterization of Porcine Multipotent Stem/Stromal Cells Derived from Skin,

Adipose, and Ovarian Tissues and Their Differentiation In Vitro into Putative Oocyte-Like Cells.

Stem Cells Dev. 20(8), 1359-1370.

Sriraman K., Bhartiya D., Anand S. and Bhutda S. (2015). Mouse Ovarian Very Small

Embryonic-Like Stem Cells Resist Chemotherapy and Retain Ability to Initiate Oocyte-Specific

Differentiation. Reprod. Sci. 22(7), 884-903.

Taddei A., Roche D., Bickmore W.A. and Almouzni, G. (2005). The effects of histone

deacetylase inhibitors on heterochromatin: implications for anticancer therapy? EMBO Rep. 6(6),

520-524.

84

Takahashi K., Tanabe K., Ohnuki M., Narita M., Ichisaka T., Tomoda K. and Yamanaka S.

(2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell

131(5), 861-872.

Takahashi K. and Yamanaka S. (2006). Induction of pluripotent stem cells from mouse

embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676.

Tanabe K., Nakamura M., Narita M., Takahashi K. and Yamanaka S. (2013). Maturation, not

initiation, is the major roadblock during reprogramming toward pluripotency from human

fibroblasts. PNAS 110(30), 12172–12179.

Tanabe K., Takahashi K. and Yamanaka S. (2014). Induction of pluripotency by defined factors.

Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 90(3), 83-96.

Telfer E.E. and Albertini D.F. (2012). The quest for human ovarian stem cells. Nat. Med. 18(3),

413-421.

Tilly J.L. and Sinclair, D.A. (2013). Germline energetics, aging, and female infertility. Cell

Metab. 17, 838-850.

Toyooka Y., Tsunekawa N., Takahashi Y., Matsui Y., Satoh M. and Noce, T. (2000). Expression

and intracellular localization of mouse Vasa-homologue protein during germ cell development.

Mech. Dev. 93, 139-149.

Virant-Klun, I., Rozman, P., Cvjeticanin, B., Vrtacnik-Bokal, E., Novakovic, S., Rülicke, T.,

Dovc P. and Meden-Vrtovec H. (2009). Parthenogenetic embryo-like structures in the human

ovarian surface epithelium cell culture in postmenopausal women with no naturally present

follicles and oocytes. Stem Cells Dev. 18(1), 137-149.

Virant-Klun I. and Skutella, T. (2010). Stem cells in aged mammalian ovaries. Aging 2, 3-6.

85

Virant-Klun I., Stimpfel M., Cvjeticanin B., Vrtacnik-Bokal E. and Skutella, T. (2013). Small

SSEA-4-positive cells from human ovarian cell cultures: related to embryonic stem cells and

germinal lineage? J. Ovarian Res. 6, 24-43.

Virant-Klun I., Stimpfel M. and Skutella T. (2011). Ovarian pluripotent/multipotent stem cells

and in vitro oogenesis in mammals. Histol. Histopathol. 26, 1071-1082.

Virant-Klun I., Zech N., Rozman P., Vogler A., Cvjeticanin B., Klemenc P., Malicev E. and

Meden-Vrtovec H. (2008). Putative stem cells with an embryonic character isolated from the

ovarian surface epithelium of women with no naturally present follicles and oocytes.

Differentiation 76(8), 843-856.

Volarevic V., Bojic S., Nurkovic J., Volarevic A., Ljujic B., Arsenijevic N., Lako M. and

Stojkovic M. (2014). Stem Cells as New Agents for the Treatment of Infertility: Current and

Future Perspectives and Challenges. Biomed Res. Int. 2014, 507234.

Warren L., Manos P.D., Ahfeldt T., Loh Y-H., Li H., Lau F., Ebina W., Mandal P.K., Smith

Z.D., Meissner A., Daley G.Q., Brack A.S., Collins J.J., Cowan C., Schlaeger T.M. and Rossi

D.J. (2010). Highly efficient reprogramming to pluripotency and directed differentiation of

human cells using synthetic modified mRNA. Cell Stem Cell 7(5), 618-630.

West J.A., Park I.H., Daley G.Q. and Geijsen N. (2006). In vitro generation of germ cells from

murine embryonic stem cells. Nat. Protoc. 1, 2026-2036.

White Y.A., Woods D.C., Takai Y., Ishihara O., Seki H. and Tilly, J.L. (2012). Oocyte formation

by mitotically active germ cells purified from ovaries of reproductive-age women. Nat. Med. 18,

413-421.

Woods D.C., Telfer E.E. and Tilly J.L. (2012). Oocyte Family Trees: Old Branches or New

Stems? PLoS Genet. 8(7), e1002848.

86

Woods D.C., White Y.A., Niikura Y., Kiatpongsan S., Lee H.J. and Tilly J.L. (2013). Embryonic

stem cell-derived granulosa cells participate in ovarian follicle formation in vitro and in vivo.

Reprod. Sci. 20, 524-535.

Wu G. and Schöler, H. R. (2014). Role of Oct4 in the early embryo development. Cell Regen.

3(1), 7.

Yakubov E., Rechavi G., Rozenblatt, S. and Givol D. (2010). Reprogramming of human

fibroblasts to pluripotent stem cells using mRNA of four transcription factors. Biochem. Biophys.

Res. Commun. 394, 189-193.

Yamanaka S. (2007). Strategies and new developments in the generation of patient-specific

pluripotent stem cells. Cell Stem Cell 1, 39-49.

Yuan L., Liu J.G., Zhao J., Brundell E., Daneholt B. and Hoog C. (2000). The murine SCP3 gene

is required for synaptonemal complex assembly, chromosome synapsis, and male fertility. Mol.

Cell 5(1), 73-83.

Yu H., Vu T.H., Cho K.S., Guo C. and Chen D.F. (2014). Mobilizing endogenous stem cells for

retinal repair. Transl. Res. 163(4), 387-398.

Yu J., Hu K., Smuga-Otto K, Tian S., Stewart R., Slukvin I.I. and Thomson J.A. (2009). Human

induced pluripotent stem cells free of vector and transgene sequences. Science 324(5928), 797-

801.

Yu J., Vodyanik M.A., Smuga-Otto K., Antosiewicz-Bourget J., Frane J.L., Tian, S., Nie J.,

Jonsdottir G.A., Ruotti V., Stewart R., Slukvin I.I. and Thomson J.A. (2007). Induced pluripotent

stem cell lines derived from human somatic cells. Science 318, 1917-1920.

Yu Z., Ji P., Cao J., Zhu S., Li Y., Zheng L., Chen X. and Feng L. (2009). Dazl Promotes Germ

Cell Differentiation from Embryonic Stem Cells. J. Mol. Cell Biol. 1, 93-103.

87

Zhang J., Nuebel E., Daley G.Q., Koehler C.M. and Teitell, M.A. (2012). Metabolic Regulation

in Pluripotent Stem Cells during Reprogramming and Self-Renewal. Cell Stem Cell 11(5), 589-

595.

Zhao W., Ji X., Zhang F., Li L. and Ma L. (2012). Embryonic Stem Cell Markers. Molecules 17,

6196-6236.

Zhou H., Wu S., Joo J.Y., Zhu S., Han D.W., Lin T., Trauger S., Bien G., Yao S., Zhu Y.,

Siuzdak G., Schöler H.R., Duan L. and Ding S. (2009). Generation of induced pluripotent stem

cells using recombinant proteins. Cell Stem Cell 4, 381-384.

Zhu Y., Hu H.L., Li P., Yang S., Zhang W., Ding H., Tian R.H., Ning Y., Zhang L.L., Guo X.Z.,

Shi Z.P., Li Z. and He Z. (2012). Generation of male germ cells from induced pluripotent stem

cells (iPS cells): an in vitro and in vivo study. Asian J. Androl. 14(4), 574-579.

Zou K., Yuan Z., Yang Z., Luo H., Sun K. and Zhou L. (2009). Production of offspring from a

germline stem cell line derived from neonatal ovaries. Nat. Cell Biol. 11, 631-636.

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Figures and tables

Table 1. Molecular markers for stem cells, PCGs, oocytes and their functions.

Figure 1. Schematic presentation of the development of stem cells into oocytes-like cells, and the

stimulating substances and markers for each stage of differentiation.

Figure 2. Procedure for restoring fertility by differentiating iPS into oocytes.

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Table 1. Molecular markers for stem cells, PCGs, oocytes and their functions.

Markers Functions References

Stem cells

OCT4 Transcription factor associated with

self-renewal of undifferentiated embryonic

stem cells, used as a marker for

undifferentiated cells.

Lavial et al., 2007;

Hu et al., 2015

NANOG Transcription regulator involved in ICM and

ESC proliferation and self-renewal, and

prevents the differentiation of ESC in towards

extraembryonic endoderm and trophectoderm

lineages.

Lavial et al., 2007;

Singhal et al., 2015

SOX2 Transcription factor associated with controls

the expression of genes involved in embryonic

development such as YES1, FGF4, UTF1 and

ZFP206. Furthermore , it is essential for early

embryogenesis and for embryonic stem cell

pluripotency.

Yu et al., 2014

REX1 Marker of pluripotency, is usually found in

undifferentiated embryonic stem cells, its

regulation is also critical in maintaining a

pluripotent state, as the cells begin to

differentiate, Rex1 is severely and abruptly

downregulated

Lavial et al., 2007

Klf4 Mediator to LIF-Stat3 signal changes, and

directly binds to the promoter of Nanog to help

Oct4 and Sox2 in regulating the expression of

Nanog


c-Myc Regulator the self-renewal and

pluripotency of mESC as well as pluripotent

cells of the early

embryo


SSEA4 Cell membrane protein of ESC Zhao et al., 2012

STAT3 Main extracellular signal that sustains mouse

ESCs self-renewal and pluripotency is the

activation of leukemia inhibitory factor

(LIF) pathway

Niwa et al., 2009

BRG1 Important role in maintaining pluripotency by

fine-tuning the expression level of Oct4 and

other pluripotency-associated genes.


Cx43 Mouse ESC express this connexin and form

functional gap junctions during growth

Dyce et al., 2014

PGC

DAZL Protein involved in early germ cell

differentiation

Panula et al., 2011;

Clark et al., 2004;

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Kerr and Cheng,

2010

BLIMP1 or

PRDM1

First indicator of germ cell fate Yu et al., 2009

STELLA Gene initial germline Hu et al., 2015;

West et al., 2006

c-KIT Migration and survival of PGCs Marqués-Marí et al.,

2009; Cai et al.,

2013

MVH Germ pre-meiotic cell-specific marker Cai et al., 2013;

Toyooka et al., 2000

FRAGILIS Early indicator of germ cell fate Medrano et al., 2012

VASA Germ pre-meiotic cell-specific marker Hu et al., 2015;

Medrano et al., 2010

Zhu et al., 2012

BOULE Genes modulate primordial germ-cell and

haploid gamete formation

Kee et al., 2009

GDF3 Protein that modulate TGF-β superfamily

members, e.g. potentiates the activity of

NODAL

Levine and

Brivanlou, 2006

Oocytes

SCP1, SCP3 Component of the synaptonemal complex, and

this complex is involved in synapsis,

recombination and segregation of meiotic

chromosomes.

Hu et al., 2015;

Yuan et al., 2000

Figa Factor required for progression of germ cells

through the pachytene stage of MI

Marqués-Marí et al.,

2009;

Liang et al., 1997

ZP1, ZP2 e

ZP3

Glycoproteins are markers of female germ cells in

post-meiotic development which are only

secreted by oocytes within primary follicles, to

form a glycocalyx known as the zona pellucida

Hu et al., 2015;

El-Mestrah et al.,

2002

GDF9 Key factor regulating germ cell development,

and act as master regulators oocyte-specific

transcription factors

Salvador et al., 2008;

Sriraman et al., 2015

GDF9B Expressed in the human fetal ovary at the time

of oocyte nest breakdown and primordial

follicle formation

Bayne et al., 2015

Nobox Essential for follicle formation and oocyte

survival, and regulates the expression of GDF9

in humans


c-MOS Specifically expressed in male and female germ cells where it appears to play a central role in

regulating the meiotic cell cycle

Cooper, 1994

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FSH-R Stimulates proliferation and differentiation of

progenitors ovarian germ stem cells to oocytes

and primordial follicle assembly

Bhartiya et al., 2012

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Figure 1. Schematic presentation of the development of stem cells into oocytes-like cells, and the stimulating substances and markers

for each stage of differentiation.

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Figure 2. Procedure for restoring fertility by differentiating iPS into oocytes.

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4 PROBLEMA

Conforme mostrado na revisão de literatura, vários estudos têm demonstrado a

diferenciação in vitro de oócitos a partir da utilização de células-tronco embrionárias, células-

tronco adultas, células-tronco ovarianas, células da linhagem germinativa isoladas de ovários de

indivíduos adultos, e ainda, células-tronco de pluripotência induzidas. Diante disto, levantou-se

os seguintes questionamentos:

1) Será que a utilização do 5-aza-citidina em fibroblastos bovinos é capaz de

promover a expressão de RNAs mensageiros para marcadores de pluripotência?

2) Será que a BMP-2, BMP-4 e fluido folicular estimulam a formação das células

germinativas primordiais e posterior diferenciação em oócitos a partir de fibroblastos tratados

com 5-aza-citidina e aumentam os níveis de RNA mensageiros para os marcadores de células

germinativas (VASA, DAZL e C-KIT), de meiose (SCP3) e de oócitos (ZPA e GDF-9) após o

cultivo in vitro?

3) Será que a BMP-2, BMP-4 e fluido folicular contribuem para a formação das

células germinativas primordiais e posterior diferenciação em oócitos a partir de células-tronco

isoladas de ovários bovinos e aumentam os níveis de RNA mensageiros para os marcadores de

células germinativas (VASA, DAZL e C-KIT), de meiose (SCP3) e de oócitos (ZPA e GDF-9)

após o cultivo in vitro?

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5 JUSTIFICATIVA

Nos últimos anos, a reprogramação de células somáticas por meio da transdução viral

de fatores de transcrição em animais de laboratório e em humanos está associada com grandes

perspectivas para o desenvolvimento da medicina regenerativa (TAKAHASHI; YAMANAKA,

2006). No entanto, há uma grande preocupação relacionada com a introdução retroviral ou

lentiviral nas células alvo, pois isto pode causar instabilidade gênica a longo prazo, bem como o

desenvolvimento de tumores (OKITA; ICHISAKA; YAMANAKA, 2007). Desta forma, é

essencial realizar a indução de pluripotência sem a introdução de modificações genéticas. Neste

contexto, a avaliação do efeito de componentes quimicamentes definidos, como o 5-aza-citidina,

pode trazer grandes benefícios para o desenvolvimento de terapias celulares que visam à cura de

doenças e também a resolução de problemas de fertilidade.

Nos países desenvolvidos tem se observado um aumento progressivo dos problemas

de concepção em humanos devido ao fato de a maioria dos casais estarem adiando cada vez mais

a concepção do primeiro filho (SCHMIDT et al., 2012). Associado a isto, a qualidade de vida dos

casais inférteis tem sido grandemente reduzida devido à ausência de capacidade de conceber

(REIJO PERA, 2013). Assim, a partir do advento da fecundação in vitro, novas intervenções

clínicas vêm sendo desenvolvidas para a resolução de problemas de fertilidade em humanos.

Recentemente, vários estudos demostraram avanços significativos na diferenciação de óocitos a

partir de células- tronco embrionárias ou adultas em animais de laboratório (PSATHAKI et al.,

2011; WHITE et al., 2012). No entanto, até o momento, a diferenciação de oócitos a partir da

reprogramação de fibroblastos ainda não foi descrita. Devido ao fato de ter mais similaridades

com humanos do que animais de laboratório, os bovinos podem ser utilizados como modelos para

o desenvolvimento desta biotecnologia que possibilitará o estudo de fatores genéticos e

epigenéticos envolvidos com a formação oocitária. Além disso, os oócitos oriundos da

diferenciação de fibroblastos podem ser utilizados para fins biomédicos, contribuindo assim para

incrementar o desenvolvimento de terapias celulares que visem à promoção da capacidade de

concepção em casais inférteis.

No tocante a reprodução animal, a obtenção de oócitos a partir de células-tronco ou

de fibroblastos reprogramados pode contribuir para a produção de um grande número de

embriões a partir de animais de alto valor zootécnico ou em via de extinção. No caso de espécies

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já em extinção, mas que deixaram tecidos criopreservados, o desenvolvimento desta técnica

possibilitará a diferenciação de gametas a partir de células somáticas, abrindo uma nova

perspectiva de recuperação de espécies extintas. Considerando que durante a meiose, ocorre a

recombinação genética, todos os embriões produzidos seriam geneticamente diferentes e evitaria

problemas de redução de variabilidade genética dos rebanhos, que é um problema da clonagem

com fins reprodutivos. Além disso, no futuro, a diferenciação de oócitos a partir de células

somáticas pode contribuir para reduzir os tratamentos hormonais ou a punção oocitária semanal

visando à produção de embriões in vitro. Apesar de estas técnicas serem largamente utilizadas,

elas apresentam várias implicações relacionadas ao bem-estar animal (FIGUEIREDO;

MOLENTO, 2008).

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6 HIPÓTESES CIENTÍFICAS

1) O 5-aza-citidina promove a expressão de RNAs mensageiros para marcadores de

pluripotência (OCT-4, NANOG, REX-1, SOX2) em fibroblastos bovinos .

2) A BMP-2, a BMP-4 e o fluido folicular influenciam positivamente na

diferenciação de fibroblastos tratados com 5-aza-citidina e de células-tronco isoladas de ovários

bovinos, em células germinativas primordiais e oócitos.

3) A BMP-2, a BMP-4 e o fluido folicular afetam positivamente a expressão de

RNAs mensageiros para os marcadores de células germinativas (VASA e DAZL), de meiose

(SCP3) e de oócitos (ZPA e GDF-9), após o cultivo in vitro de fibroblastos tratados com 5-aza-

citidina e de células-tronco isoladas de ovários bovinos.

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7 OBJETIVOS

7.1 Objetivos gerais

1) Induzir a expressão de marcadores de pluripotência em fibroblastos bovinos e

diferenciá-los em células germinativas primordiais e em oócitos.

2) Isolar células-tronco da linhagem germinativa a partir de ovários de bovinos

adultos e diferenciá-las em células germinativas primordiais e em oócitos.

7.2 Objetivos específicos

1) Estudar os efeitos da 5-aza-citidina na indução da aquisição pluripotência em

fibroblastos bovinos, através da expressão de marcadores de pluripotência (OCT-4, NANOG,

REX-1, SOX2).

2) Avaliar os efeitos da BMP-2, BMP-4 e do fluido folicular na diferenciação de

fibroblastos tratados com 5-aza-citidina em células germinativas primordiais e em oócitos.

3) Identificar a capacidade de células-tronco isoladas de ovários de animais adultos

se diferenciarem em células germinativas primordiais e em oócitos após estímulo com BMP-2,

BMP-4 e fluido folicular.

4) Quantificar a expressão de marcadores de células germinativas (VASA e DAZL),

de meiose (SCP3) e de oócitos (ZPA e GDF-9) após o cultivo in vitro de fibroblastos tratados

com 5-aza-citidina e de células-tronco isoladas de ovários bovinos em meio suplementado com

BMP-2, BMP-4 e fluido folicular.

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8 ARTIGO II

Expression of markers for germ cells and oocytes in cow dermal fibroblast treated with 5-

azacytidine and cultured in presence of BMP-2, BMP-4 or follicular fluid

(Expressão de marcadores de células germinativas e oócitos em fibroblastos da pele bovina

tratados com 5-aza-citidina e cultivados na presença de BMP-2, BMP-4 ou fluido folicular)

Artigo submetido ao periódico Molecular Biology Reports

(Qualis B1 - Biotecnologia)

100

Expression of markers for germ cells and oocytes in cow dermal fibroblast treated with 5-

azacytidine and cultured in presence of BMP-2, BMP-4 and follicular fluid

José Jackson do Nascimento Costa1, Glaucinete Borges de Souza1, Joyla Maria Pires Bernardo1,

Regislane Pinto Ribeiro1, José Renato de Souza Passos1, Francisco Taiã Gomes Bezerra1, Márcia

Viviane Alves Saraiva1, José Roberto Viana Silva1*


Sobral, CE, Brazil.




Abstract

I. Background: This study aims to investigate the effect 5-azacytidine during induction of

pluripotency in bovine fibroblast skin, evaluate the effects of culture for 7 or 14 days in a

medium containing BMP-2, BMP-4 or follicular fluid in the differentiation of reprogrammed

fibroblasts in primordial germ cells and oocytes, as well as to analyze the mRNA levels for OCT-

4, NANOG, REX-1, SOX2, VASA, DAZL, c-KIT, SCP3, ZPA and GDF-9, after induction of

pluripotency or differentiation in bovine fibroblasts.

II. Methods and Results: Dermal fibroblasts were cultured and exposed to 0.5, 1.0 or 2.0 μM of

5-Aza for 18 h, 36 h or 72 h. Then, cultured in DMEM/F12 supplemented with with 10 ng/mL

BMP-2, or 10 ng/mL BMP-4 or 5% follicular fluid. After the cell culture, were evaluated

101

morphological characteristics, viability and gene expression by qPCR. Treatment of skin

fibroblasts at 2.0 μM 5-Aza for 72 h results in changes in the morphology (oval or round shape)

and cellular proliferation rate, and significantly increased expression of pluripotency factors. The

culture in medium supplemented with BMP-2, BMP-4 or follicular fluid, for 7 or 14 days,

altering cellular morphology, inducing formation of cell, and gene expression of germ cells and

oocytes markers.

III. Conclusions: This study describes the possibility to convert bovine skin fibroblasts into

another cell type, oocyte-likes cells, contribute to increase the development of therapies aimed at

solving the problems of infertility in humans, as well as enhance the reproductive efficiency of

animals.

Keywords: Gene expression. Fibroblast. 5-Azacytidine. BMP-2. BMP-4. Follicular fluid.

Introduction

The development of new regenerative therapies adapted to reproductive needs of female

has been challenging in the last years [1]. Stem cell-based strategies for ovarian regeneration and

oocyte production have been proposed as future clinical therapies for treating infertility in women

[2]. Various studies have been conducted to identify, characterize, and differentiate cells from

various sources [3-5], that can potentially be used for clinical studies, including embryonic stem

cells (ESC) [6], stem cells isolated from adult tissues like the mesenchymal stem cells (MSC) [7],

and induced pluripotent stem cells (iPS) which are adult somatic cells reprogrammed to

pluripotency [8].

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In 2006, Takahashi and Yamanaka [9] generated pluripotent cells by reprogramming

somatic cells, this discovery it is now possible to convert differentiated somatic cells into

pluripotent stem cells that have the capacity to generate all cell types of adult tissues [10]. iPS

were generated by using a combination of 4 reprogramming factors, including OCT4 (Octamer

binding transcription factor-4), SOX2 (Sex determining region Y)-box 2, KLF4 (Kruppel Like

Factor-4), and c-MYC and were demonstrated both self-renewing and differentiating like ESCs

[11]. Conventional reprogramming techniques depend on the stable integration of transgenes,

using viral vectors, such as retroviruses [9] and lentiviruses [12], but it can introduce the current

risk of insertional mutagenesis [13]. A chemical reprogramming is a promising strategy for safety

and efficiency of iPS generation, many small molecules have been identified that can be used in

place of exogenous transcription factors and significantly improve iPS reprogramming efficiency

and quality [14]. Small molecules promote a chemical reprogramming, accompanied by

remodeling of the epigenome, modulations of the epigenetic processes may facilitate such

conversion of cell fate by making cells more permissive to these epigenomic changes [15].

To this purpose, 5-Azacytidine (5-Aza), a DNA methyltransferase inhibitor, can also

improve reprogramming efficiency, by activating the expression of silent genes and altering the

differentiation state of cells [16, 17]. Taylor and Jones, (1979) [18], used the 5-Aza in the

conversion of a mesenchymal cell line into muscle cells, adipocytes and chondroblasts.

Pennarossa et al. [19, 20] reprogrammed human and pig dermal fibroblast into insulin secreting

cells by a brief exposure to 5-Aza. However, the use of 5-Aza to reprogram bovine skin fibroblast

followed by a differentiation protocol, have not yet been reported.

The efficiency of differentiation of iPS cells into female germ cells has been considered

low [21], since Eguizabal et al. (2011) [22] observed between 1.0%–2.0% haploid cells

differentiated from a human female iPS cell line. The formation of iPS-derived germ cells

103

requires a strategy that involves differentiating iPS cells into primordial germ cells (PGCs), and

subsequently directing the PGCs to undergo meiosis to form functional gametes [23, 21]. Several

studies indicate that efficiency of differentiation to PGC can be increased with the addition of

bone morphogenetic proteins (BMP-4, -7 and -8b) in human [24] and murine [25, 26] species.

Günesdogan, et al. (2014) [27] indicated the BMP signals acts through a receptor complex

including BMP receptor type II and ALK3/6, which results in SMAD1/5 phosphorylation, which

form a complex with SMAD4 and translocate to the nucleus to control target gene expression.

Mice carrying null mutations for BMP4, BMP8B, SMAD1 and SMAD5 showed impaired PGC

development [28-30]. Ying et al. (2001) [31], Ying and Zhao (2001) [32] indicated mutation in

BMP2 affects the size of the PGC founding population rather than PGC proliferation and/or

survival, besides that, BMP2 produced by the endoderm (together with BMP4 and BMP8B) acts

as a part of the first signal in PGC precursor generation. In human, Kee et al. (2006) [24] founded

that addition of BMP-4 increased the expression of the germ cell-specific markers VASA and

SYCP3 during differentiation of hES cells. These authors also showed that BMP-7 and BMP8b

have additive effects on germ cell induction when added together with BMP4. West et al. (2010)

[33] reported that BMP-4 increased the expression of pre-migratory (OCT-4, NANOG, c-KIT)

and post-migration (DAZL, VASA) markers in PGC differentiated from ESC. Studies have also

shown that follicular fluid contains many bioactives factors such as GDF9b, stem cell factor,

basic fibroblast growth factor and oestrogen [34-36]. Dyce et al. (2006) [37] used follicular fluid

in porcine skin-derived cells, and observed the induction of germ-cell formation and supported

the expression of the markers, as OCT4, GDF9B, VASA and DAZL. In woman, the addition of

follicular fluid, rich in substances for oocyte growth and maturation, to the culture medium

triggers development of putative stem cells from the ovarian surface epithelium of women, and

express several genes related to pluripotency and oocytes [38].

104

The aims of the present study were to investigate the expression of pluripotency markers

(OCT-4, NANOG, REX-1, SOX2) in bovine fibroblast treated with different concentration of 5-

Aza, as well as to evaluate the effects of BMP-2, BMP-4 or follicular fluid on mRNA levels for

germ cell (VASA, DAZL, c-KIT), meiosis (SCP3) and oocytes (ZPA and GDF-9) markers in 5-

Aza treated fibroblasts cultured for 7 and 14 days.

Materials and Methods

Skin Fibroblasts

All biological material were collected from animals at the local abattoir. Primary bovine

skin fibroblast cultures were established from fresh biopsies from fetal ear skin. Fragments of

tissues were washed twice in saline solution (0.9% NaCl) that contained antibiotics (100 IU/mL

penicillin and 100 mg/mL streptomycin) (Sigma, St. Louis, MO, USA) and then, transported

within 1 h to the laboratory in this same solution.

In the laboratory, fragments of tissues of approximately 2 mm3 were transferred into 24-

well culture dishes (Corning, Lowell, MA, USA) that contained 1 mL of culture media. The basic

culture medium consisted of D-MEM (pH 7.2–7.4) supplemented with 20% FBS (Sigma), 2 mM

glutamine (Sigma) and antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin) (Sigma).

Cells were cultured at 38.5 °C in 5% CO2 in a humidified incubator. After 7 days, primary

fibroblast cultures started to grow out of the tissue fragments which were carefully removed and

passaged twice a week in a 1:3 ratio. All experiments were performed on at least three lines.

Treatment of Skin Fibroblasts with 5-Aza and cell viability

105

Dermal fibroblasts (1.5×105 cells/well) were cultured in 0.1 % gelatin (Sigma) precoated

4-well multidish (Nunc) and exposed to 0.5, 1.0 or 2.0 μM of 5-Aza (Sigma) for 18 h, 36 h or 72

h. The basic culture medium consisted of D-MEM (pH 7.2–7.4) supplemented with 20% FBS

(Sigma), 2 mM glutamine (Sigma) and antibiotics (100 IU/mL penicillin and 100 mg/mL

streptomycin) (Sigma). Cells were cultured at 38.5 °C in 5% CO2 in a humidified incubator. At

the end of the culture period, the proportion of living and dead cells was assessed with calcein

AM (Molecular Probes) and ethidium homodimer-1 (Molecular Probes). Calcein AM (4 µM) and

ethidium homodimer-1 (2 µM) was added to wells and incubated protected from light for a

period of 15 min at room temperature. Determination of calcein and ethidium fluorescence at

excitation/emission wave lengths of 488/568 nm was performed using an inverted microscopy

(NIKON, Eclipse, TS100). Besides morphological evaluation, from each treatment, samples of

cells were collected and stored at –80 ºC until RNA extraction to analyze the expression of

markers for pluripotency (OCT4, NANOG, SOX2 and REX1).

Influence of follicular fluid and BMPs on the differentiation of germ cells

After treatment with 5-Aza (concentration and time of incubation determined previously),

the bovine fibroblast cells were cultured in DMEM/F12 medium supplemented with 0.1 mM β-

mercaptoetanol (Sigma), 2 mM glutamine (Sigma), 1 mM sodium pyruvate (Sigma), 1 mM Non-

Essential Amino Acids (Sigma ), antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin)

(Sigma), 20% Knockout Serum Replacement (KSR) (Life, Grand Sland, NY, USA). For the

treatments, this medium was supplemented with 10 ng/mL BMP-2 (R&D systems, Minneapolis,

MN, USA), or 10 ng/mL BMP-4 (R&D systems) or 5% follicular fluid [39]. Cells were cultured

106

at 38.5 °C in 5% CO2 in a humidified incubator. Every 2 days, the culture medium was replaced

with fresh medium. After 7 and 14 days of culture, morphological analysis was performed and

cellular viability was determined by immunofluorescence analysis (Calcein AM and ethidium

homodimer-1) as described previously. Besides morphological evaluation, from each treatment,

samples of cells were collected and stored at –80 ºC until RNA extraction to analyze the

expression of markers for germ cells (VASA, DAZL and c-KIT), and oocytes (GDF-9, SCP3 and

ZPA).

RNA extraction and cDNA synthesis

Isolation of total RNA was performed using the Trizol® Plus purification kit (Invitrogen,

São Paulo, Brazil). According to the manufacturer’s instructions, 800 µL of Trizol solution was

added to each frozen samples and the lysate was aspirated through a 20-gauge needle before

centrifugation at 10,000 g for 3 min at room temperature. Thereafter, all lysates were diluted 1:1

with 70% ethanol and subjected to a mini-column. After binding of the RNA to the column, DNA

digestion was performed using RNAse-free DNAse (340 Kunitz units/mL) for 15 min at room

temperature. After washing the column three times, the RNA was eluted with 30 µL RNAse-free

water. The RNA concentration was estimated by reading the absorbance at 260 nm and was

checked for purity at 280 nm in a spectrophotometer (Amersham Biosciences, Cambridge,

England). For each sample, RNA concentrations were adjusted and used to synthesize cDNA.

Before the reverse transcription reaction, samples of RNA were incubated for 5 min at 70 ºC and

then cooled in ice. The reverse transcription was performed in a total volume of 20 µL composed

of 10 µL of sample RNA, 4 µL reverse transcriptase buffer (Invitrogen, São Paulo, Brazil), 8

units RNase out, 150 units of reverse transcriptase Superscript III, 0036 U random primers, 10

107

mM DTT and 0.5mMof each dNTP (Invitrogen, São Paulo, Brazil). The mixture was incubated at

42 ºC for 1 h, subsequently at 80 ºC for 5 min, and finally stored at –20ºC. The negative control

was prepared under the same conditions, but without the addition of reverse transcriptase.

qPCR

Quantification of mRNA was performed using GoTaq® qPCR Master Mix. PCR

reactions were composed of 1μL cDNA as a template in 7.5 μL of GoTaq® qPCR Master Mix

(Promega Corporation, Madison, WI, USA), 5.5 µL of ultra-pure water, and 0.5 μM of each

primer. The primers were designed by using the PrimerQuestSM program

(http://www.idtdna.com). Primers used in this study are shown in table 1. The specificity of each

primer pair was confirmed by melting curve analysis of PCR products. The thermal cycling

profile for the first round of PCR was: initial denaturation and activation of the polymerase for 10

min at 95 oC, followed by 40 cycles of 15 sec at 95 oC, 30 sec at 58 oC, and 30 sec at 72 oC. The

final extension was for 10 min at 72 oC. All reactions were performed in StepOne Real-Time

PCR (Applied Biosystems, Foster, CA, USA). Relative quantifications of mRNA were carried

out using the comparative threshold (Ct) cycle method. The delta-delta-Ct method was used to

transform the Ct values into normalized relative expression levels [40].

Statistical analysis

Levels of mRNA for pluripotency (SOX2, NANOG, OCT4, REX1), germ cells (VASA,

DAZL, c-KIT) and oocytes (ZPA, GDF9, SCP3) genes were analyzed by using the non-parametric

Kruskal–Wallis test and Dunn’s test for post hoc pair-wise comparisons (P<0.05). Data were

expressed as mean ± s.e.m.

http://www.idtdna.com/

108

Results

Cellular morphology and viability after treating fibroblasts with 5-Aza

Fibroblasts obtained from biopsies from ear skin fetuses grew out of the original explants

within 7 days and formed a monolayer, displaying a standard elongated morphology and a

vigorous growth in culture typical of this cell population. After the exposure to 5-Aza, cell

phenotype changed and fibroblast elongated morphology (Figure 1A) was replaced by an oval or

round shape (Figure 1B), furthermore, there was a reduction in cell number.

During the exposure to 5-Aza, the total cell number remained substantially unstable,

resulting from the effect of different concentrations of 5-Aza at different times, in manner time x

concentration-dependent, it can be observed in immunofluorescence analysis (calcein AM and

ethidium homodimer-1) (Figure 1C and Figure 1D). Cell proliferation rapidly decreased after

exposure to 5-Aza, and can observed a high apoptotic index, especially with the treatment with

2.0 μM of 5-Aza for 72 h showed a greater reduction in the number of cells when compared to

other treatments.

Expression of mRNA for markers of pluripotency after treating fibroblasts with 5-aza-cytidine

After treatment with 5-Aza for 18 h, real-time PCR analysis demonstrated that the levels

of mRNA for SOX2 (Figure 2A) and REX (Figure 2D) did not significant changes in any of the

treatments tested. However, NANOG expression was significantly higher after treatment with 2.0

μM of 5-Aza, when compared to 0.5 μM (Figure 2B). While the treatment with 1.0 μM of 5-Aza

109

significantly increased the levels of mRNA for OCT4, when compared to 0.5 μM of 5-Aza

(Figure 2C).

Figure 3 shows the expression of pluripotency genes after treatment with different

concentrations of 5-Aza for 36 h. The expression of mRNA for SOX2 (Figure 3A) did not

significant change in any of the treatments tested. The expression of mRNA for NANOG (Figure

3B), was significantly increased after treatment with 1.0 μM of 5-Aza when compared to 0.5 μM

of 5-Aza. The expression of mRNA for OCT4 (Figure 3C) was significantly increased after

treatment with 2.0 μM of 5-Aza when compared to 0.5 μM of 5-Aza. In addition, expression of

mRNA for REX (Figure 3D) was significantly increased after treatment with 1.0 or 2.0 μM of 5-

Aza when compared to 0.5 μM of 5-Aza.

After culture for 72 h in different treatments with 5-Aza, the expression of SOX2 (Figure

4A), NANOG (Figure 4B), OCT4 (Figure 4C), and REX (Figure 4D) was significantly higher

after supplementation of the medium with 2.0 μM of 5-Aza compared to 0.5 μM of 5-Aza.

Regarding the effects of incubation time on the expression of pluripotency genes, the

culture with 0.5 (Figure 5Ai), 1.0 (Figure 5Aii) or 2.0 µM (Figure 5Aiii) 5-Aza for 72 h increased

SOX2 mRNA levels, when compared to those seen after 18h or 36h. Fibroblasts cultured with 5-

Aza (0.5 μM or 2.0 μM) for 72 h had higher levels of mRNA for NANOG than those cultured for

18 and 36 h (Figure 5Bi and 5Bii). However, no effect of incubation time on expression of

NANOG was observed after culturing in presence of 1.0 μM of 5-Aza (Figure 5Bii).

After culturing cells with 0.5 μM and 1.0 μM of 5-Aza, a significant reduction in OCT4

expression was observed when the incubation time was increased from 18 to 72h (Figures 5Ci

and 5Cii). On the other hand, fibroblasts cultured with 2.0 μM of 5-Aza for 72h had higher levels

of OCT4 mRNA than those cultured for 18 and 36h a (Figure 5Ciii). The mRNA levels for REX1

in fibroblasts cultured either with 0.5 μM or 2.0 μM of 5-Aza for 72h were significantly higher

110

than those observed after 18 and 36h (Figures 5Di and 5Diii). When the cells were cultured with

1.0 μM of 5-Aza, a progressive and significant increase in REX1 expression was observed when

the incubation time was increased from 18 to 36 and 72h (Figure 5Dii).

Cellular morphology and viability after culturing 5-Aza treated cells with BMP-2, BMP-4 or

follicular fluid

After a series of preliminary experiments, we established that 2.0 μM of 5-Aza for 72 h

represented the optimal combination for bovine skin fibroblasts. At the end of this treatment,

cells were cultured in differentiation medium supplemented with BMP-2, or BMP-4 or follicular

fluid, for 7 or 14 days.

Initially, growing in differentiation medium with BMP-2, BMP-4 or follicular fluid,

induced the cells to present the ability to form colony. The culture in differentiation medium

associated with 10 ng/mL of BMP-2 (Figure 6A, B, C, D) or BMP-4 (Figure 6E, F, G, H) for 14

days, resulted in a resumption of proliferative index, and morphological changes, gradually cells

organized in clusters. The culture in BMP-2 or BMP-4 for 7 days did not affect the cell

proliferation rate. The culture in 5% follicular fluid for 7 or 14 days (Figure 6I, J, K, L),

promoted an large increase in cell proliferation rate, with vigorous growth and large rate of

apoptosis.

Expression of mRNA for markers of germ cells and oocytes after culturing 5-Aza treated cells

with BMP-2, BMP-4 or follicular fluid

111

Since exposure of fibroblasts to 2.0 µM of 5-Aza for 72h promoted higher expression of

pluripotency genes, this concentration and incubation time were chosen to treat the cells before

culture in differentiation medium containing BMP-2, BMP-4 or follicular fluid for 7 or 14 days.

After 7 days of culture, BMP-4 stimulated a significant increase in the expression of

VASA, when compared to control medium. However, BMP-4 or follicular fluid had no effect on

VASA expression (Figure 7A). While the culture for 14 days in medium containing follicular

fluid, significantly increased the expression of VASA, compared to the culture in medium control

(Figure 7B). DAZL expression was significantly increased after culture in medium containing

BMP-2 for 7 or 14 days (Figure 7C, D), when compared to other treatments. The culture for 7

days in medium supplemented with BMP-2, stimulated mRNA expression of c-KIT (Figure 7E),

when compared to the culture in to other treatments. The culture for 14 days, did not significantly

modify the expression of c-KIT (Figure 7F).

Regarding the expression of oocyte markers, after 7 days of the culture, ZPA expression

was significantly reduced when cells were cultured in medium containing BMP-2 (Figure 8A).

Already, the culture in medium containing follicular fluid for 14 days, significantly increased

ZPA expression, when compared to the culture in medium containing BMP-4 or control medium

only (Figure 8B). Follicular fluid increased significantly mRNA levels for GDF-9 (Figure 8C),

when compared to culture in control medium. After 14 days of culture in medium supplemented

with BMP-2, increased significantly GDF-9 expression, when compared to other treatments

(Figure 8D). An increase in SCP3 mRNA expression was observed after culture in presence of

BMP-4 for 7 days, when compared to other treatments (Figure 8E). After 14 days culture in

medium supplemented with BMP-2, promoted an increase in expression of SCP3 (Figure 8F),

when compared to other treatments.

112

Figures 9, 10, 11 and 12, showed the expression of makers for germ cells and oocyte after

0 h, 7 or 14 days in the different treatments. For cells cultured in control medium, VASA

expression was significantly increased after 7 days, when compared to the time 0 or after 14 days

(Figure 9A). DAZL expression was not altered after 7 or 14 days culture (Figure 9B). An increase

in the mRNA levels for c-KIT was observed after 7 or 14 days of culture, when compared to 0h

(Figure 9C). Culture for 14 days significantly increased ZPA (Figure 9D) and GDF-9 (Figure 9E)

expression, compared to culture for 0 h or 7 days. A progressive and significant increased in

SCP3 expression was observed when increasing culture time from 0 h to 7 and 14 days (Figure

9F). Regarding the cells cultured in medium supplemented with BMP-2, an increase in the

mRNA levels for VASA (Figure 10A), DAZL (Figure 10B), c-KIT (Figure 10C) and GDF-9

(Figure 10E) was observed after 7 days of culture, when compared to 0 h and 14 days. While,

after 14 days of culture in medium supplemented with BMP-2, the levels of mRNA for ZPA

(Figure 10D) and SCP3 (Figure 10F) were significantly higher when compared to other times of

culture.

For cells cultured in presence of BMP-4, an increased in the mRNA levels for VASA

(Figure 11A), c-KIT (Figure 11C), GDF-9 (Figure 11E) and SCP3 (Figure 11F) was observed

after 7 days of culture, when compared to 0h or 14 days. However, a reduction in DAZL levels

(Figure 11B), was seen when increasing culture time from 7 or 14 days. A progressive and

significant increased in mRNA levels for ZPA (Figure 11D) was observed when increasing

culture time from 0 h to 7 or 14 days.

The culture for 7 days in medium containing 5% follicular fluid, significantly increased

VASA expression (Figure 12A), when compared to 0 h or 14 days. After 14 days of culture, and

significantly increased in the levels of mRNA for DAZL (Figure 12B) were observed, when

compared with 0 h or 7 days. A progressive and significant increased in mRNA levels for c-KIT

113

(Figure 12C), ZPA (Figure 12D) and SCP3 (Figure 12F) was observed when increasing culture

time from 0 h to 7 or 14 days. The culture with follicular fluid for 7 or 14 days, significantly

increased the levels of mRNA for GDF-9 (Figure 12E).

Discussion

This study shows for the first time that 5-Aza promotes changes in cell morphology and

induces expression of genes related with pluripotency in cultured bovine fibroblasts. The 5-Aza is

able to modify cell morphology [18, 41] in tumor-derived cell lines [18, 42], embryonic and adult

cells [41]. Pennarossa et al. (2013) [19] used 5-Aza to induce conversion of human skin

fibroblasts into insulin-secreting cells. 5-Aza is a chemical derivative of the DNA nucleoside

cytidine that causes DNA demethylation or hemi-demethylation, binds covalently and

irreversibly to DNA methyltransferase 1 (DNMT1), and regulate gene expression by relaxing

chromatin structure. Consequently, 5-Aza allows transcription factors to bind to the promoter

regions of genes associated with cell pluripotency [43-45]. Previous studies have reported that 5-

Aza alter cell, as mouse fibroblasts and B lymphocytes [46], human and porcines fibroblasts [19,

20, 44], as well as phenotype and gene expression and promotes the generation of iPS in mouse

[15, 47]. Efficient reprogramming methods have been explored since the first report of the

generation of human induced pluripotent stem cells [9, 48, 49]. Currently, new methods have

been applied in the production of iPS cells, which include the use of episomal plasmids [50] or

excisable expression systems [51], recombinant cell-penetrating reprogramming proteins [52,

53], reprogramming mRNAs [55, 56, or microRNAs [57, 58]. Besides 5-Aza, a growing number

of compounds have been identified that can functionally replace reprogramming transcription

factors, enhancing efficiency of iPS generation and accelerating the reprogramming process [15].

114

Among these compounds, it can be highlighted N-Phthalyl-L-Tryptophan (RG108), valproic acid

(VPA), trichostatin A (TSA), sodium butyrate (NaB) and other molecules [44].

In this study we demonstrate that exposure of bovine skin fibroblasts to 2.0μM of 5-Aza

for 72h lead to highest expression of mRNA for SOX2, NANOG, OCT4 and REX. OCT4,

NANOG and REX are considered the main pluripotency markers, and the expression of these

genes supported the pluripotency of iPS in goat species [59]. Exposure of in porcine fetal

fibroblasts to 5-Aza reduced the expression of DNA methyltransferase 1 (DNMT1) and altered the

expression of genes involved in imprinting (IGF2) and apoptosis (BAX, BCL2L1) [60]. In

addition, Pennarosa et al. (19, 20] exposed human and pig dermal fibroblasts to 5-Aza for 18 h,

followed by a protocol for the induction of endocrine pancreatic differentiation. The results

demonstrated changes in cell morphology and expression of OCT4, NANOG, SOX2 and REX, and

after differentiation, the cells expressed insulin and was able to release it in response to a

physiological glucose challenge [19, 20]. OCT4, a regulator of pluripotency, it is expressed in

pluripotent stem cells, in iPS and in germ cells (during embryogenesis) [9, 61, 62]. It has been

demonstrated that germline stem cells derived from ovaries also possessed pluripotency as

evidenced by the expression of OCT4 [54, 63]. The transcription factor NANOG is essential for

the establishment of pluripotency during the derivation of ESC and iPS. However, NANOG is not

essential to maintain pluripotency [64, 65]. The repression of OCT4 or SOX2 in ESCs promotes

differentiation [66, 67], besides that, OCT4 and SOX2 proteins dimerize on DNA to activate their

target genes in order to specify pluripotency [68]. NANOG directly transactivates the REX1

promoter and positively regulates REX expression. OCT4 and SOX2 can either activate or repress

the REX promoter, depending on the cellular environment [69-71]. REX is a stringent

pluripotency marker that specifies a subpopulation of the most undifferentiated ESC [72].

115

The culture of 5-Aza treated fibroblasts in differentiation medium supplemented with

BMP-2 and BMP-4 induced the expression of mRNA for germ cell and oocyte markers. In vivo

and in vitro evidences demonstrate that BMP4 induce the differiantion of PGC in mouse

developing embryos [28, 73, 74]. BMP signals through ACVRI in the visceral endoderm ant is

necessary for normal induction of PGCs in the epiblast [75]. In vitro differentiation of human

PGCs was increased by addition of BMP-4, BMP-7 and BMP-8b, in co-culture with human or

mouse fetal gonad stromal cells [24, 76-79). Several studies have PGC absence in BMP4 mutant

mouse embryos and a reduction in these cells in BMP2 and BMP8 mutant embryos [25, 28, 31,

80-81]. Addionally, BMP-4 promotes mammalian oogonial stem cell differentiation in humans

[82-84], buffaloes [85], and goats [11]. BMP-2 also plays a crucial role in the differentiation of

primordial germ cells [86].

The presence of follicular fluid in differentiation medium also influences the expression

of mRNA for germ cell and oocyte markers in cultured 5-Aza treated fibroblasts. The follicular

fluid is a select ultrafiltrate of plasma that has been modified by secretion and uptake of specific

components by the cells within the follicle itself [87, 88]. The follicular fluid may contain several

proteins, such as BMP that are involved in the formation of germ cells [37, 89-91]. In porcine

species, follicular fluid successfully simulate differentiation of oocyte during culture of stem

cells obtained from skin, adipose and ovarian tissues [37, 39, 92], and this cells expressed DAZL,

VASA and SCP3 [39]. Follicle-like structures were successfully differentiated in vitro from

hESCs in the presence of follicular fluid [94]. Cheng et al. (2012) [93] promoted differentiation

human amniotic fluid stem cells (hAFSCs) into oocyte-like cells after culturing those cells in

presence of porcine follicular fluid. The differentiated OLCs expressed EGFP under the BMP15

promoter which was co-localized with ZP2 expression [93].

116

Our results indicate that BMP-2, BMP-4 or bovine follicular fluid influenced the

expression of markers for germ cells (VASA, DAZL and c-KIT), meiosis (SCP3) and oocyte

(GDF-9, ZPA). Studies indicated that VASA is expressed in post-migratory PGCs until the post-

meiotic stage of oocytes [95, 96]. DAZL is also considered essential for PGC development, since

DAZL knockout mice lack a germ cell population [76, 97]. DAZL and VASA have been used as

markers for germ cell differentiation in various species (human: [76, 98], murine: [99], and

porcine [37]. Clark et al. (2004) [100] indicated that c-KIT is one of the most used markers for

PGCs, and this receptor is important for migration and survival of PGC [101]. GDF-9 is

expressed specifically in oocytes [102], and have been used as oocyte marker after its

differentiation from stem cells [37, 91]. GDF-9 was expressed in populations of cultured mouse

embryonic stem cells with an oocyte-like phenotype [103]. SCP3 expression is frequently used as

a meiotic marker [104]. The zona pellucida glycoprotein is only expressed in oocytes [105], and

earlier studies reported ZP-like structures surrounding OLCs differentiated in vitro [106, 107].

Singhal et al. (2015) [108] recently showed that expression of germ cell markers, like zona

pellucida and VASA, were stimulated by BMP-4.

Conclusions

In conclusion, treatment of bovine skin derived fibroblasts with 2.0 μM 5-Aza for 72h

induces the expression of mRNAs for SOX2, OCT4, NANOG and REX. The cultured of these

cells in differentiation medium culture supplemented with BMP-2, BMP -4 or follicular fluid

induce morphological changes and promotes expression of markers for germ cells (VASA, DAZL

and c-KIT), meiosis (SCP3) and oocyte (GDF-9, ZPA). This method can be effective for

production of oocytes from differentiation of fibroblasts and can thus contribute, in the future, for

117

the development of new biotechnologies to solve fertility problems in human species, and to

increase the reproductive potential of genetically superior animals or endangerous species.

Acknowledgements

This work was financially supported by CNPq (Grant N° 478198/2013-2), and the

authors thank the members of the Laboratory of Animal Reproduction of the Biotechnology

Nucleus of Sobral.

Compliance with ethical standards

Conflict of interest: The authors declare that they have no conflict of interest.

References

1. Schenke-Layland K, Brucker SY (2015) Prospects for regenerative medicine approaches in

women's health. J Anat 227(6):781-785.

2. Volarevic V, Bojic S, Nurkovic J, Volarevic A, Ljujic B, Arsenijevic N (2014) Stem Cells as

New Agents for the Treatment of Infertility: Current and Future Perspectives and Challenges.

Biomed Res Int (2014):8.

3. De Bari C, Dell'Accio F, Tylzanowski P, Luyten FP (2001) Multipotent mesenchymal stem

cells from adult human synovial membrane. Arthritis Rheumatol 44(8):1928-1942.

4. Beyer Nardi N, da Silva Meirelles L (2006) Mesenchymal stem cells: isolation, in vitro

expansion and characterization. Hand Exp Pharmacol 174:249-282.

118

5. Zomer HD, Vidane AS, Gonçalves NN, Ambrósio CE (2015) Mesenchymal and induced

pluripotent stem cells: general insights and clinical perspectives. Stem Cells Cloning 8:125–

134.

6. Keefer CL, Panta D, Blomberg L, Talbot NC (2007) Challenges and prospects for the

establishment of embryonic stem cell lines of domesticated ungulates. Ani Reprod Sci 98:147-

168.

7. Ratajczak MZ, Zuba-Surma E, Kucia M, Poniewierska A, Suszynska M, Ratajczak J (2012)

Pluripotent and multipotent stem cells in adult tissues. Adv Med Sci 57(1):1-17.

8. Angelos MG, Kaufman DS (2015) Pluripotent stem cell applications for regenerative

medicine. Curr Opin Organ Transplant 20(6):663-670.

9. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic

and adult fibroblast cultures by defined factors. Cell 126(4):663-676.

10. Hirschi KK, Li S, Roy K (2014) Induced Pluripotent Stem Cells for Regenerative Medicine.

Annu Rev Biomed Eng 16:277–294.

11. Singh VK, Kalsan M, Kumar N, Saini A, Chandra R (2015) Induced pluripotent stem cells:

applications in regenerative medicine, disease modeling, and drug discovery. Front Cell Dev

Biol 3:(2).

12. Picanço-Castro V, Russo-Carbolante E, Reis LC, Fraga AM, de Magalhães DA, Orellana

MD (2011) Pluripotent reprogramming of fibroblasts by lentiviral mediated insertion of

SOX2, C-MYC, and TCL-1A. Stem Cells Dev 20(1):169-180.

13. Diecke S, Jung SM, Lee J, Ju JH (2014) Recent technological updates and clinical

applications of induced pluripotent stem cells. Korean J Intern Med 29(5):547–557.

119

14. Lin T, Wu S (2015) Reprogramming with Small Molecules instead of Exogenous

Transcription Factors. Stem Cells Int 2015:794632.

15. Zhang Y, Li W, Laurent T, Ding S (2012) Small molecules, big roles – the chemical

manipulation of stem cell fate and somatic cell reprogramming. J Cell Sci 125(23):5609-

5620.

16. Constantinides PG, Jones PA, Gevers W (1977) Functional striated muscle cells from non-

myoblast precursors following 5-azacytidine treatment. Nature 267(5609):364–366.

17. Jones PA (1985) Effects of 5-azacytidine and its 2′-deoxyderivative on cell differentiation

and DNA methylation. Pharmacol Ther 28(1):17-27.

18. Taylor SM, Jones PA (1979) Multiple new phenotypes induced in 10T1/2 and 3T3 cells

treated with 5-azacytidine. Cell 17(4):771–779.

19. Pennarossa G, Maffei S, Campagnol M, Tarantini L, Gandolfi F, Brevini TAL (2013) Brief

demethylation step allows the conversion of adult human skin fibroblasts into insulin-

secreting cells. Proc Natl Acad Sci USA 110(22):8948-8953.

20. Pennarossa G, Maffei S, Campagnol M, Rahman MM, Brevini TAL, Gandolfi F (2014)

Reprogramming of Pig Dermal Fibroblast into Insulin Secreting Cells by a Brief Exposure to

5-aza-cytidine. Stem Cell Rev 10(1):31-43.

21. Ishii T (2014) Human iPS Cell-Derived Germ Cells: Current Status and Clinical Potential. J

Clin Med Res 3:1064-1083.

22. Eguizabal C, Montserrat N, Vassena R, Barragan M, Garreta E, Garcia-Quevedo L (2011)

Complete meiosis from human induced pluripotent stem cells. Stem Cells 29:1186-1195.

23. Park T, Galic Z, Conway A, Lindgren A, van Handel B, Magnusson M (2009) Derivation of

primordial germ cells from human embryonic and induced pluripotent stem cells is

significantly improved by coculture with human fetal gonadal cells. Stem Cells 27:783-795.

120

24. Kee K, Gonsalves J, Clark A, Reijo Pera RA (2006) Bone morphogenetic proteins induce

germ cell differentiation from human embryonic stem cells. Stem Cells Dev 15:831–837.

25. Young JC, Dias VL, Loveland KL (2010) Defining the window of germline genesis in vitro

from murine embryonic stem cells. Biol Reprod 82:390-401.

26. Panula S, Medrano JV, Kee K, Bergström R, Nguyen HN, Byers B (2011) Human germ cell

differentiation from fetal- and adult-derived induced pluripotent stem cells. Hum Mol Gen

20:752–762.

27. Günesdogan U, Magnúsdóttir E, Surani MA (2014) Primoridal germ cell specification: a

context-dependent cellular differentiation event. Philos Trans R Soc Lond B Biol Sci

369:(1657).

28. Lawson KA, Dunn NR, Roelen BAJ, Zeinstra LM, Davis AM, Wright CVE (1999) Bmp4 is

required for the generation of primordial germ cells in the mouse embryo. Gen Dev 13:424-

36.

29. Chang H, Matzuk MM (2001) Smad5 is required for mouse primordial germ cell

development. Mech Dev 104:61-67.

30. Hayashi K, Kobayashi T, Umino T, Goitsuka R, Matsui Y, Kitamura D (2002) SMAD1

signaling is critical for initial commitment of germ cell lineage from mouse epiblast. Mech

Dev 118:99-109.

31. Ying Y, Qi X, Zhao G-Q (2001) Induction of primordial germ cells from murine epiblasts by

synergistic action of BMP4 and BMP8B signaling pathways. Proc Natl Acad Sci USA

98:7858-7862.

32. Ying Y, Zhao GQ (2001) Cooperation of endoderm-derived BMP2 and extraembryonic

ectoderm-derived BMP4 in primordial germ cell generation in the mouse. Dev Biol 232:484-

492.

121

33. West FD, Machacek DW Boyd NL, Pandiyan K, Robbins KR, Stice SL (2008) Enrichment

and differentiation of human germ-like cells mediated by feeder cells and basic fibroblast

growth factor signaling. Stem Cells 26:2768-2776.

34. Prochazka R, Nemcova L, Nagyova E, Kanka J (2004) Expression of growth differentiation

factor 9 messenger RNA in porcine growing and preovulatory ovarian follicles. Biol Reprod

71:1290-1295.

35. Wang J, Roy SK (2004) Growth differentiation factor-9 and stem cell factor promote

primordial follicle formation in the hamster, p. modulation by follicle-stimulating hormone.

Biol Reprod 70(3):577–585.

36. Bertoldo MJ, Nadal-Desbarats L, Gérard N, Dubois A, Holyoake PK, Grupen CG (2013)

Differences in the metabolomic signatures of porcine follicular fluid collected from

environments associated with good and poor oocyte quality. Reproduction 146:221-231.

37. Dyce PW, Wen L, Li J (2006) In vitro germline potential of stem cells derived from fetal

porcine skin. Nat Cell Biol 8:384-390.

38. Virant-Klun I, Skutella T, Kubista M, Vogler A, Sinkovec J, Meden-Vrtovec H (2013)

Expression of pluripotency and oocyte-related genes in single putative stem cells from

human adult ovarian surface epithelium cultured in vitro in the presence of follicular fluid.

Biomed Res Int 2013:861460.

39. Dyce PW, Liu J, Tayade C, Kidder GM, Betts DH, Li J (2011) In Vitro and In Vivo Germ

Line Potential of Stem Cells Derived from Newborn Mouse Skin. PLoS ONE, 6(5):e20339.

40. Livak JK, Schmittgen TD (2001) Analysis of relative gene expression data using real-time

quantitative PCR and the 2-ΔΔCt method. Methods 25:402-408.

41. Taylor SM, Jones PA (1982) Changes in phenotypic expression in embryonic and adult cells

treated with 5-azacytidine. J. Cell. Physio. 111(2):187-194.

122

42. Enjoji M, Nakashima M, Honda M, Sakai H, Nawata H (1997) Hepatocytic phenotypes

induced in sarcomatous cholangiocarcinoma cells treated with 5-azacytidine. Hepatology,

26(2):288-294.

43. Shi Y, Desponts C, Do JT, Hahm HS, Scholer HR, Ding S (2008) Induction of pluripotent

stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule

compounds. Cell Stem Cell 3:568-574.

44. Federation, AJ, Bradner JE, Meissner A (2014) The use of small molecules in somatic-cell

reprogramming. Trends Cell Biol 24(3):179-187.

45. Okita K, Yamanaka S (2011) Induced pluripotent stem cells: opportunities and challenges.

Philos Trans R Soc Lond B Biol Sci 366(1575):2198-2207.

46. Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, Schorderet P (2008) Dissecting direct

reprogramming through integrative genomic analysis. Nature 454:49-55.

47. Hou P, Li Y, Zhang X, Liu C, Guan J, Li H (2013) Pluripotent stem cells induced from

mouse somatic cells by small-molecule compound. Science 341(6146):651-654.

48. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K (2007) Induction of

pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861-872.

49. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S (2007)

Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917-

1920.

50. Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II (2009) Human induced pluripotent

stem cells free of vector and transgene sequences. Science 324(5928):797-801.

51. Soldner F, Hockemeyer D, Beard C, Gao Q, Bell GW, Cook EG (2009) Parkinson's disease

patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell

136:964-977.

123

52. Kim D, Kim CH, Moon JI, Chung YG, Chang MY, Han BS (2009) Generation of human

induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell

4(6):472–476.

53. Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T (2009) Generation of induced pluripotent

stem cells using recombinant proteins. Cell Stem Cell 4:381-384.

54. Zou K, Yuan Z, Yang Z, Luo H, Sun K, Zhou L (2009) Production of offspring from a

germline stem cell line derived from neonatal ovaries. Nat Cell Biol 11:631-636.

55. Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F (2010) Highly efficient

reprogramming to pluripotency and directed differentiation of human cells with synthetic

modified mRNA. Cell Stem Cell 7(5):618-630.

56. Yakubov E, Rechavi G, Rozenblatt S, Givol D (2010) Reprogramming of human fibroblasts

to pluripotent stem cells using mRNA of four transcription factors. Biochem Biophy Res

Commun 394(1):189-193.

57. Anokye-Danso F, Trivedi CM, Juhr D, Gupta M, Cui Z, Tian Y (2011) Highly efficient

miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell

Stem Cell 8:376-388.

58. Miyoshi N, Ishii H, Nagano H, Haraguchi N, Dewi DL, Kano Y. (2011) Reprogramming of

mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell 8(6):633-

638.

59. Singhal DK, Singhal R, Malik HN, Singh S, Kumar S, Kaushik JK (2015) Generation of

Germ Cell-Like Cells and Oocyte-Like Cells from Goat Induced Pluripotent Stem Cells.

Stem Cell Res Ther 5:(5).

124

60. Mohana Kumar B, Jin HF, Kim JG, Song HJ, Hong Y, Balasubramanian S (2006) DNA

methylation levels in porcine fetal fibroblasts induced by an inhibitor of methylation, 5-

azacytidine. Cell Tissue Res 325(3):445-454.

61. Shi G, Jin Y (2010) Role of Oct4 in maintaining and regaining stem cell pluripotency. Stem

Cell Res Ther 1:39.

62. Sterneckert J, Hoing S, Scholer HR (2012) Concise review: Oct4 and more: the

reprogramming expressway. Stem Cells 30:15-21.

63. Pacchiarotti J, Maki C, Ramos T, Marh J, Howerton K, Wong J (2010) Differentiation

potential of germ line stem cells derived from the postnatal mouse ovary. Differentiation

79:159-170.

64. Carter AC, Davis-Dusenbery BN, Koszka K, Ichida JK, Eggan K (2014) Nanog-Independent

Reprogramming to iPSCs with Canonical Factors. Stem Cell Rep 2:119-126.

65. Marthaler AG, Tubsuwan A, Schmid B, Poulsen UB, Hyttel P, Nielsen JE (2016) Generation

of spinocerebellar ataxia type 2 patient-derived iPSC line H271. Stem Cell Res 16(1):159-

161.

66. Masui S, Nakatake Y, Toyooka Y, Shimosato D, Yagi R, Takahashi K (2007) Pluripotency

governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat

Cell Biol 9:625-635.

67. Niwa H, Miyazaki J, Smith AG (2000) Quantitative expression of Oct-3/4 defines

differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24:372-376.

68. Remenyi A, Lins K, Nissen LJ, Reinbold R, Schöler HR, Wilmanns M (2003) Crystal

structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and

Sox2 on two enhancers. Gen Dev 17:2048-2059.

125

69. Hosler BA, Rogers MB, Kozak CA, Gudas LJ (1993) An octamer motif contributes to the

expression of the retinoic acid-regulated zinc finger gene Rex-1 (Zfp-42) in F9

teratocarcinoma cells. Mol Cell Biol 13:2919-2928.

70. Shi W, Wang H, Pan G, Geng Y, Guo Y, Pei D (2006) Regulation of the pluripotency

marker Rex-1 by Nanog and Sox2. J Biol Chem 281:23319-23325.

71. Son M-Y, Choi H, Han Y-M, Cho YS (2013) Unveiling the critical role of REX1 in the

regulation of human stem cell pluripotency. Stem Cells 31(11):2374-2387.

72. Kopper O, Giladi O, Golan-Lev T, Benvenisty N (2010) Characterization of gastrulation-

stage progenitor cells and their inhibitory crosstalk in human embryoid bodies. Stem Cells

28:75-83.

73. Okamura D, Hayashi K, Matsui Y (2005) Mouse epiblasts change responsiveness to BMP4

signal required for PGC formation through functions of extraembryonic ectoderm. Mol

Reprod Dev 70:20-29.

74. Ohinata Y, Ohta H, Shigeta M, Yamanaka K, Wakayama T (2009) A signaling principle for

the specification of the germ cell lineage in mice. Cell 137:571-584.

75. de Sousa Lopes SM, Roelen BA, Monteiro RM, Emmens R, Lin HY, Li E (2004) BMP

signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of

primordial germ cells in the mouse embryo. Genes Dev 18:1838–1849.

76. Kee K, Angeles V, Flores M, Nguyen H, Reijo Pera RA (2009) Human DAZL, DAZ and

BOULE genes modulate primordial germ cell and haploid gamete formation. Nature

462:222–225.

77. West FD, Roche-Rios MI, Abraham S, Rao RR, Natrajan MS, Bacanamwo M (2010) KIT

ligand and bone morphogenetic protein signaling enhances human embryonic stem cell to

germ-like cell differentiation. Hum Reprod 25(1):168-178.

126

78. Bucay N, Yebra M, Cirulli V, Afrikanova I, Kaido T, Hayek A (2009) A novel approach for

the derivation of putative primordial germ cells and Sertoli cells from human embryonic

stem cells. Stem Cells 27:68-77.

79. Park TS, Galic Z, Conway AE, Lindgren A, Van Handel BJ, Magnusson M (2009)

Derivation of primordial germ cells from human embryonic and induced pluripotent stem

cells is significantly improved by coculture with human fetal gonadal cells. Stem Cells

27(4):783-795.

80. Ying YL, Marble XM, Lawson A, Zhao GQ (2000) Requirement of Bmp8b for the

generation of primordial germ cells in the mouse. Mol Endocrinol 14:1053-1063.

81. Kishigami S, Mishina Y (2005) BMP signaling and early embryonic patterning. Cytokine

Growth Factor Rev 16(3):265-278.

82. Chuang CY, Lin KI, Hsiao M, Stone L, Chen HF, Huang YH (2012) Meiotic competent

human germ cell-like cells derived from human embryonic stem cells induced by

BMP4/WNT3A signaling and OCT4/EpCAM (epithelial cell adhesion molecule) selection. J

Biol Chem 287:14389–14401.

83. Park ES, Woods DC, Tilly JL (2013) Bone morphogenetic protein 4 promotes mammalian

oogonial stem cell differentiation via Smad1/5/8 signaling. Fertil Steril 100(5):1468-1475.

84. Yu H, Vu TH, Cho KS, Guo C, Chen DF (2014) Mobilizing endogenous stem cells for

retinal repair. Transl Res 163(4):387-398.

85. Shah SM, Saini N, Ashraf S, Singh MK, Manik RS, Singla SK (2015) Bone morphogenetic

protein 4 (BMP4) induces buffalo (Bubalus bubalis) embryonic stem cell differentiation into

germ cells. Biochimie 119:113-124.

127

86. Pera MF, Andrade J, Houssami S, Reubinoff B, Trounson A, Stanley EG (2004) Regulation

of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J Cell Sci

117:1269-1280.

87. Shalgi R, Kraicer P, Rimon A, Pinto M, Soferman N (1973) Proteins of human follicular

fluid: the blood-follicle barrier. Fertil Steril 24(6):429-434.

88. Rodgers RJ, Irving-Rodgers HF (2010) Formation of the ovarian follicular antrum and

follicular fluid. Biol Reprod 82(6):1021–1029.

89. Dyce PW, Zhu H, Craig J, Li J (2004) Stem cells with multilineage potential derived from

porcine skin. Biochem Biophy Res Commun 316(3):651–658.

90. Wu YT, Tang L, Cai J, Lu XE, Xu J, Zhu XM (2007) High bone morphogenetic protein-15

level in follicular fluid is associated with high quality oocyte and subsequent embryonic

development. Hum Reprod 22(6):1526-1531.

91. Linher K, Dyce P, Li J (2009) Primordial germ cell-like cells differentiated in vitro from

skin-derived stem cells. PLoS ONE 4(12):e8263.

92. Song S-H, Kumar BM, Kang E-J, Lee Y-M, Kim T-H, Ock S-A (2011) Characterization of

Porcine Multipotent Stem/Stromal Cells Derived from Skin, Adipose, and Ovarian Tissues

and Their Differentiation In Vitro into Putative Oocyte-Like Cells. Stem Cells Dev

20(8):1359-1370.

93. Cheng X, Chen S, Yu X, Zheng P, Wang H (2012) BMP15 gene is activated during human

amniotic fluid stem cell differentiation into oocyte-like cells. DNA Cell Biol 31:1198–1204.

94. Aflatoonian B, Ruban L, Jones M, Aflatoonian R, Fazeli A, Moore HD (2009) In vitro post-

meiotic germ cell development from human embryonic stem cells. Hum Reprod 24:3150-

3159.

128

95. Toyooka Y, Tsunekawa N, Akasu R, Noce T (2003) Embryonic stem cells can form germ

cells in vitro. Proc Natl Acad Sci USA 100:11457-11462.

96. Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA (2003) Suppression of ovarian

follicle activation in mice by the transcription factor Foxo3a. Science 301:215-218.

97. Ruggiu M, Speed R, Taggart M, McKay SJ, Kilanowski F, Saunders P (1997) The mouse

Dazla gene encodes a cytoplasmic protein essential for gametogenesis. Nature 389(6646):

73-77.

98. Chen HF, Kuo HC, Chien CL, Shun CT, Yao YL, Ip PL (2007) Derivation, characterization

and differentiation of human embryonic stem cells: comparing serum-containing versus

serum-free media and evidence of germ cell differentiation. Hum Reprod 22:567-577.

99. Niikura Y, Niikura T, Tilly JL (2009) Aged mouse ovaries possess rare premeiotic germ

cells that can generate oocytes following transplantation into a young host environment.

Aging 1(12): 971-978.

100. Clark AT, Bodnar MS, Fox M, Rodriquez RT, Abeyta MJ, Firpo MT (2004) Spontaneous

differentiation of germ cells from human embryonic stem cells in vitro. Hum Mol Gen

13(7):727-739.

101. Cai L, Rothbart SB, Lu R, Xu B, Chen W-Y, Tripathy A (2013) An H3K36 methylation-

engaging Tudor motif of polycomb-like proteins mediates PRC2 complex targeting. Mol

Cell 49:571-582.

102. Kona SS, Praveen Chakravarthi V, Siva Kumar AV, Srividya D, Padmaja K, Rao VH (2016)

Quantitative expression patterns of GDF9 and BMP15 genes in sheep ovarian follicles grown

in vivo or cultured in vitro. Theriogenology 85(2):315-322.

129

103. Salvador LM, Silva CP, Kostetskii I, Radice GL, Strauss JF (2008) The promoter of the

oocyte-specific gene, Gdf9, is active in population of cultured mouse embryonic stem cells

with an oocyte-like phenotype. Methods 45:172-181.

104. Lin CY, Chen CY, Yu CH, Yu IS, Lin SR, Wu JT (2016) Human X-linked Intellectual

Disability Factor CUL4B Is Required for Post-meiotic Sperm Development and Male.

Fertility. Sci Rep 6:20227.

105. Lefièvre L, Conner SJ, Salpekar A, Olufowobi O, Ashton P, Pavlovic B (2004) Four zona

pellucida glycoproteins are expressed in the human. Hum Reprod 19:1580–1586.

106. Hong SH, Maghen L, Kenigsberg S, Teichert AM, Rammeloo AW, Shlush E (2013)

Ontogeny of human umbilical cord perivascular cells: molecular and fate potential changes

during gestation. Stem Cells Dev 22(17):2425-2439.

107. Hu X, Lu H, Cao S, Deng Y-L, Li Q-J, Wan Q (2015) Stem cells derived from human first-

trimester umbilical cord have the potential to differentiate into oocyte-like cells in vitro. Int J

Mol Med 35:1219-1229.

108. Singhal VK, Kalsan M, Kumar N, Saini A, Chandra R (2015) Induced pluripotent stem cells:

applications in regenerative medicine, disease modeling, and drug discovery. Front Cell Dev

Biol 3:(2).

130

List of tables and figures

Table 1. Primer pairs used in real-time PCR for quantification of markers of pluripotency, germ

cells and oocytes genes expressed in cells cultured.

Figure 1. Representative pictures of the morphological changes in bovine skin fibroblasts

exposed to 5-Aza. (A) The fibroblast cells isolated from bovine fetal ear skin (untreated cells).

(B) Fibroblasts exposed to 2.0 µM of 5-Aza for 72 h. (C) Fluorescence staining of viable cells for

Calcein AM. (D) Fluorescence staining of apoptotic cells for ethidium homodimer-1. Scale bar =

100 µm.

Figure 2. Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in fibroblasts

cultured for 18 h in with different concentrations of 5-Aza (0.5, 1.0 or 2.0 µM).





Figure 5. Levels of mRNA for of pluripotency genes, (A) SOX2, (B) NANOG, (C) OCT4 and (D)

REX, in fibroblasts cultured for 18 h, 36 h or 72 h in with different concentrations of 5-Aza (0.5,

1.0 or 2.0 µM).

Figure 6. Representative pictures of the morphological characterization in bovine skin fibroblasts

exposed to 5-Aza and cultured in differentiation medium for 14 days. Fibroblast cultured for 14

days in differentiation medium supplemented with 10 ng/mL of BMP-2 (line 1), 10 ng/mL of

BMP-4 (line 2), 5% follicular fluid (line 3), (A, D, G) cell analyzed by light microscopy, (B, E,

H) Fluorescence staining of viable cells for Calcein AM; (C, F, I) Fluorescence staining of

apoptotic cells for ethidium homodimer-1. Scale bar = 100 µm.

Figure 7. Levels of mRNA for markers of germ cells [VASA (A, B), DAZL (C, D), C-KIT (E, F)]

in cells cultured for 7 (A, C, E) or 14 (B, D, F) days in control medium or supplemented with

BMP-2 (10 ng/mL), BMP-4 (10 ng/mL) and 5% follicular fluid.

Figure 8. Levels of mRNA for markers of oocytes [ZPA (A, B), GDF-9 (C, D), SCP3 (E, F)] in

cells cultured for 7 (A, C, E) or 14 (B, D, F) days in control medium or supplemented with BMP-

2 (10 ng/mL), BMP-4 (10 ng/mL) and 5% follicular fluid.

Figure 9. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-KIT (C)] and

oocytes [ZPA (D), GDF-9 (E), C-KIT (F)] after culture cells for 0 h (5-Aza), 7 or 14 days in

control medium.



medium supplemented with 10 ng/mL of BMP-2.

131



medium supplemented with 10 ng/mL of BMP-4.


oocytes [ZPA (D), GDF-9 E), C-KIT (F)] after culture cells for 0 h, 7 or 14 days in medium

supplemented with 5% follicular fluid.

132

Table 1. Primer pairs used in real-time PCR for quantification of markers of pluripotency, germ

cells and oocytes genes expressed in cells cultured.

Target

gene

Primer sequence (5´ 3´)

Sense (s),

anti-sense

(As)

GenBank number

GAPDH

CACCCTCAAGATTGTCAGCA

GGTCATAAGTCCCTCCACGA

S

As

NM_001034034.2

SOX2

TGGATCGGCCAGAAGAGGAG

CAGGCGAAGAATAATTTGGGGG

S

As

NM_001105463.2

NANOG

CGTGTCCTTGCAAACGTCAT

CTGTCTCTCCTCTTCCCTCCTC

S

As

DQ069776

OCT-4

GAGAAAGACGTGGTCCGAGTG

GACCCAGCAGCCTCAAAATC

S

As

NM_174580.2

REX

CCTGTGAGGGGAGCTCTAGT

TTTTCAGCAAGCACCCATGC

S

As

XM_003587951.1

VASA

TGGTCCTGGCTTCAGTGGTA

TCTTGCCGGGGTAATTCTTTCT

S

As

NM_001007819.1

DAZL

TACCCGCCTCTGACTCTCTC

GTGTTCACTCAGAGGGGCTC

S

As

EF501823.2

c-KIT

TCGTGGATGGCTGTGAATACAA

CAAGTGAGAGAATGCCGGGT

S

As

GI: 16580734

ZPA



S

As

NM_173973.2

GDF-9

ACAACACTGTTCGGCTCTTCACCC

CCACAACAGTAACACGATCCAGGTT

S

As

GI:51702523

SCP3

GTTGGCAAAACCATCCGTGG

GGGGTCTTCTCTTCAATGGCA

S

As

NM_001040588.2

133

Figure 1. Representative pictures of the morphological changes in bovine skin fibroblasts

exposed to 5-Aza. (A) The fibroblast cells isolated from bovine fetal ear skin (untreated cells).

(B) Fibroblasts exposed to 2.0 µM of 5-Aza for 72 h. (C) Fluorescence staining of viable cells for

Calcein AM. (D) Fluorescence staining of apoptotic cells for ethidium homodimer-1. Scale bar =

100 µm.

134

Figure 2. Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in fibroblasts cultured for 18 h in with different

concentrations of 5-Aza (0.5, 1.0 or 2.0 µM).

135



136



137

Figure 5. Levels of mRNA for of pluripotency genes, (A) SOX2, (B) NANOG, (C) OCT4 and (D) REX, in fibroblasts cultured for 18

h, 36 h or 72 h in with different concentrations of 5-Aza (0.5, 1.0 or 2.0 µM).

138

Figure 6. Representative pictures of the morphological characterization in bovine skin fibroblasts exposed to 5-Aza and cultured in

differentiation medium for 14 days. Fibroblast cultured for 14 days in differentiation medium supplemented with 10 ng/mL of BMP-2

(line 1), 10 ng/mL of BMP-4 (line 2), 5% follicular fluid (line 3), (A, D, G) cell analyzed by light microscopy, (B, E, H) Fluorescence

staining of viable cells for Calcein AM; (C, F, I) Fluorescence staining of apoptotic cells for ethidium homodimer-1. Scale bar = 100

µm.

139

Figure 7. Levels of mRNA for markers of germ cells [VASA (A, B), DAZL (C, D), C-KIT (E, F)] in cells cultured for 7 (A, C, E) or 14

(B, D, F) days in control medium or supplemented with BMP-2 (10 ng/mL), BMP-4 (10 ng/mL) and 5% follicular fluid.

140

Figure 8. Levels of mRNA for markers of oocytes [ZPA (A, B), GDF9 (C, D), SCP3 (E, F)] in cells cultured for 7 (A, C, E) or 14 (B,

D, F) days in control medium or supplemented with BMP-2 (10 ng/mL), BMP-4 (10 ng/mL) and 5% follicular fluid.

141

Figure 9. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-KIT (C)] and oocytes [ZPA (D), GDF-9 (E), C-KIT

(F)] after culture cells for 0 h (5-Aza), 7 or 14 days in control medium.

142

Figure 10. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-KIT (C)] and oocytes [ZPA (D), GDF-9 (E), C-

KIT(F)] after culture cells for 0 h (5-Aza), 7 or 14 days in medium supplemented with 10 ng/mL of BMP-2.

143


KIT (F)] after culture cells for 0 h (5-Aza), 7 or 14 days in medium supplemented with 10 ng/mL of BMP-4.

144


KIT(F)] after culture cells for 0 h, 7 or 14 days in medium supplemented with 5% follicular fluid.

145

9 ARTIGO III

Bovine ovarian stem cells differentiate into germ cells and oocyte-like structures after

culture in vitro

(Células-tronco ovarianas bovinas diferenciam-se em células germinativas e estruturas

semelhantes a oócitos após cultivo in vitro)

Artigo a ser submetido ao periódico Reproduction in Domestic Animals

(Qualis B2 - Biotecnologia)

146

Bovine ovarian stem cells differentiate into germ cells and oocyte-like structures after

culture in vitro

Oocyte-like structures formation from from ovarian stem cells bovine

G B. de Souza1, J J N Costa1, E V da Cunha1, J R S Passos1, R P Ribeiro1, M V A Saraiva1, R van

den Hurk2, J R V Silva1*


Sobral, CE, Brazil; 2Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht

University, Utrecht, The Netherlands




Contents

The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the

effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation

of these stem cells into oocyte-like structures. The ovarian stem cells were isolated and cultured

in α-MEM+ supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were

147

evaluated for their morphological appearance, viability, expression of alkaline phosphatase, and

for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and

SCP3) by qPCR. Levels of mRNA were analysed by using ANOVA and Bonferroni test

(P<0.05). The results showed that at day 0, ovarian stem cells expressed specific markers of

pluripotency (OCT4, SOX). In addition, these cells were positive for alkaline phosphatase. After

the period of differentiation, cells had morphological features that resemble primordial germ cells

(PGCs) and oocyte-like cells (OLCs). The PGC-like cells expressed VASA and DAZL, while the

OLCs expressed mRNAs for GDF9, ZPA and SCP3. In conclusion, OLCs can be differentiated in

vitro from ovarian stem cells and BMPs and follicular fluid are effective in stimulating the

expression of mRNAs for germ cell and oocyte markers.

Keywords: BMP2, BMP4, gene expression, follicular fluid

Introduction

In recent years, reproductive biotechnology has been developed in order to solve female

reproductive problems or to increase reproductive efficiency in several species of domestic

animals or in endangered species. In humans, it is well-known that between 10 and 15% of

couples are infertile, the main cause being associated with the default of appropriate release of

fertilizable oocytes (Nicholas et al. 2009a). This information shows that in-vitro differentiation of

germ cells in oocytes from stem cells may have an important therapeutic use. In addition, it may

provide a valuable model for identifying factors involved in germ cell formation and

differentiation.

148

In the last years, researches have provided evidences for the existence of female germ

line cells that contribute to postnatal oogenesis (Pan et al. 2015). Several other studies have

shown the presence of germ line cells and potential postnatal oogenesis using various techniques

in mice as well as in other species, including human (Parte et al. 2011). The isolation and

manipulation of ovarian stem cells have tremendous application in medical, veterinary and

animal production fields (Mooyottu et al. 2011).The stem cells have been isolated using different

strategies, propagated in vitro for several generations and their ability to differentiate into oocytes

in vivo has been demonstrated (White et al. 2012). Multiple attempts have been made over the

past decade to determine whether murine or human embryonic stem cells (ESC) are able to

differentiate into primordial germ cells (PGCs) or oocyte-like cells (OLCs) in vitro (Parte et al.

2011). Moreover, it has been reported that germ cell-like cells can be derived in vitro from

mesenchymal stem cells (MSCs) derived from porcine fetal skins (Dyce et al. 2011), mouse bone

marrow (Johnson et al. 2005), or human adult ovaries (Bukovsky et al. 2005). Additionally,

certain studies have reported that human or murine ESCs can spontaneously differentiate into

OLCs (Clark et al. 2004).

Several studies show the role of exogenous factors on the formation of germ cells and

oocyte differentiation. Bone morphogenetic protein 4 (BMP4) promotes mammalian oogonial

stem cell differentiation in humans (Park et al. 2013), buffalo (Shah et al. 2015) and goat (Singhal

et al. 2015). BMP2 also plays a crucial role in the differentiation of primordial germ cells (Pera et

al. 2004). In rats, this protein plays an important role in the formation of alkaline phosphatase

positive primordial germ cells and induces expression of markers for PGCs (Saitou and Yamaji,

2010). Other studies have shown that porcine follicular fluid effectively promotes the formation

of germ-like cells from stem cells (Cheng et al. 2012). Hong et al. (2014) reported that human

follicular fluid stimulated differentiation into oocyte-like cells in vitro from stem cells (Hong et

149

al. 2014). However, the differentiation of OLCs from ovarian stem cells isolated from adult

bovine ovaries has not yet been demonstrated. Thus, we hypothesize that bovine BMP2, BMP4

and bovine follicular fluid stimulated ovarian stem cells to form germ cells and OLCs in vitro.

The aim of the study was to evaluate the effects of BMP2, BMP4 and follicular fluid on

differentiation of ovarian stem cells into germ cells and oocytes, and to evaluate the effects on

expression of markers for primordial germ cells (VASA, DAZL) and oocytes (SCP3, GDF9, ZPA)

in vitro.

Materials and Methods

Unless otherwise stated, all chemicals and reagents used in this study were purchased

form from Sigma Chemical Co. (St Louis, MO, USA). All the animal procedures and protocols

were approved by the Local Ethics Committee for Animal Experiments of the Federal University

of Ceara (2015/18).

Ovary Collection

Fresh ovaries (n = 20) from adult mixed breed cows (Bos taurus) were collected at a

slaughterhouse. Immediatelly, the ovaries were washed (for approximately 10 sec) in 70%

alcohol, then washed twice in 0.9% saline solution, and transferred to minimum essential

medium-alpha (α-MEM – Sigma-Aldrich, Saint-Louis, Missouri, USA) supplemented with 100

IU/mL penicillin and 100 mg/mL streptomycin. Subsequently, ovarian pairs from each cow were

transported to the laboratory within 1 h at 4 ºC (Chaves et al. 2008).

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Primary ovarian cortical tissue culture

In the laboratory, to perform a primary culture, ovarian cortical tissue from the same

ovarian pair was cut (1 mm × 1 mm × 1 mm) using a scissor and scalpel under sterile conditions.

To isolate ovarian stem cells, cortical slices were transferred to 24-well culture dishes (Corning,

Germany) that contained 1 mL of culture medium. The basic culture medium consisted of α-

MEM (pH 7.2–7.4) supplemented with ITS (10 µg/mL insulin, 5.5 µg/mL transferrin, and 5

ng/mLselenium), 2 mM glutamine (Sigma-Aldrich), antibiotics (100 IU/mL penicillin and 100

mg/mL streptomycin) (Sigma), 2 mM pyruvate and 3.0 mg/mL of bovine serum albumin (α-

MEM+) as described by Garor et al. (2009). Culture was performed at 38.5 °C in 5% CO2 in a

humidified incubator. The cortical tissue pieces were discarded from the cell culture, where after

ovarian stem cells grow and migrate from the margin of the ovarian tissue piece and adhered onto

the plastic surface of the culture plates to form cell colonies. These ovarian stem cells were

processed to analyse the morphology, viability, activity of alkaline phosphatase and expression of

specific markers of pluripotency stem cells or placed in culture for 14 days in the presence of

BMP2, BMP4, both BMP2 and BMP4, and follicular fluid.

Influence of follicular fluid and BMPs on cellular differentiation

In order to induce differentiation into germ cells and oocytes, ovarian stem cells

obtained from ovarian tissues were plated onto 24-well culture plates (Corning, Germany), and

cultured in five treatments, i.e., in control medium (α-MEM+) alone or supplemented with 50

ng/mL BMP2 (R&D Systems, Inc., Minneapolis, MN), 50 ng/mL BMP4 (R&D Systems, Inc.,

Minneapolis, MN), both BMP2 (50 ng/mL) plus BMP4 (50 ng/mL) (Panula et al. 2011), or 5%

151

cow follicular fluid (Dyce et al. 2011) at 38.5 °C in 5% CO2 in a humidified incubator. The

culture medium was replaced every 4 days with fresh medium. After 14 days of culture in

differentiation medium, the cells were destined to analyse viability and expression of alkaline

phosphatase or stored at –80 ºC until RNA extraction to evaluate the expression of specific

markers for germ cells and oocytes.

Viability analysis

On days 0 and 14 of culture, the proportion of living and dead cells was assessed with

calcein AM (Molecular Probes) and ethidium homodimer-1 (Molecular Probes). Calcein AM (12

µM) and ethidium homodimer-1 (120 µM) was added to wells and the cells were incubated

protected from light for a period of 10 min at room temperature. Determination of calcein and

ethidium fluorescence at excitation/emission wave lengths of 488/568 nm a fluorescence

microplate reader was used, using an inverted microscopy (NIKON, Eclipse, TS100).

Alkaline Phosphatase Assay

Alkaline phosphatase (AP) activity was evaluated according to manufacturer instructions

(Alkaline Phosphatase Detection Kit, Sigma-Aldrich) in ovarian stem cells before (day 0) and

after differentiation (day 14) in the different treatments. In short, cells were fixed (citrate,

buffered acetone 60%) for 30 sec and then incubated in a working solution of reagents which

consisted of Salt RR Blue, Naphtol AS-MX phosphate solution and water in a 2:1:1 ratio. After

30 min, the cells were rinsed with deionized water, stained with hematoxylin, rinsed with

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deionized water and after 15 min the cells were observed under an inverted microscope (NIKON,

Eclipse, TS100). The cells or cell colonies expressing AP activity were stained in red.

RNA extraction and cDNA synthesis

To analyze the expression of markers for pluripotency (OCT4 and SOX2), in germ cells

(DAZL and VASA) and oocytes (SCP3, GDF9, ZPA), isolation of total RNA was performed using

the TRIzol® Reagent Plus purification kit (Invitrogen, São Paulo, Brazil). According to the

manufacturer’s instructions, 800 µL of TRIzol® solution was added to each of the frozen samples

and the respective lysates were aspirated through 20-gauge needles before centrifugation at

10,000 g for 3 min at room temperature. Thereafter, all lysates were diluted 1:1 with 70% ethanol

and subjected to a mini-column. After binding of the RNA to the column, DNA digestion was

performed using RNAse-free DNAse (340 Kunitz units/mL) for 15 min at room temperature.

After washing the column three times, the RNA was eluted with 30 µL RNAse-free water. The

RNA concentration was estimated by reading the absorbance at 260 nm and was checked for

purity at 280 nm in a spectrophotometer (Amersham Biosciences, Cambridge, England). For each

sample, RNA concentrations were adjusted and used to synthesize cDNA. Before the reverse

transcription reaction, samples of RNA were incubated for 5 min at 70 ºC and then cooled in ice.

The reverse transcription was performed in a total volume of 20 µL composed of 10 µL of

sample RNA, 4 µL reverse transcriptase buffer (Invitrogen, São Paulo, Brazil), 8 units RNase

out, 150 units of reverse transcriptase Superscript III, 0036 U random primers, 10 mM DTT and

0.5 mM of each dNTP (Invitrogen, São Paulo, Brazil). The mixture was incubated at 42 ºC for 1

h, subsequently at 80 ºC for 5 min, and finally stored at –20 ºC. The negative control was

prepared under the same conditions, but without the addition of reverse transcriptase.

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Real time PCR

The quantification of mRNA was performed with the use of SYBR® Green. Each

reaction in real time (20 µL) contained 10 µL of SYBR® Green Master Mix (Applied

Biosystems, Warrington, UK), 7,3 µL of ultra-pure water, 1 µL of cDNA and 0.5 μM of each

primer, and real-time PCR was performed in StepOne Real-Time PCR (Applied Biosystems,

Warrington, UK) thermocycler. The thermal cycling profile for the first round of PCR was initial

denaturation and activation of the polymerase for 10 min at 95 oC, followed by 40 cycles of 15

sec at 95 oC, 30 sec at 58 oC, and 30 sec at 72 oC. The final extension was for 10 min at 72 oC.

The primers were designed by using the primerquestsm program (http://www.idtdna.com) primers

used in this study are shown in Table 1. The specificity of each primer pair was confirmed by

melting curve analysis of PCR products. Relative quantifications of mRNA were carried out

using the comparative threshold (CT) cycle method. The delta-delta-Ct method was used to

transform the Ct values into normalized relative expression levels (Livak and Schmittgen, 2001).

Statistical analysis

Levels of mRNA were analysed by using ANOVA and Bonferroni test (P<0.05). Data

were expressed as mean ± standard error of the mean (SEM).

Results

http://www.idtdna.com/

154

Morphological characteristics of cells before and after culture with BMP-2, BMP-4 and

follicular fluid

During culture of ovarian cortical tissue, cells migrated from the margin of the ovarian

tissue piece and adhered onto the plastic surface of the culture plates to form cell colonies. These

cells showed fusiform shapes and, morphologically, resembled undifferentiated stem cells (Fig.

1A-B). As illustrated in Figure 2, alkaline phosphatase activity was detected in these cells. After

seven days of culture in differentiation medium, some subpopulations of the cells became

morphologically different from the starting cultures, increased their volume and formed

aggregates (Fig. 1C-D). These aggregates gradually became structures similar to oocytes after 14

days of culture in medium supplemented with BMP2 (Fig. 1E-F), BMP4 (Fig. 1G-H), BMP2 and

BMP4 (Fig. 1I-J) and follicular fluid (Fig. 1K-L).

On days 0 and 14 of culture, the proportion of living and dead cells was assessed with

calcein AM and ethidium homodimer-1 staining of cells, respectively. Fluorescence analysis

shows ovoid cells clusters positive for calcein (green) and ethidium homodimer (red) in control

medium (α-MEM), suggesting that the ovarian stem cells proliferated and form clusters (Fig. 1B).

After 14 days of culture in control medium and other treatments, cells became morphologically

distinct, suggesting that they are germ cell clusters. These cells aggregated and gradually became

structures similar to oocytes that were labeled positive for calcein (Fig.1. D, F, H, L).

Quantification of mRNA for specific markers of pluripotency, germ cells and oocytes

At day 0, real-time PCR showed that cells that migrated from the margin of the ovarian

tissue piece expressed mRNA for OCT4 and SOX2 (Fig. 3A). Melting curve analysis confirmed

155

the specificity of the products after amplification. After 14 days of culture, OLCs formed after

culture in medium, supplemented with BMP2, both BMP2 and BMP4, and follicular fluid, had

significantly higher (P<0.05) levels of mRNA for VASA than those cultured in control medium

(α-MEM+, Fig. 4A). Addition of BMP-4 alone to cell cultures had no effect on VASA expression

(Fig. 4A). Cells cultured in presence of follicular fluid had significantly increased levels of

mRNA for VASA compared to those cultured with BMP2, BMP4 or both BMP2 and BMP4 (Fig.

3A). Presence of BMP2, BMP4, both BMP2 and BMP4, and follicular fluid in culture medium

significantly (P<0.05) increased the levels of mRNA DAZL in OLCs, compared to those found in

control medium (Fig. 4B). Additionaly, lower levels of mRNA for DAZL were observed in OLCs

cultured with both BMP2 and BMP4 than in those cultured in presence of both BMP4 alone or

follicular fluid (Fig. 4B). Regarding the oocyte-specific genes, mRNA levels for SCP3 were

significantly increased in OLCs cultured in medium containing BMP2, BMP4, both BMP2 and

BMP4 or follicular fluid, when compared to control medium (Fig. 4C). Furthermore, compared to

control medium, BMP4, both BMP2 and BMP4 and follicular fluid significantly increased GDF9

mRNA expression in OLCs (Fig. 4D). Moreover, OLCs cultured with follicular fluid had higher

levels of mRNA for GDF9 than those cultured in presence of BMP2 alone (Fig. 4D). Both BMP2

and BMP4, as well as follicular fluid increased the expression of mRNA for ZPA, compared to

those cultured in control medium alone or in medium supplemented with BMP2 (Fig. 4E). In

addition, OLCs cultured in presence of follicular fluid had higher levels of mRNA for ZPA than

those cultured in control medium supplemented with BMP2, BMP4 or both (Fig. 4E).

Discussion

156

This study shows for the first time that bovine ovarian stem cells have an intrinsic ability

to differentiate into OLCs. Isolated ovarian stem cells expressed mRNAs for OCT4, and SOX2,

which are transcription factors that act in maintainance of cellular pluripotency (Takahashi and

Yamanaka, 2006). Foregoing studies have shown that OCT4 and SOX2 regulate the expression of

NANOG (Rodda et al. 2005), while NANOG regulates REX1 expression (Shi et al. 2006). OCT4

and NANOG activate transcription of non encoding RNAs that promotes self-renewal of stem

cells and avoid differentiation (Mohamed et al. 2010).

Bovine ovarian stem cells also had shown alkaline phosphatase activity. This enzyme is a

key marker to identify pluripotent embryonic stem, since blastocysts present alkaline phosphatase

activity strictly in inner cell mass, but not in trophoblast cells (Štefková et al. 2015). Various

studies have also reported that alkaline phosphatase is an enzyme commonly used to identify

primordial germ cells (Clark et al. 2004; Parte et al. 2011).

As previously has been demonstrated, germ cell development requires a series of

multiple well-orchestrated steps, which involve up and down regulation of the expression of

specific genes (Takai et al. 2003). Earlier studies have also shown that PGC-like cells and OLCs

can be generated from embryonic, differentiated pluripotent and adult stem cells in vitro (Clark et

al. 2004; Panula et al. 2011). In mouse, pigs and human, the differentiation of ESCs or somatic

stem cells into OLCs was generally performed by culturing the cells with growth factors (Hübner

et al. 2003; Wei et al. 2008), estrogenic stimuli (Clark et al. 2004), conditioned medium from

testicular cell cultures (Lacham-Kaplan et al. 2006), follicular fluid, gonadotrophins (Hong et al.

2014) or with ovarian granulosa cells (Qing et al. 2007). The present study shows that BMP2 and

follicular fluid increase the expression of VASA in bovine OLCs formed in vitro. VASA is

expressed in post-migratory PGCs until the post-meiotic stage of oocytes (Castrillon et al. 2003).

Previous studies did show that follicular fluid promotes VASA expression and diffentiation of

157

skin-derived stem cells into OLCs (Dyce et al. 2011; Linher et al. 2009). Apart from VASA

expression, that of DAZL appears currently increased after culture of stem cells in presence of

BMP2 and follicular fluid, but also in presence of BMP4. DAZL is considered essential for PGC

development, as knockout mice lack a germ cell population (Kee et al. 2009). DAZL and VASA

have been used as markers for germ cell differentiation in various species (human: Kee et al.

2006; murine: Niikura et al. 2009).

Expression of SCP3 is frequently used as a meiotic marker, while GDF9 is expressed

specifically in oocytes. This study showed that their expression in in-vitro differentiated bovine

OLCs is promoted by BMPs and follicular fluid. SCP3-positive oocytes were previously

considered to be direct evidence of postnatal oogenesis through differentiation of germline stem

cells in mice (Pan et al. 2015). GDF9 is expressed in bovine oocytes (Vasconcelos et al. 2013)

and it has been used as marker to identify oocytes differentiated from stem cells cultured in vitro

(Linher et al. 2009). Both BMP2 and BMP4, as well as follicular fluid are currently found to

stimulate expression of ZPA in OLCs. The latter zona pellucida glycoprotein is only expressed in

oocytes (Lefièvre et al. 2004). Earlier studies reported ZP-like structures surrounding OLCs

differentiated in vitro (Hong et al. 2014). Singhal et al. (2015) recently showed that expression of

germ cell markers, like ZP1, ZP2, ZP3 and VASA, were stimulated by BMP4. In the present

study, BMP4 and BMP2 had no synergistic interaction to stimulate mRNA expression of genes

related to germ cells and oocytes. This is probably due to binding of these BMPs to the same

receptor, i.e., the BMP type II receptor.

In this study, it can be emphasized that follicular fluid stimulates the expression of

markers for both PGCs and oocytes, while most of these markers are also activated by BMPs.

Various studies have previously shown that BMP4 promotes oogonial stem cell differentiation in

human (Park et al. 2013), buffalo (Shah et al. 2015) and goat (Singhal et al. 2015). BMP2 also

158

plays a crucial role in the differentiation of primordial germ cells (Pera et al. 2004) and influence

the expression of oocyte-specific genes, like GDF9 in bovine follicles (Rossi et al. 2016).

Follicular fluid contains numerous bioactive factors secreted from granulosa cells, theca

cells, and oocytes and play important roles in regulating folliculogenesis and oogenesis (Jiang et

al. 2003; Webb et al. 2003). As oocytes secrete soluble paracrine growth factors that can regulate

granulosa cell development, and granulosa cells in turn regulate oocyte growth during follicle

formation (Van den Hurk and Zhao, 2005), it is apparent that the biochemical substances from

granulosa cells may play a key role in the initiation of germ cell formation and oocyte

development. The inducing role of follicular fluid in the formation of germ cells from stem cells

was demonstrated in swine species (Cheng et al. 2012; Linher et al. 2009). Dyce et al. (2011)

showed that follicular fluid induces the expression of markers of germ cells (DAZL and VASA),

meiosis (SCP3) and oocyte (GDF9, ZP1, ZP2, ZP3).

Conclusions

OLCs can be differentiated in vitro from ovarian stem cells, while BMPs and follicular

fluid are effective in stimulating the expression of mRNAs for germ cell and oocyte markers.

This study opens new possibilities for in-vitro production of bovine oocytes from ovarian stem

cells, which can have a positive impact on the production of embryos from genetically superior

animals or endangered species.

Acknowledgements

159

This work was financially supported by CNPq (Grant N° 478198/2013-2), and the

authors thank the members of the Laboratory of Animal Reproduction of the Biotechnology

Nucleus of Sobral.

Conflict of interest

None of the authors have any conflict of interest to declare.

References

Bukovsky A, Svetlikova M, Caudle MR, 2005: Oogenesis in cultures derived from adult human

ovaries. Reprod Biol Endocrinol 3, 17.

Castrillon DH, Miao L, Kollipara R, Horner JW, Depinho RA, 2003: Suppression of ovarian

follicle activation in mice by the transcription factor Foxo3a. Science 11, 301(5630), 215-8.

Chaves RN, Martins FS, Saraiva MVA, Celestino JJH, Lopes CAP, Correia JC, Lima-Verde IB,

Matos MHT, Báo SN, Name KPO, Campello CC, Silva JRV, Figueiredo JR, 2008: Chilling

ovarian fragments during transportation improves viability and growth of goat preantral follicles

cultured in vitro. Reprod Fert Dev 20, (5), 640-647.

Cheng X, Chen S, Yu X, Zheng P, Wang H, 2012: BMP15 gene is activated during human

amniotic fluid stem cell differentiation into oocyte-like cells. DNA Cell Biol 31, 1198-1204.

Clark AT, Bodnar MS, Fox M, Rodriquez RT, Abeyta MJ, Firpo MT, Pera RA, 2004:

Spontaneous differentiation of germ cells from human embryonic stem cells in vitro. Hum Mol

Genet 13, (7), 727–739.

160

Dyce PW, Shen W, Huynh E, Shao H, Villagómez DA, Kidder GM, King WA, Li J, 2011:

Analysis of oocyte-like cells differentiated from porcine fetal skin-derived stem cells. Stem Cells

Dev 20, (5), 809-819.

Garor R, Abir R, Erman A, Felz C, Nitke S, Fisch B, 2009: Effects of basic fibroblast growth

factor on in vitro development of human ovarian primordial follicles. Fertil Steril 91, (5), 1967-

1975.

Hong J, Jin H, Han J, Hu H, Liu J, Li L, Huang Y, Wang D, Wu M, Qiu L, Qian Q, 2014:

Infusion of human umbilical cord-derived mesenchymal stem cells effectively relieves liver

cirrhosis in DEN-induced rats. Mol Med Rep 9, (4), 1103-1111.

Hübner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, Wood J,

Strauss JF, Boiani M, Scholer HR, 2003: Derivation of oocytes from mouse embryonic stem

cells. Science 300, (5623), 1251–1256.

Jiang Y, Henderson D, Blackstad M, Chen A, Miller RF, Verfaillie CM, 2003: Neuroectodermal

differentiation from mouse multipotent adult progenitor cells. Proc Natl Acad Sci USA 100, (1),

11854-11860.

Johnson J, Bagley J, Skaznik-Wikiel M, Lee HJ, Adams GB, Niikura Y, Tschudy KS, Tilly, JC,

Cortes ML, Forkert R, Spitzer T, Iacomini J, Scadden DT, Tilly JL, 2005: Oocyte generation in

adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood. Cell 122,

(2), 303-315.

Kee K, Angeles VT, Flores M, Nguyen HN, Reijo Pera RA, 2009: Human DAZL, DAZ and

BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature 462, 222–

225.

Kee K, Gonsalves JM, Clark AT, Pera RA, 2006: Bone morphogenetic proteins induce germ cell

differentiation from human embryonic stem cells. Stem Cells and Development 15, (6), 831–837.

161

Lacham-Kaplan O, Chy H, Trounson A, 2006: Testicular cell conditioned medium supports

differentiation of embryonic stem cells into ovarian structures containing oocytes. Stem Cells 24,

(2), 266–273.

Lefièvre L, Conner SJ, Salpekar A, Olufowobi O, Ashton P, Pavlovic B, Lenton W, Afnan M,

Brewis IA, Monk M, Hughes DC, Barratt CL, 2004: Four zona pellucida glycoproteins are

expressed in the human. Hum Reprod 19, (7), 1580–1586.

Linher K, Dyce P, Li J, 2009: Primordial germ cell-like cells differentiated in vitro from skin-

derived stem cells. PLoS ONE 4, (12).

Livak KJ, Schmittgen TD, 2001: Analysis of Relative Gene Expression Data Using Real-Time

Quantitative PCR and the 2-ΔΔCT Method. Methods 25, 402–408.

Mohamed JS, Gaughwin PM, Lim B, Robson P, Ipovich L, 2010: Conserved long noncoding

RNAs transcriptionally regulated by Oct4 and Nanog modulate pluripotency in mouse embryonic

stem cells. RNA 16, (2), 324–337.

Mooyottu S, Anees S, Cherian S, 2011: Ovarian stem cells and neo-oogenesis: A breakthrough in

reproductive biology research. Vet World 4, (2), 89-91.

Nicholas CR, Chavez SH, Baker VL, Reijo Pera RA, 2009a: Instructing an embryonic stem cell-

derived oocyte fate: lessons from endogenous oogenesis. Endo Rev 30, (3), 264–283.

Niikura Y, Niikura T, Tilly JL, 2009: Aged mouse ovaries possess rare premeiotic germ cells that

can generate oocytes following transplantation into a young host environment. Aging (Albany

NY) 1, (12), 971-978.

Pan Z, Sun M, Li J, Zhou F, Liang X, Huang J, Zheng T, Zheng L, Zheng Y, 2015: The

Expression of Markers Related to Ovarian Germline Stem Cells in the Mouse Ovarian Surface

Epithelium and the Correlation with Notch Signaling Pathway. Cell Physiol Biochem 37, (6),

2311-2322.

162

Panula S, Medrano JV, Kee K, Bergström R, Nguyen HN, Byers B, Wilson KD, Wu JC, Simon

C, Hovatta O, Reijo Pera RA, 2011: Human germ cell differentiation from fetal- and adult-

derived induced pluripotent stem cells. Hum Mol Genet 20, (4), 752 – 762.

Park ES, Woods DC, Tilly JL, 2013: Bone morphogenetic protein 4 promotes mammalian

oogonial stem cell differentiation via Smad1/5/8 signaling. Fertil Steril 100, (5), 1468-1475.

Parte S, Bhartiya D, Telang J, Daithankar V, Salvi V, Zaveri K, Hinduja I, 2011: Detection,

characterization, and spontaneous differentiation in vitro of very small embryonic-like putative

stem cells in adult mammalian ovary. Stem Cells Dev 20, (8), 1451-1464.

Pera MF, Trounson AO, 2004: Human embryonic stem cells: prospects for development.

Development 131, (22), 5515-5525.

Qing T, ShiY, Qin H, Ye X, Wei W, Liu H, Ding M, Deng H, 2007: Induction of oocyte-like

cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells. Differentiation

75, (10), 902–911.

Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, Robson P, 2005: Transcriptional

regulation of nanog by OCT4 and SOX2. J Biol Chem 280, (26), 24731-24737.

Rossi RODS, Cunha EV, Portela AMLR, Passos JRS, Costa JJN, Silva AWB, Saraiva MVA,

Peixoto CA, Donato MAM, Hurk R Van den, Silva JRV, 2016: Influence of BMP-2 on early

follicular development and mRNA expression of oocyte specific genes in bovine preantral

follicles cultured in vitro. Histol Histopathol 31, 339-348.

Saitou M, Yamaji M, 2010: Germ cell specification in mice: Signaling, transcription regulation,

and epigenetic consequences. Reproduction 139, 931-942.

Shah SM, Saini N, Ashraf S, Singh MK, Manik R, Singla SK, Palta P, Chauhan MS, 2015:

Development of buffalo (Bubalus bubalis) embryonic stem cell lines from somatic cell nuclear

transferred blastocysts. Stem Cell Res 15, (3), 633-639.

http://www.ncbi.nlm.nih.gov/pubmed/21291304



http://www.ncbi.nlm.nih.gov/pubmed/?term=Rodda%20DJ%5BAuthor%5D&cauthor=true&cauthor_uid=15860457

http://www.ncbi.nlm.nih.gov/pubmed/?term=Chew%20JL%5BAuthor%5D&cauthor=true&cauthor_uid=15860457

http://www.ncbi.nlm.nih.gov/pubmed/?term=Lim%20LH%5BAuthor%5D&cauthor=true&cauthor_uid=15860457

http://www.ncbi.nlm.nih.gov/pubmed/?term=Loh%20YH%5BAuthor%5D&cauthor=true&cauthor_uid=15860457

http://www.ncbi.nlm.nih.gov/pubmed/?term=Wang%20B%5BAuthor%5D&cauthor=true&cauthor_uid=15860457

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ng%20HH%5BAuthor%5D&cauthor=true&cauthor_uid=15860457

http://www.ncbi.nlm.nih.gov/pubmed/?term=Robson%20P%5BAuthor%5D&cauthor=true&cauthor_uid=15860457


http://lattes.cnpq.br/5393329585491839

163

Shi W, Wang H, Pan G, Geng Y, Guo Y, Pei D, 2006: Regulation of the pluripotency marker

Rex-1 by Nanog and Sox2. J Biol Chem 18, (281), 23319-23325.

Singhal DK, Singhal R, Malik HN, Singh S, Kumar S, Kaushik JK, Mohanty AK, Malakar D,

2015: Generation of Germ Cell-Like Cells and Oocyte-Like Cells from Goat Induced Pluripotent

Stem Cells. J Stem Cell Res Ther 5:5.

Štefková K, Procházková J, Pacherník J, 2015: Alkaline Phosphatase in Stem Cells. Stem Cells

Int 2015 (2015), 11, http://dx.doi.org/10.1155/2015/628368

Takahashi K, Yamanaka S, 2006: Induction of pluripotent stem cells from mouse embryonic and

adult fibroblast cultures by defined factors. Cell 126, 663-676.

Takai H, Smogorzewska A, De Lange T, 2003: DNA damage foci at dysfunctional telomeres.

Curr Biol 13, (17), 1549–1556.

Van den Hurk R, Zhao J, 2005: Formation of mammalian oocytes and their growth,

differentiation and maturation within ovarian follicles. Theriogenology 63, 1717–1751.

Vasconcelos GL, Saraiva MV, Costa JJ, Passos MJ, Silva AW, Rossi RO, Portela AML, Duarte

AB, Magalhães-Padilha DM, Campelo CC, Figueiredo JR,Van den Hurk R, Silva JRV, 2013:

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA

expression in bovine preantral follicles. Reprod Fertil Develop 25, (8), 1194-1203.

Webb R, Nicholas B, Gong JG, Campbell BK, Gutierrez CG, Garverick HA, Armstrong DG,

2003: Mechanisms regulating follicular development and selection of the dominant follicle.

Reprod Suppl 61, 71-90.

Wei W, Qing T, Ye X, Liu H, Zhang D, Yang W, Deng H, 2008: Primordial germcell

specification from embryonic stem cells. PLoS One 3, 12.

http://www.hindawi.com/84219129/



http://dx.doi.org/10.1155/2015/628368

164

White YAR, Woods DC, Takai Y, Ishihara O, Seki H, Tilly JL, 2012: Oocyte formation by

mitotically active germ cells purified from ovaries of reproductive-age women. Nat Med 18, 413-

421.



165

List of tables and figures

Table 1. Primer pairs used in real-time PCR.

Figure 1. Fluorescence staining of ovarian stem cells before (A-B) and after 14 days culture in

minimum essential medium (α-MEM) (C-D) or supplemented with BMP2 (E-F), BMP4 (G-H),

both BMP2 and BMP4 (I-J) and follicular fluid (K-L). Scale bar = 100 µm. Green (calcein)

staining cells are viable and red (ethidium homodimer) staining cells are not.

Figure 2. Alkaline phosphatase activity in ovarian stem cells at day 0. Scale bar = 100 µm.

Figure 3. Cycle threshold (CT) after amplification of mRNA for pluripotency stem cell markers

(OCT4 and SOX2) by real time RT-PCR.

Figure 4. Levels of mRNA for specific germline cell [VASA (A), DAZL (B)] and oocyte [SCP3

(C), GDF9 (D) and ZPA (E)] markers in ovarian stem cells cultured for 14 days in MEM

supplemented with BMP2, BMP4, BMP2 and BMP4 and follicular fluid. Significant differences

at P<0.05.

166

Table 1. Primer pairs used in real-time PCR.

Target

gene

Primer sequence (5´ 3´)

Sense (s),

anti-sense

(As)

GenBank number

GAPDH

CACCCTCAAGATTGTCAGCA

GGTCATAAGTCCCTCCACGA

S

As

NM_001034034.2

SOX2

TGGATCGGCCAGAAGAGGAG

CAGGCGAAGAATAATTTGGGGG

S

As

NM_001105463.2

OCT-4

GAGAAAGACGTGGTCCGAGTG

GACCCAGCAGCCTCAAAATC

S

As

NM_174580.2

VASA

TGGTCCTGGCTTCAGTGGTA

TCTTGCCGGGGTAATTCTTTCT

S

As

NM_001007819.1

DAZL

TACCCGCCTCTGACTCTCTC

GTGTTCACTCAGAGGGGCTC

S

As

EF501823.2

ZPA



S

As

NM_173973.2

GDF-9

ACAACACTGTTCGGCTCTTCACCC

CCACAACAGTAACACGATCCAGGTT

S

As

GI:51702523

SCP3

GTTGGCAAAACCATCCGTGG

GGGGTCTTCTCTTCAATGGCA

S

As

NM_001040588.2

s: sense; as: anti-sense.

167

Figure 1. Fluorescence staining of ovarian stem cells before (A-B) and after 14 days culture in

minimum essential medium (α-MEM) (C-D) or supplemented with BMP2 (E-F), BMP4 (G-H),

both BMP2 and BMP4 (I-J) and follicular fluid (K-L). Scale bar = 100 µm. Green (calcein)

staining cells are viable and red (ethidium homodimer) staining cells are not.

168

Figure 2. Alkaline phosphatase activity in ovarian stem cells at day 0. Scale bar = 100 µm.

Figure 3. Cycle threshold (CT) after amplification of mRNA for pluripotency stem cell markers

(OCT4 and SOX2) by real time qRT-PCR.

169

Figure 4. Levels of mRNA for specific germline cell [VASA (A), DAZL (B)] and oocyte [SCP3 (C), GDF9 (D) and ZPA (E)] markers

in ovarian stem cells cultured for 14 days in MEM supplemented with BMP2, BMP4, BMP2 and BMP4 and follicular fluid.

Significant differences at P<0.05.

170

10 CONCLUSÕES

O tratamento de fibroblastos com 2.0 μM de 5-aza-citidina por 72 h induz mudanças

na morfologia e na taxa de proliferação celular, e induz a expressão de RNAs mensageiros para

fatores de pluripotência (OCT-4, SOX2, NANOG e REX1).

O cultivo de fibroblastos, previamente tratados com 5-aza-citidina, em meio de

diferenciação suplementado com BMP-2, BMP-4 ou fluido folicular, durante 7 ou 14 dias,

promove alterações na morfologia celular e induz a expressão de RNAs mensageiros para para

marcadores de células germinativas (VASA, DAZL e C-KIT) e de oócitos (SCP3, GDF-9 e ZPA).

Células-tronco da linhagem germinativas, que expressam marcadores de

pluripotência (OCT-4 e SOX2) e fosfatase alcalina, podem ser isoladas eficientemente do epitélio

ovariano de bovinos, e podem ser utilizadas para a diferenciação em estruturas semelhantes a

oócitos.

O cultivo de células-tronco da linhagem germinativas em meio de diferenciação

suplementado com BMP-2, BMP-4 ou fluido folicular promove modificações na morfologia

celular e estimula a expressão de marcadores par células germinativas primordiais (VASA e

DAZL) e de oócitos (SCP3, GDF-9 e ZPA).

171

11 PERSPECTIVAS

Os resultados obtidos no presente trabalho poderão ser utilizados para o

desenvolvimento de novas tecnologias que podem revolucionar a reprodução assistida, tanto em

humanos, como em animais de alto padrão zootécnico. Nos últimos anos, a diferenciação de

células somáticas está associada com grandes perspectivas para o desenvolvimento da medicina

regenerativa. Adicionalmente, este trabalho contribuirá para a formação de gametas a partir de

células somáticas, abrindo uma nova perspectiva de recuperação de espécies em via de extinção.

A reprogramação celular e a presença de células-tronco mitoticamente ativas no

epitélio da superfície ovariana podem viabilizar a formação de uma quantidade de oócitos

suficientes para iniciar um novo capítulo na biotecnologia e na medicina reprodutiva. Com base

na disponibilidade dessas novas tecnologias no campo da medicina reprodutiva, os resultados

deste trabalho poderão contribuir para a solução dos problemas relacionados com a infertilidade

no futuro.

172

REFERÊNCIAS

ABIR, R.; NITKE, S.; BEN-HAROUSH, A.; FISCH, B. In vitro maturation of human primordial

ovarian follicles: Clinical significance, progress in mammals, and methods for growth evaluation.

Histology and Histopathology, v. 21, n. 8, p. 887-898, 2006.

ADAMS, G. P.; JAISWAL, R.; SINGH, J.; MALHI, P. Progress in understanding ovarian

follicular dynamics in cattle. Theriogenology, v. 69, n. 1, p. 72-80, 2008.

AFLATOONIAN, B.; RUBAN, L.; JONES, M.; AFLATOONIAN, R.; FAZELI, A.; MOORE,

H. D. In vitro post-meiotic germ cell development from human embryonic stem cells. Human

Reproduction, v. 24, p. 3150-3159, 2009.

ALBERTINI, D. F.; COMBELLES, C. M.; BENECCHI, E.; CARABATSOS, M. J. Cellular

basis for paracrine regulation of ovarian follicle development. Reproduction, v. 121, n. 5, p.

647-653, 2001.

ALMUTAWAA, W.; KANG, N. H.; PAN, Y.; NILES, L. P. Induction of Neurotrophic and

Differentiation Factors in Neural Stem Cells by Valproic Acid. Basic & Clinical Pharmacology

& Toxicology, v. 115, n. 2, p. 216-221, 2014.

ANDERSON, R.; COPELAND, T. K.; SCHOLER, H.; HEASMAN, J.; WYLIE, C. The onset of

germ cell migration in the mouse embryo. Mechanisms of Development, v. 91, n. 1-2, p. 61-68,

2000.

ANDERSON, R.; FÄSSLER, R.; GEORGES-LABOUESSE, E.; HYNES, R. O.; BADER, B. L.;

KREIDBERG, J. A.; SCHAIBLE, K.; HEASMAN, J.; WYLIE, C. Mouse primordial germ cells

lacking 𝛽1 integrins enter the germline but fail to migrate normally to the gonads. Development, v. 126, n. 8, p. 1655-1664, 1999.

ANGELOS, M. G.; KAUFMAN, D. S. Pluripotent stem cell applications for regenerative

medicine. Current Opinion in Organ Transplantation, v.20, n. 6, p. 663-670, 2015.

ANOKYE-DANSO, F.; TRIVEDI, C. M.; JUHR, D.; GUPTA, M.; CUI, Z.; TIAN, Y.; ZHANG,

Y.; YANG, W.; GRUBER, P. J.; EPSTEIN, J. A.; MORRISEY, E. E. Highly efficient miRNA-

mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell, v.

8, p. 376-388, 2011.

ARAKI, Y. Formation and Structure of Mammalian Ovaries. In: TULSIANI, D. (Ed.)

Introduction to Mammalian Reproduction, 418p. NewYork: Springer, 2003, p 141-153.

AURICH, H.; SGODDA, M.; KALTWASSER, P.; VETTER, M.; WEISE, A.; LIEHR, T.;

BRULPORT, M.; HENGSTLER, J. G.; DOLLINGER, M. M.; FLEIG, W. E.; CHRIST, B.

Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro

promotes hepatic integration in vivo. Gut, v. 58, n. 4, p. 570-581, 2009.

173

BALEN, A. H. Infertility in practice. Fourth edition. London: CRC Press, 2011.

BALTUS, A. E.; MENKE, D. B.; HU, Y. C.; GOODHEART, M. L.; CARPENTER, A. E.; DE

ROOIJ, D. G.; PAGE, D. C. In germ cells of mouse embryonic ovaries, the decision to enter

meiosis precedes premeiotic DNA replication. Nature Genetics, v. 38, p. 1430-1434, 2006.

BARNETT, K. R.; SCHILLING, C. C. R.; GREENFELD, C. R.; TOMIC, D.; FLAWS, J. A.

Ovarian follicle development and transgenic mouse models. Human Reproduction, v.10, p. 1-

19, 2006.

BAYNE, R. A. L.; KINNELL, H. L.; COUTTS, S. M.; HE, J.; CHILDS, A. J.; ANDERSON, R.

A. GDF9 is Transiently Expressed in Oocytes before Follicle Formation in the Human Fetal

Ovary and is Regulated by a Novel NOBOX Transcript. PLoS One, v. 10, n. 3, e0119819, 2015.

BECK, B.; BLANPAIN, C. Mechanisms regulating epidermal stem cells. The EMBO Journal,

v. 31, p. 2067-2075, 2012.

BENDEL-STENZEL, M. R.; GOMPERTS, M.; ANDERSON, R.; HEASMAN, J.; WYLIE, C.

The role of cadherins during primordial germ cell migration and early gonad formation in the

mouse. Mechanisms of Development, v. 91, n. 1-2, p. 143-152, 2000.

BERTOLDO, M. J; NADAL-DESBARATS, L; GÉRARD, N; DUBOIS, A; HOLYOAKE, P. K;

GRUPEN, C. G. Differences in the metabolomic signatures of porcine follicular fluid collected

from environments associated with good and poor oocyte quality. Reproduction, v. 146, n. 3, p.

221-231, 2013.

BEYER NARDI, N.; DA SILVA MEIRELLES, L. Mesenchymal stem cells: isolation, in vitro

expansion and characterization. Handbook of Experimental Pharmacology, v. 174, p. 249-282,

2006.

BHARTIYA, D.; HINDUJA, I.; PATEL, H.; BHILAWADIKAR, R. Making gametes from

pluripotent stem cells – a promising role for very small embryonic-like stem cells. Reproductive

Biology and Endocrinology, v. 12, p. 114. 2014.

BHARTIYA, D.; KASIVISWANATHAN, S.; UNNI, S. K.; PETHE, P.; DHABALIA, J. V.;

PATWARDHAN, S.; TONGAONKAR, H. B. Newer insights into pre-meiotic development of

germ cells in adult human testis using Oct-4 as a stem cell marker. Journal of Histochemistry &

Cytochemistry, v. 58, p. 1093-1106, 2010.

BHARTIYA, D.; SRIRAMAN, K.; GUNJAL, P.; MODAK, H. Gonadotropin treatment

augments postnatal oogenesis and primordial follicle assembly in adult mouse ovaries? Journal

of Ovarian Research, v. 5, n. 1, p. 32, 2012.

BHARTIYA, D.; SRIRAMAN, K.; PARTE, S.; PATEL, H. Ovarian stem cells: absence of

evidence is not evidence of absence. Journal of Ovarian Research, v. 6, p. 1-6, 2013.

174

BIANCHI, G.; MURAGLIA, A.; DAGA, A.; CORTE, G.; CANCEDDA, R.; QUARTO, R.

Microenvironment and stem properties of bone marrow-derived mesenchymal cells. Wound

Repair and Regeneration, v. 9, n. 6, p. 460-466, 2001.

BOIANI, M.; SCHÖLER, H. R. Regulatory networks in embryo derived pluripotent stem cells.

Nature Reviews Molecular Cell Biology, v. 6, p. 872-884, 2005.

BOIVIN, J.; BUNTING, L.; COLLINS, J. A.; NYGREN, K. G. International estimates of

infertility prevalence and treatment-seeking: potential need and demand for infertility medical

care. Human Reproduction, v. 22, n. 6, p. 1506-1512, 2007.

BOWLES, J.; KNIGHT, D.; SMITH, C.; WILHELM, D.; RICHMAN, J. M.; MAMIYA, S.;

YASHIRO, K.; CHAWENGSAKSOPHAK, K.; WILSON, M. J.; ROSSANT, J. Sex-specific

regulation of retinoic acid levels in developing mouse gonads determines germ cell fate. Science,

v. 312, p. 596-600, 2006.

BOWLES, J.; KOOPMAN, P. Retinoic acid, meiosis and germ cell fate in mammals.

Development, v. 134, p. 3401-3411, 2007.

BOYER, L. A.; LEE, T. I.; COLE, M. F.; JOHNSTONE, S. E.; LEVINE, S. S.; ZUCKER, J. P.;

GUENTHER, M. G.; KUMAR, R. M.; MURRAY, H. L.; JENNER, R. G.; GIFFORD, D. K.;

MELTON, D. A.; JAENISCH, R.; YOUNG, R. A. Core transcriptional regulatory circuitry in

human embryonic stem cells. Cell, v. 122, p. 947-956, 2005.

BOYER, L. A.; PLATH, K.; ZEITLINGER, J.; BRAMBRINK, T.; MEDEIROS, L. A.; LEE, T.

I.; LEVINE, S. S.; WERNIG, M.; TAJONAR, A.; RAY, M. K.; BELL, G. W.; OTTE, A. P.;

VIDAL, M.; GIFFORD, D. K.; YOUNG, R. A.; JAENISCH, R. Polycomb complexes repress

developmental regulators in murine embryonic stem cells. Nature, v. 441, p. 349-353, 2006.

BRISTOL-GOULD, S. K.; KREEGER, P. K.; SELKIRK, C. G.; KILEN, S. M.; MAYO, K. E.;

SHEA, L. D.; WOODRUFF, T. K. Fate of the initial follicle pool: empirical and mathematical

evidence supporting its sufficiency for adult fertility. Developmental Biology, v. 298, p. 149-

154, 2006.

BUCAY, N.; YEBRA, M.; CIRULLI, V.; AFRIKANOVA, I.; KAIDO, T.; HAYEK, A.;

MONTGOMERY, A. M. A novel approach for the derivation of putative primordial germ cells

and Sertoli cells from human embryonic stem cells. Stem Cells, v. 27, p. 68-77, 2009.

BUKOVSKY, A. Ovarian stem cell niche and follicular renewal in mammals. The Anatomical

Record, v. 294, p. 1284-1306, 2011.

BUKOVSKY, A.; CAUDLE, M. R. Immunoregulation of follicular renewal, selection, POF, and

menopause in vivo, vs. neo-oogenesis in vitro, POF and ovarian infertility treatment, and a

clinical trial. Reproductive Biology and Endocrinology, v. 10, p. 97, 2012.

175

BUKOVSKY, A.; CAUDLE, M. R.; SVETLIKOVA, M.; WIMALASENA, J.; AYALA, M. E.;

DOMINGUEZ, R. Oogenesis in adult mammals, including humans: a review. Endocrine, v. 26,

n. 3, p. 301-316, 2005.

BUKOVSKY, A.; GUPTA, S. K.; VIRANT-KLUN, I.; UPADHYAYA, N. B.; COPAS, P.; VAN

METER, S. E.; SVETLIKOVA, M.; AYALA, M. E.; DOMINGUEZ, R. Study origin of germ

cells and formation of new primary follicles in adult human and rat ovaries. Methods in

Molecular Biology, v. 450, p. 233-265, 2008.

BYDLOWSKI, S. P.; DEBES, A. A.; MASELLI, L. M. F.; JANZ, F. L. Características

biológicas das células-tronco mesenquimais [Biological characteristics of the mesenchymal stem

cells]. Revista Brasileira de Hematologia e Hemoterapia, v. 31, p. 25-35, 2009.

CAI, L.; JOHNSTONE, B. H.; COOK, T. G.; TAN, J.; FISHBEIN, M. C.; CHEN, P. S.;

MARCH, K. L. IFATS collection: Human adipose tissue-derived stem cells induce angiogenesis

and nerve sprouting following myocardial infarction, in conjunction with potent preservation of

cardiac function. Stem Cells, v. 27, p. 230-237, 2009.

CAI, L.; ROTHBART, S. B.; LU, R.; XU, B.; CHEN, W-Y.; TRIPATHY, A.; ROCKOWITZ,

S.; ZHENG, D.; PATEL, D. J.; ALLIS, C. D.; STRAHL, B. D.; SONG, J.; WANG, G. G. An

H3K36 methylation-engaging Tudor motif of polycomb-like proteins mediates PRC2 complex

targeting. Molecular Cell, v. 49, p. 571-582, 2013.

CANDIDO, E. P. M.; REEVES, R.; DAVIE, J. R. Sodium butyrate inhibits histone deacetylation

in cultured cells. Cell, v. 14, p. 105-113, 1978.

CARMO, D. D. D.; SANTOS JÚNIOR, A. R. Aplicação clínica de células-tronco adultas. In:

SIMPÓSIO DE INICIAÇÃO CIENTÍFICA DA UNIVERSIDADE FEDERAL DO ABC, 2009,

Santo André. Anais do Simpósio de Iniciação Científica Da Universidade Federal do ABC.

Santo André: UFABC, 2009.

CARTER, A. C.; DAVIS-DUSENBERY, B. N.; KOSZKA, K.; ICHIDA, J. K.; EGGAN, K.

Nanog-Independent Reprogramming to iPSCs with Canonical Factors. Stem Cell Reports, n. 2,

p. 119-126, 2014.

CASTRILLON, D. H.; MIAO, L.; KOLLIPARA, R.; HORNER, J. W.; DEPINHO, R. A.

Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science, v.

301, n. 5630, p. 215-218, 2003.

CASTRILLON, D. H.; QUADE, B. J.; WANG, T. Y.; QUIGLEY, C.; CRUM, C. P. The human

VASA gene is specifically expressed in the germ cell lineage. Proceedings of the National

Academy of Sciences of the United States of America, v. 97, n. 17, p. 9585-9590, 2000.

CEDAR, H.; BERGMAN, Y. Linking DNA methylation and histone modification: patterns and

paradigms. Nature Reviews Genetics, v. 10, p. 295-304, 2009.

176

CHANG, H.; MATZUK, M. M. Smad5 is required for mouse primordial germ cell development.

Mechanisms of Development, v. 104, n. 61-67, 2001.

CHAVES, R. N.; MARTINS, F. S.; SARAIVA, M. V. A.; CELESTINO, J. J. H.; LOPES, C. A.

P.; CORREIA, J. C.; LIMA-VERDE, I. B.; MATOS, M. H. T.; BÁO, S. N.; NAME, K. P. O.;

CAMPELLO, C. C.; SILVA, J. R. V.; FIGUEIREDO, J. R. Chilling ovarian fragments during

transportation improves viability and growth of goat preantral follicles cultured in vitro.

Reproduction, Fertility and Development, v. 20, n. 5, p. 640-647, 2008.

CHEN, A. E.; EGLI, D.; NIAKAN, K.; DENG, J.; AKUTSU, H.; YAMAKI, M.; COWAN, C.;

FITZ-GERALD, C.; ZHANG, K.; MELTON, D. A.; EGGAN, K. Optimal timing of inner cell

mass isolation increases the efficiency of human embryonic stem cell derivation and allows

generation of sibling cell lines. Cell Stem Cell, v. 4, p. 103-106, 2009.

CHEN, H. F.; KUO, H. C.; CHIEN, C. L.; SHUN, C. T.; YAO, Y. L.; IP, P. L.; CHUANG, C.

Y.; WANG, C. C.; YANG, Y. S.; HO, H. N. Derivation, characterization and differentiation of

human embryonic stem cells: comparing serum-containing versus serum-free media and evidence

of germ cell differentiation. Human Reproduction, v. 22, p. 567-577, 2007.

CHEN, H-H.; WELLING, M.; BLOCH, D. B.; MUÑOZ, J.; MIENTJES, E.; CHEN, X.;

TRAMP, C.; WU, J.; YABUUCHI, A.; CHOU, Y-F.; BUECKER, C.; KRAINER, A.;

WILLEMSEN, R.; HECK, A. J.; GEIJSEN, N. DAZL limits pluripotency, differentiation, and

apoptosis in developing primordial germ cells. Stem Cell Reports, v. 3, n. 5, p. 892-904, 2014.

CHEN, S-R.; ZHENG, Q-S.; ZHANG, Y.; GAO, F.; LIU, Y-X. Disruption of genital ridge

development causes aberrante primordial germ cell proliferation but does not affect their

directional migration. BMC Biology, v. 11, n. 22, 2013.

CHENG, X.; CHEN, S.; YU, X.; ZHENG, P.; WANG, H. BMP15 gene is activated during

human amniotic fluid stem cell differentiation into oocyte-like cells. DNA and Cell Biology, v.

31, p. 1198-1204, 2012.

CHERUBINO, M.; RUBIN, J. P.; MILJKOVIC, N.; KELMENDI-DOKO, A.; MARRA, K. G.

Adipose-derived stem cells for wound healing applications. Annals of Plastic Surgery, v. 66, n.

2, p. 210-215, 2011.

CHILDS, A. J.; KINNELL, H. L.; COLLINS, C. S.; HOGG, K.; BAYNE, R. A.; GREEN, S. J.;

MCNEILLY, A. S.; ANDERSON, R. A. BMP signaling in the human fetal ovary is

developmentally regulated and promotes primordial germ cell apoptosis. Stem Cells, v. 28, n. 8,

p. 1368-1378, 2010.

CHIN, M. H.; MASON, M. J.; XIE, W.; VOLINIA, S.; SINGER, M.; PETERSON, C.;

AMBARTSUMYAN, G.; AIMIUWU, O.; RICHTER, L.; ZHANG, J.; KHVOROSTOV, I.;

OTT, V.; GRUNSTEIN, M.; LAVON, N.; BENVENISTY, N.; CROCE, C. M.; CLARK, A. T.;

BAXTER, T.; PYLE, A. D.; TEITELL, M. A.; PELEGRINI, M.; PLATH, K.; LOWRY, W. E.

Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression

signatures. Cell Stem Cell, v. 5, p. 111-123, 2009.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Eggan%20K%5BAuthor%5D&cauthor=true&cauthor_uid=19200798

177

CHIN, M. H.; PELLEGRINI, M.; PLATH, K.; LOWRY, W. E. Molecular analyses of human

induced pluripotent stem cells and embryonic stem cells. Cell Stem Cell, v. 7, p. 263-269, 2010.

CHIQUOINE, D. The identification, origin, and migration of the primordial germ cells in the

mouse embryo. The Anatomical Record, v. 118, n. 2, p. 135-146, 1954.

CHIRPUTKAR, R.; VAIDYA, A. Understanding Infertility and the Potential Role of Stem Cells

in Infertility Treatment: A Short Communication. International Journal of Reproduction,

Fertility & Sexual Health, v. 2, n. 1, p. 37-40, 2015.

CHUANG, C. Y.; LIN, K. I.; HSIAO, M.; STONE, L.; CHEN, H. F.; HUANG, Y. H. Meiotic

competent human germ cell-like cells derived from human embryonic stem cells induced by

BMP4/WNT3A signaling and OCT4/EpCAM (epithelial cell adhesion molecule) selection. The

Journal of Biological Chemistry, v. 287, p. 14389-14401, 2012.

CHUMA, S.; KANATSU-SHINOHARA, M.; INOUE, K.; OGONUKI, N.; MIKI, H.;

TOYOKUNI, S.; HOSOKAWA, M.; NAKATSUJI, N.; OGURA, A.; SHINOHARA, T.

Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal

mouse testis. Development, v. 132, n. 1, p. 117-122, 2005.

CHUNG, Y.; KLIMANSKAYA, I.; BECKER, S.; MARH, J.; LU, S. J.; JOHNSON, J.;

MEISNER, L.; LANZA, R. Embryonic and extraembryonic stem cell lines derived from single

mouse blastomeres. Nature, v. 439, p. 216-219, 2006.

CHRISTMAN, J. K. 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA

methylation: mechanistic studies and their implications for cancer therapy. Oncogene, v. 21, n.

35, p. 5483-5491, 2002.

CLARK, A. T.; BODNAR, M. S.; FOX, M.; RODRIQUEZ, R. T.; ABEYTA, M. J.; FIRPO, M.

T.; PERA R. A. Spontaneous differentiation of germ cells from human embryonic stem cells in

vitro. Human Molecular Genetics, v. 13, n. 7, p. 727-739, 2004a.

CLARK, A. T.; RODRIGUEZ, R. T.; BODNAR, M. S.; ABEYTA, M. J.; CEDARS, M. I.;

TUREK, P. J.; FIRPO, M. T.; REIJO PERA, R. A. Human STELLAR, NANOG, and GDF3

genes are expressed in pluripotent cells and map to chromosome 12p13, a hotspot for

teratocarcinoma. Stem Cells. v. 22, n. 2, p. 169-179, 2004b.

CLARKE, H. G.; HOPE, S. A.; BYERS, S.; RODGERS, R. J. Formation of ovarian follicular

fluid may be due to the osmotic potential of large glycosaminoglycans and proteoglycans.

Reproduction, v. 132, n. 1, p. 119-131, 2006.

CLERMONT, Y.; LEBLOND, C. P. Definition of the stages of the cycle of the seminiferous

epithelium in the rat. Annals of the New York Academy of Sciences, v. 55, p. 548-573, 1952.

CLERMONT, Y.; LEBLOND, C. P. Renewal of spermatogonia in the rat. The American

Journal of Anatomy, v. 93, p. 475-501, 1953.

178

CONSTANTINIDES, P. G.; JONES, P. A.; GEVERS, W. Functional striated muscle cells from

non-myoblast precursors following 5-azacytidine treatment. Nature, v. 267, n. 5609, p. 364-366,

1977.

COOKE, H. J.; LEE, M.; KERR, S.; RUGGIU, M. A murine homologue of the human DAZ gene

is autosomal and expressed only in male and female gonads. Human Molecular Genetics, v. 5,

n. 4, p. 513-516, 1996.

COOPER, G.M. Expression and Function of c-MOS in Mammalian Germ Cells. Advances in

Developmental Biochemistry, v. 3, p. 127-148, 1994.

COVELLO, K. L.; KEHLER, J.; YU, H.; GORDAN, J. D.; ARSHAM, A. M.; HU, C. J.;

LABOSKY, P. A.; SIMON, M. C.; KEITH, B. HIF-2a regulates Oct-4: Effects of hypoxia

on stem cell function, embryonic development, and tumor growth. Genes & Development, v. 20,

p. 557-570, 2006.

CHRISTOFOROU, N.; LIAU, B.; CHAKRABORTY, S.; CHELLAPAN, M.; BURSAC, N.;

LEONG, K. W. Induced pluripotent stem cell-derived cardiac progenitors differentiate to

cardiomyocytes and form biosynthetic tissues. PLoS One, v. 8, n. 6, e65963, 2013.

DA SILVA MEIRELLES, L.; CAPLAN, A. I.; NARDI, N. B. In search of the in vivo identity of

mesenchymal stem cells. Stem Cells, v. 26, p. 2287-2299, 2008.

DE BARI, C.; DELL'ACCIO, F.; TYLZANOWSKI, P.; LUYTEN, F. P. Multipotent

mesenchymal stem cells from adult human synovial membrane. Arthritis & Rheumatology, v.

44, n. 8, p. 1928-1942, 2001.

DE PAEPE, C.; KRIVEGA, M.; CAUFFMAN, G.; GEENS, M.; VAN DE VELDE, H.

Totipotency and lineage segregation in the human embryo. Molecular Human Reproduction, v.

20, n. 7, p. 599-618, 2014.

DE SOUSA LOPES, S. M.; ROELEN, B. A.; MONTEIRO, R. M.; EMMENS, R.; LIN, H. Y.;

LI, E.; LAWSON, K. A.; MUMMERY, C. L. BMP signaling mediated by ALK2 in the visceral

endoderm is necessary for the generation of primordial germ cells in the mouse embryo. Genes

and Devolopment, v. 18, n. 15, p. 1838-1849, 2004.

DE SOUSA LOPES, S. M.; HAYASHI, K.; SURANI, M. A. Proximal visceral endoderm and

extraembryonic ectoderm regulate the formation of primordial germ cell precursors. BMC

Developmental Biology, v. 7, p. 140, 2007.

DEY, D.; EVANS, G. R. Generation of induced pluripotent stem (iPS) cells by nuclear

reprogramming. Stem Cells International, v. 2011, 619583, 2011.

DIECKE, S.; JUNG, S. M.; LEE, J.; JU, J. H. Recent technological updates and clinical

applications of induced pluripotent stem cells. The Korean Journal of Internal Medicine, v.

29, n. 5, p. 547-557, 2014.

179

DOKMANOVIC, M.; CLARKE, C.; MARKS, P. A. Histone deacetylase inhibitors: overview

and perspectives. Molecular Cancer Research, v. 5, p. 981-989, 2007.

DU, Y.; ROH, D. S.; FUNDERBURGH, M. L, MANN, M. M.; MARRA, K. G.; RUBIN, J. P.;

LI, X.; FUNDERBURGH, J. L. Adipose-derived stem cells differentiate to keratocytes in vitro.

Molecular Vision, v. 16, p. 2680-2689, 2010.

DUDLEY, B. M.; RUNYAN, C.; TAKEUCHI, Y.; SCHAIBLE, K.; MOLYNEAUX, K. BMP

signaling regulates PGC numbers and motility in organ culture. Mechanisms of Development, v.

124, p. 68-77, 2007.

DUNLOP, C. E.; TELFER, E. E.; ANDERSON, R. A. Ovarian germline stem cells. Stem Cell

Research and Therapy. v. 5, n. 98, p. 10.1186, 2014.

DYCE, P. W.; LI, D.; BARR, K. J.; KIDDER, G. M. Connexin43 is required for the maintenance

of multipotency in skin-derived stem cells. Stem cells and development, v. 23, n. 14, p. 1636-

1646, 2014.

DYCE, P. W.; LIU, J.; TAYADE, C.; KIDDER, G. M.; BETTS, D. H.; LI, J. In Vitro and In

Vivo Germ Line Potential of Stem Cells Derived from Newborn Mouse Skin. PLoS ONE, v. 6,

n. 5, e20339, 2011.

DYCE, P. W.; SHEN, W.; HUYNH, E.; SHAO, H.; VILLAGÓMEZ, D. A.; KIDDER, G. M.;

KING, W. A.; LI, J. Analysis of oocyte-like cells differentiated from porcine fetal skin-derived

stem cells. Stem Cells and Development, v. 20, n. 5, p. 809-819, 2011.

DYCE, P. W.; WEN, L.; LI, J. In vitro germline potential of stem cells derived from fetal porcine

skin. Nature Cell Biology, v. 8, p. 384-390, 2006.

DYCE, P. W.; ZHU, H.; CRAIG, J.; LI, J. Stem cells with multilineage potential derived from

porcine skin. Biochemical and Biophysical Research Communications, v. 316, n, 3, p. 651–

658, 2004.

EDSON, M. A.; NAGARAJA, A. K.; MATZUK, M. M. The mammalian ovary from genesis to

revelation. Endocrine Reveviews, v. 30, n. 6, p. 624-712, 2009.

EGUIZABAL, C.; MONTSERRAT, N.; VASSENA, R.; BARRAGAN, M.; GARRETA, E.;

GARCIA-QUEVEDO, L.; VIDAL, F.; GIORGETTI, A.; VEIGA, A. IZPISUA BELMONTE, J.

C. Complete meiosis from human induced pluripotent stem cells. Stem Cells, v. 29, n. 8, p. 1186-

1195, 2011.

EL-MESTRAH, M.; CASTLE, P.E.; BOROSSA, G.; KAN, F. W. Subcellular distribution of

ZP1, ZP2 and ZP3 glycoproteins during folliculogenesis and demonstration of their topographical

disposition within the zona matrix of mouse ovarian oocytes. Biology of reproduction, v. 66, n.

4, p. 866-876, 2002.

180

ENJOJI, M.; NAKASHIMA, M.; HONDA, M.; SAKAI, H.; NAWATA, H. Hepatocytic

phenotypes induced in sarcomatous cholangiocarcinoma cells treated with 5-azacytidine.

Hepatology, v. 26, n. 2, p. 288-294, 1997.

EVANS, M. J.; KAUFMAN, M. H. Establishment in culture of pluripotential cells from mouse

embryos. Nature, v. 292, n. 5819, p. 154-156, 1981.

FAIR, T. Follicular oocyte growth and acquisition of developmental competence. Animal

Reproduction Science, v. 78, p. 203-216, 2003.

FEDERATION, A. J.; BRADNER, J. E.; MEISSNER, A. The use of small molecules in somatic-

cell reprogramming. Trends in Cell Biology, v. 24, n. 3, p. 179-187, 2014.

FEKI, A.; BOSMAN, A.; DUBUISSON, J. B.; IRION, O.; DAHOUN, S.; PELTE, M. F.;

HOVATTA, O.; JACONI, M. E. Derivation of the first Swiss human embryonic stem cell line

from a single blastomere of an arrested four-cell stage embryo. Swiss Medical Weekly, v. 138, p.

540-550, 2008.

FIGUEIREDO, J. R.; MOLENTO, C. F. M. Bioética e bem-estar animal aplicados às biotécnicas

reprodutivas. In: Biotécnicas aplicadas à reprodução animal. 2 ed. São Paulo: Roca, 2008. p.

1-16.

FILIOLI URANIO, M.; VALENTINI, L.; LANGE-CONSIGLIO, A.; CAIRA, M.; GUARICCI,

A. C.; L'ABBATE, A.; CATACCHIO, C. R.; VENTURA, M.; CREMONESI, F.;

DELL'AQUILA, M. E. Isolation, proliferation, cytogenetic, and molecular characterization and

in vitro differentiation potency of canine stem cells from foetal adnexa: A comparative study of

amniotic fluid, amnion, and umbilical cord matrix. Molecular Reproduction and Development,

v. 78, n. 5, p. 361-373, 2011.

FORTUNE, J. E. Ovarian follicular growth and development in mammals. Biology of

Reproduction, v. 50, n. 2, p. 225-232, 1994.

FUCHS, E.; CHEN, T. A matter of life and death: self-renewal in stem cells. EMBO Reports, v.

14, p. 39-48, 2013.

GALAN, A.; DIAZ-GIMENO, P.; POO, M. E.; VALBUENA, D.; SANCHEZ, E.; RUIZ, V.;

DOPAZO, J.; MONTANER, D.; CONESA, A.; SIMON, C. Defining the genomic signature of

totipotency and pluripotency during early human development. PLoS One, 8:e62135, 2013.

GAROR, R.; ABIR, R.; ERMAN, A.; FELZ, C.; NITKE, S.; FISCH, B. Effects of basic

fibroblast growth factor on in vitro development of human ovarian primordial follicles. Fertility

and Sterility, v. 91, n 5, p. 1967-1975, 2009.

GARTNER, L. P.; HIATT, J. L. Sistema Reprodutor Feminino. In: GARTNER L. P.; HIATT, J.

L. Tratado de Histologia em Cores, 3 ed. 576p. Rio de Janeiro: Elsevier Brasil, 2007, p. 469-

494.

181

GEENS, M.; MATEIZEL, I.; SERMON, K.; DE RYCKE, M.; SPITS, C.; CAUFFMAN, G.;

DEVROEY, P.; TOURNAYE, H.; LIEBAERS, I.; VAN DE VELDE, H. Human embryonic stem

cell lines derived from single blastomeres of two 4-cell stage embryos. Human Reproduction, v.

24, p. 2709-2717, 2009.

GEIJSEN, N.; HOROSCHAK, M.; KIM, K.; GRIBNAU, J.; EGGAN, K.; DALEY, G. Q.

Derivation of embryonic germ cells and male gametes from embryonic stemcells. Nature, v. 427,

p. 148-154, 2004.

GERAMI-NAINI, B.; DOVZHENKO, O. V.; DURNING, M.; WEGNER, F. H.; THOMSON, J.

A.; GOLOS, T. G. Trophoblast differentiation in embryoid bodies derived from human

embryonic stem cells. Endocrinology, v. 145, p. 1517-1524, 2004.

GHEORGHISAN-GALATEANU, A.A., HINESCU, M.E; ENCIU, A.M. Ovarian adult stem

cells: hope or pitfall? Journal of ovarian research, v. 7, n. 1, p. 1, 2014.

GILL, M. E.; HU, Y-C.; LIN, Y.; PAGE, D. C. Licensing of gametogenesis, dependent on RNA

binding protein DAZL, as a gateway to sexual differentiation of fetal germcells. Proceedings

of the National Academy of Sciences of the United States of America, v. 108, n. 18, p. 7443-

7448, 2011.

GINSBURG, M.; SNOW, M. H.; McLAREN, A. Primordial germ cells in the mouse embryo

during gastrulation. Development, v. 110, n. 2, p. 521-528, 1990.

GIRITHARAN, G.; ILIC, D.; GORMLEY, M.; KRTOLICA, A. Human embryonic stem cells

derived from embryos at different stages of development share similar transcription profiles.

PLoS One, 6:e26570, 2011.

GILCHRIST, R. B.; RITTER, L. J.; ARMSTRONG, D. T. Oocyte-somatic cell interactions

during follicle development in mammals. Animal Reproduction Science, v. 82-83, p. 431-446,

2004.

GONG, S. P.; LEE, S. T.; LEE, E. J.; KIM, D. Y.; LEE, G.; CHI, S. G.; RYU, B. K.; LEE, C. H.;

YUM, K. E.; LEE, H. J.; HAN, J. Y.; TILLY, J. L.; LIM, J. M. Embryonic stem cell-like cells

established by culture of adult ovarian cells in mice. Fertility and Sterility, v. 93, n. 8, p. 2594-

2601, 2010.

GORDON, I. Storage and cryopreservation of oocytes and embryos. In: GORDON, I.

Laboratory production of cattle embryos, 548p. Cambridge: CAB International, Raven Press,

1994, p. 293-328.

GOTFRYD, K.; HANSEN, M.; KAWA, A; ELLERBECK, U.; BEREZIN, V.; BOCK, E.;

WALMOD, P. S. The teratogenic potencies of valproic acid derivatives and their effects on

biological end-points are related to changes in histone deacetylase and Erk1/2 activities. Basic &

clinical pharmacology & toxicology, v. 109, n. 3, p. 164-174, 2011.

182

GOUGEON, A. Dynamics for human follicular growth: morphologic, dynamic, and functional

aspects. In: LEUNG P. C. K.; ADASHI E. Y. (Eds.), The ovary, 2 ed., 664p. San Diego: Elsevier

Academic Press, 2004, p. 25-43.

GÜNESDOGAN, U.; MAGNÚSDÓTTIR, E.; SURANI, M. A. Primoridal germ cell

specification: a context-dependent cellular differentiation event. Philosophical Transactions of

the Royal Society B: Biological Sciences, v. 369, p. 20130543, 2014.

HAFEZ, B.; HAFEZ, E. S. E. Anatomia da reprodução feminina. In: HAFEZ, B.; HAFEZ, E. S.

E. Reprodução Animal, 7 ed., 513p. São Paulo: Manole, 2004, p. 13-29.

HANNA, C. B.; HENNEBOLD, J. D. Ovarian germline stem cells: an unlimited source of

oocytes? Fertility and sterility, v. 101, n. 1, p. 20-30, 2014.

HARUN, R.; RUBAN, L.; MATIN, M.; DRAPER, J.; JENKINS, N. M.; LIEW, G. C.;

ANDREWS, P. W.; LI, T. C.; LAIRD, S. M.; MOORE, H. D. Cytotrophoblast stem cell lines

derived from human embryonic stem cells and their capacity to mimic invasive implantation

events. Human Reproduction, v. 21, p. 1349-1358, 2006.

HAWKINS, R. D.; HON, G. C.; LEE, L. K.; NGO, Q.; LISTER, R.; PELIZZOLA, M.;

EDSALL, L. E.; KUAN, S.; LUU, Y.; KLUGMAN, S.; ANTOSIEWICZ-BOURGET, J.; YE, Z.;

ESPINOZA, C.; AGARWAHL, S.; SHEN, L.; RUOTTI, V.; WANG, W.; STEWART, R.;

THOMSON, J. A.; ECKER, J. R.; REN, B. Distinct epigenomic landscapes of pluripotent and

lineage-committed human cells. Cell Stem Cell, v. 6, n. 5, p. 479-491, 2010.

HAYASHI, K.; KOBAYASHI, T.; UMINO, T.; GOITSUKA, R.; MATSUI, Y.; KITAMURA,

D. SMAD1 signaling is critical for initial commitment of germ cell lineage from mouse epiblast.

Mechanisms of Development, v. 118, p. 99-109, 2002.

HAYASHI, K.; DE SOUSA LOPES, S. M. C.; SURANI, M. A. Germ cell specification in mice.

Science, v. 316, n. 5823, p. 394-396, 2007.

HAYASHI, K.; OGUSHI, S.; KURIMOTO, K.; SHIMAMOTO, S.; OHTA, H.; SAITOU, M.

Offspring from oocytes derived from in vitro primordial germ cell–like cells in mice. Science, n.

338, p. 971-975, 2012.

HAYASHI, K., OHTA, H., KURIMOTO, K., ARAMAKI, S.; SAITOU, M. Reconstitution of the

mouse germ cell specification pathway in culture by pluripotent stem cells. Cell, v. 146, n. 4, p.

519-532, 2011.

HEILKER, R.; TRAUB, S.; REINHARDT, P.; SCHÖLER, H. R.; STERNECKERT, J. iPS cell

derived neuronal cells for drug discovery. Trends in Pharmacological Sciences, v. 35, n. 10, p.

510-519, 2014.

HILSCHER, B.; HILSCHER, W.; BULTHOFF-OHNOLZ, B.; KRAMER, U.; BIRKE, A.;

PELZER, H.; GAUSS, G. Kinetics of gametogenesis I. Comparative histological and

183

autoradiographic studies of oocytes and transitional prospermatogonia during oogenesis and

prespermatogenesis. Cell and Tissue Research, v. 154, p. 443-470, 1974.

HIRSCHI, K. K.; LI, S.; ROY, K. Induced Pluripotent Stem Cells for Regenerative Medicine.

Annual Review of Biomedical Engineering, v. 16, p. 277–294, 2014.

HONG, J.; JIN, H.; HAN, J.; HU, H.; LIU, J.; LI, L.; HUANG, Y.; WANG, D.; WU, M.; QIU,

L.; QIAN, Q. Infusion of human umbilical cord-derived mesenchymal stem cells effectively

relieves liver cirrhosis in DEN-induced rats. Molecular Medicine Reports, v. 9, n. 4, p. 1103-

1111, 2014.

HONG, S. H.; MAGHEN, L.; KENIGSBERG, S.; TEICHERT, A. M.; RAMMELOO, A. W.;

SHLUSH, E.; SZARAZ, P.; PEREIRA, S.; LULAT, A.; XIAO, R.; YIE, S. M.; GAUTHIER-

FISHER, A.; LIBRACH, C. L. Ontogeny of human umbilical cord perivascular cells: molecular

and fate potential changes during gestation. Stem Cells and Development, v. 22, n. 17, p. 2425-

2439, 2013.

HOTTA, A.; ELLIS, J. Retroviral vector silencing during iPS cell induction: an epigenetic

beacon that signals distinct pluripotent states. Journal of Cellular Biochemistry, v. 105, n. 4, p.

940-948, 2008.

HOU, P.; LI, Y.; ZHANG, X.; LIU, C.; GUAN, J.; LI, H.; ZHAO, T.; YE, J.; YANG, W.; LIU,

K.; GE, J.; XU, J.; ZHANG, Q.; ZHAO, Y.; DENG, H. Pluripotent stem cells induced from

mouse somatic cells by small-molecule compound. Science, v. 341, n. 6146, p. 651-654, 2013.

HOSLER, B. A.; ROGERS, M. B.; KOZAK, C. A.; GUDAS, L. J. An octamer motif contributes

to the expression of the retinoic acid-regulated zinc finger gene Rex-1 (Zfp-42) in F9

teratocarcinoma cells. Molecular and Cellular Biology, v. 13, p. 2919-2928, 1993.

HOUSMAN, T. S.; LAWRENCE, N.; MELLEN, B. G.; GEORGE, M. N.; FILIPPO, J. S.;

CERVENY, K. A.; DEMARCO, M.; FELDMAN, S. R.; FLEISCHER, A. B. The safety of

liposuction: results of a national survey. Dermatologic Surgery, v. 28, n. 11, p. 971-978, 2002.

HU, X.; LU, H.; CAO, S.; DENG, Y-L.; LI, Q.-J.; WAN, Q.; YIE, S. M. Stem cells derived from

human first-trimester umbilical cord have the potential to differentiate into oocyte-like cells in

vitro. International Journal of Molecular Medicine, v. 35, p. 1219-1229, 2015.

HUANGFU, D.; OSAFUNE, K.; MAEHR, R.; GUO, W.; EIJKELENBOOM, A.; CHEN, S.;

MUHLESTEIN, W.; MELTON, D. A. Induction of pluripotent stem cells from primary human

fibroblasts with only Oct4 and Sox2. Nature Biotechnology, v. 26, n. 11, p. 1269-1275, 2008.

HÜBNER, K.; FUHRMANN, G.; CHRISTENSON, L.K.; KEHLER, J.; REINBOLD, R.; DE LA

FUENTE, R.; WOOD, J.; STRAUSS, J.F.; BOIANI, M.; SCHÖLER, H.R. Derivation of oocytes

from mouse embryonic stem cells. Science, v. 300, n. 5623, p. 1251-1256, 2003.

184

HUMMITZSCH, K.; ANDERSON, R. A.; WILHELM, D.; WU, J.; TELFER, E. E.; RUSSELL,

D. L.; ROBERTSON, S. A.; RODGERS, R. J. Stem Cells, Progenitor Cells, and Lineage

Decisions in the Ovary. Endocrine Reviews, v. 36, n. 1, 65-91, 2015.

HU, W.; QIU, B.; GUAN, W.; WANG, Q.; WANG, M.; LI, W.; GAO, L.; SHEN, L.; HUANG

Y.; XIE, G.; ZHAO, H.; JIN, Y.; TANG, B.; YU, Y.; ZHAO, J.; PEI, G. Direct Conversion of

Normal and Alzheimer's Disease Human Fibroblasts into Neuronal Cells by Small Molecules.

Cell Stem Cell, v. 17, n. 2, p. 204-212, 2015.

ILIC, D.; GIRITHARAN, G.; ZDRAVKOVIC, T.; CACERES, E.; GENBACEV, O.; FISHER,

S. J.; KRTOLICA, A. Derivation of human embryonic stem cell lines from biopsied blastomeres

on human feeders with minimal exposure to xenomaterials. Stem Cells and Development, v. 18,

p. 1343-1350, 2009.

INSAUSTI, C. L.; BLANQUER, M.; GARCÍA-HERNÁNDEZ, A. M.; CASTELLANOS, G.;

MORALEDA, J. M. Amniotic membrane-derived stem cells: immunomodulatory properties and

potential clinical application. Stem Cells Cloning, v. 7, p. 53-63, 2014.

ISHII, T. Human iPS Cell-Derived Germ Cells: Current Status and Clinical Potential. Journal of

Clinical Medicine, v. 3, p. 1064-1083, 2014.

ISHII, T.; PERA, R.A.; GREELY, H.T. Ethical and legal issues arising in research on inducing

human germ cells from pluripotent stem cells. Cell Stem Cell, v. 13, n. 2, p. 145-148, 2013.

ITOH, F.; WATABE, T.; MIYAZONO, K. Roles of TGF-β family signals in the fate

determination of pluripotent stem cells.Seminars in cell & developmental biology. v. 32, p. 98-

106.

JAENISCH, R.; YOUNG, R. Stem cells, the molecular circuitry of pluripotency and nuclear

reprogramming. Cell, v. 132, n. 4, p. 567-582, 2008.

JIANG, Y.; HENDERSON, D.; BLACKSTAD, M.; CHEN, A.; MILLER, R. F.; VERFAILLIE,

C. M. Neuroectodermal differentiation from mouse multipotent adult progenitor cells.

Proceedings of the National Academy of Sciences of the United States of America, v. 100, n.

1, p. 11854-11860, 2003.

JOHNSON, J.; BAGLEY, J.; SKAZNIK-WIKIEL, M.; LEE, H. J.; ADAMS, G. B.; NIIKURA,

Y.; TSCHUDY, K. S.; TILLY, J. C.; CORTES, M. L.; FORKERT, R.; SPITZER, T.;

IACOMINI, J.; SCADDEN, D. T.; TILLY, J. L. Oocyte generation in adult mammalian ovaries

by putative germ cells in bone marrow and peripheral blood. Cell, v. 122, p. 303-15, 2005.

JOHNSON, J.; CANNING, J.; KANEKO, T.; PRU, J. K.; TILLY, J. L. Germline stem cells and

follicular renewal in the postnatal mammalian ovary. Nature, v. 428, p. 145-150, 2004.

JONES, P. A.; TAYLOR, S. M. Cellular differentiation, cytidine analogs and DNA methylation.

Cell, v. 20, p. 85-93, 1980.

185

JONES, P. A. Effects of 5-azacytidine and its 2′-deoxyderivative on cell differentiation and DNA

methylation. Pharmacology & Therapeutics, v. 28, n. 1, p. 17-27, 1985.

JUENGEL, J. L.; SAWYER, H. R.; SMITH, P. R.; QUIRKE, L. D.; HEATH, D. A.; LUN, S.;

WAKEFIELD, S. J.; MCNATTY, K. P. Origins of follicular cells and ontogeny of

steroidogenesis in ovine fetal ovaries. Molecular and Cellular Endocrinology, v. 191, p. 1-10,

2002.

JUTTERMANN, R.; LI, E.; JAENISCH, R. Toxicity of 5-aza-2′-deoxycytidine to mammalian

cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA

demethylation. Proceedings of the National Academy of Sciences of the United State of

America, v. 91, p. 11797-11801, 1994.

KEE, K.; ANGELES, V. T.; FLORES, M.; NGUYEN, H. N.; REIJO PERA, R. A. Human

DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation.

Nature, v. 462, p. 222-225, 2009.

KEE, K.; GONSALVES, J. M.; CLARK, A. T.; REIJO PERA, R. A. Bone Morphogenetic

Proteins Induce Germ Cell Differentiation from Human Embryonic Stem Cells. Stem Cells and

Development, v. 15, p. 831-837, 2006.

KEEFER, C. L.; PANTA, D.; BLOMBERG, L.; TALBOT, N. C. Challenges and prospects for

the establishment of embryonic stem cell lines of domesticated ungulates. Animal Reproduction

Science, v. 98, p. 147-168, 2007.

KERKIS, A.; FONSECA, S.A.; SERAFIM, R.C.; LAVAGNOLLI, T.M.; ABDELMASSIH, S.;

ABDELMASSIH, R.; KERKIS, I. In vitro differentiation of male mouse embryonic stem cells

into both presumptive sperm cells and oocytes. Cloning and stem cells, v. 9, n. 4, p. 535-548,

2007.

KERR, C.L.; CHENG, L. The dazzle in germ cell differentiation. Journal of molecular cell

biology, v. 2, n. 1, p. 26-29, 2010.

KERN, S.; EICHLER, H.; STOEVE, J.; KLUTER, H.; BIEBACK, K. Comparative analysis of

mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells,

v. 24, p. 1294-1301, 2006.

KIM, D.; KIM, C. H.; MOON, J. I.; CHUNG, Y. G.; CHANG, M. Y.; HAN, B. S.; KO, S.;

YANG, E.; CHA, K. Y.; LANZA, R.; KIM, K. S. Generation of human induced pluripotent stem

cells by direct delivery of reprogramming proteins. Cell Stem Cell, v. 4, n. 6, p. 472–476, 2009.

KIM, J. H.; JUNG, M.; KIM, H. S.; KIM, Y. M.; CHOI, E. H. Adipose-derived stem cells as a

new therapeutic modality for ageing skin. Experimental Dermatology, v. 20, n. 5, p. 383-387,

2011.

KISHIGAMI, S.; MISHINA, Y. BMP signaling and early embryonic patterning. Cytokine

Growth Factor Reviews, v. 16, n. 3, p. 265-278, 2005.

186

KISIEL, A. H.; MCDUFFEE, L. A.; MASAOUD, E.; BAILEY, T. R.; ESPARZA GONZALEZ,

B. P.; NINO-FONG, R. Isolation, characterization, and in vitro proliferation of canine

mesenchymal stem cells derived from bone marrow, adipose tissue, muscle, and periosteum.

American Journal of Veterinary Research, v. 73, n. 8, 1305-1317, 2012.

KLIMANSKAYA, I.; CHUNG, Y.; BECKER, S.; LU, S. J.; LANZA, R. Derivation of human

embryonic stem cells from single blastomeres. Nature Protocols, v. 2, p. 1963-1972, 2007.

KLIMANSKAYA, I.; CHUNG, Y.; BECKER, S.; LU, S. J.; LANZA, R. Human embryonic stem

cell lines derived from single blastomeres. Nature, v. 444, p. 481-485, 2006.

KONA, S. S.; PRAVEEN CHAKRAVARTHI, V.; SIVA KUMAR, A. V.; SRIVIDYA, D.;

PADMAJA, K.; RAO, V. H. Quantitative expression patterns of GDF9 and BMP15 genes in

sheep ovarian follicles grown in vivo or cultured in vitro. Theriogenology, v. 85, n. 2, p. 315-

322, 2016.

KOPPER, O.; GILADI, O.; GOLAN-LEV, T.; BENVENISTY, N. Characterization of

gastrulation-stage progenitor cells and their inhibitory crosstalk in human embryoid bodies. Stem

Cells, v. 28, p. 75-83, 2010.

KOUBOVA, J.; HU, Y. C.; BHATTACHARYYA, T.; SOH, Y. Q.; GILL, M. E.;

GOODHEART, M. L.; HOGARTH, C. A.; GRISWOLD, M. D.; PAGE, D. C. Retinoic acid

activates two pathways required for meiosis in mice. PLoS Genetics, v. 10(8): e1004541, 2014.

KOUBOVA, J.; MENKE, D. B.; ZHOU, Q.; CAPEL, B.; GRISWOLD, M. D.; PAGE, D. C.

Retinoic acid regulates sex-specific timing of meiotic initiation in mice. Proceedings of the

National Academy of Sciences of the United States of America. v. 103, n. 8, p. 2474-2479,

2006.

KOŹLIK, M.; WÓJCICKI, P. The Use of Stem Cells in Plastic and Reconstructive Surgery.

Advances in Clinical and Experimental Medicine, v. 23, n. 6, p. 1011–1017, 2014.

KRAMPERA, M.; MARCONI, S.; PASINI, A.; GALIÈ, M.; RIGOTTI, G.; MOSNA, F.;

TINELLI, M.; LOVATO, L.; ANGHILERI, E.; ANDREINI, A.; PIZZOLO, G.; SBARBATI, A.;

BONETTI, B. Induction of neural-like differentiation in human mesenchymal stem cells derived

from bone marrow, fat, spleen and thymus. Bone, v. 40, n. 2, p. 382-490, 2007.

KRIEGER, T.; SIMONS, B. D. Dynamic stem cell heterogeneity. Development, v. 142, p. 1396-

1406, 2015.

KURIMOTO, K.; YABUTA, Y.; OHINATA, Y.; SHIGETA, M.; YAMANAKA, K.; SAITOU,

M. Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of

the germ cell lineage in mice. Genes & Development, v. 22, p. 1617-1635, 2008.

187

LACHAM-KAPLAN, O.; CHY, H.; TROUNSON, A. Testicular cell conditioned medium

supports differentiation of embryonic stem cells into ovarian structures containing oocytes. Stem

cells, v. 24, n. 2, p. 266-273, 2006.

LAGE-CONSIGLIO, A.; ROSSI, D.; TASSAN, S.; PEREGO, R.; CREMONESI, F.;

PAROLINI, O. Conditioned medium from horse amniotic membrane-derived multipotent

progenitor cells: immunomodulatory activity in vitro and first clinical application in tendon and

ligament injuries in vivo. Stem Cells and Development, v. 22, n. 22, p. 3015-3024, 2013.

LAM, M. T.; LONGAKER, M. T. Comparison of several attachment methods for human iPS,

embryonic and adipose-derived stem cells for tissue engineering. Tissue Engineering and

Regenerative Medicine, v. 6, p. s80-s86, 2012.

LANGE, U. C.; ADAMS, D. J.; LEE, C.; BARTON, S.; SCHNEIDER, R.; BRADLEY, A.;

SURANI, M. A. Normal germ line establishment in mice carrying a deletion of the Ifitm/Fragilis

gene family cluster. Molecular and Cellular Biology, v. 28, p. 4688-4696, 2008.

LASKO, P. F.; ASHBURNER, M. Posterior localization of vasa protein correlates with, but is

not sufficient for, pole cell development. Genes and Development, v. 4, n. 6, p. 905-921, 1990.

LAVIAL, F.; ACLOQUE, H.; BERTOCCHINI, F.; MACLEOD, D.J.; BOAST, S.;

BACHELARD, E.; MONTILLET, G.; THENOT, S.; SANG, H.M.; STERN, C.D.; SAMARUT,

J.; PAIN, B. The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic

stem cells.Development, v. 134, n. 19, p. 3549-3563, 2007.

LAWSON, K. A.; DUNN, N. R.; ROELEN, B. A.; ZEINSTRA, L. M.; DAVIS, A. M.;

WRIGHT, C. V.; KORVING, J. P.; HOGAN, B. L. Bmp4 is required for the generation of

primordial germ cells in the mouse embryo. Genes & Development, v. 13, p. 424-436, 1999.

LAWSON, K. A.; HAGE, W. J. Clonal analysis of the origin of primordial germ cells in the

mouse. Ciba Foundation Symposia, v. 182, p. 68-84, 1994.

LEBLOND, C. P.; STEVENS, C. E. The constant renewal of the intestinal epithelium in the

albino rat. The Anatomical Record, v. 100, p. 357-377, 1948.

LEE, H.J.; SELESNIEMI, K.; NIIKURA, Y.; NIIKURA, T.; KLEIN, R.; DOMBKOWSKI,

D.M.; TILLY, J.L. Bone marrow transplantation generates immature oocytes and rescues long-

term fertility in a preclinical mouse model of chemotherapy-induced premature ovarian failure.

Journal of Clinical Oncology, v. 25, n. 22, p. 3198-3204, 2007.

LEE, J. H.; HONG, K. S.; MANTEL, C.; BROXMEYER, H. E.; LEE, M. R.; KIM, K. S.

Spontaneously differentiated GATA6-positive hESCs represent an important cellular step in

human embryonic development; they are not just an artifact of in vitro culture. Stem Cells and

Development, v. 22, p. 2706-2713, 2013.

LEFIÈVRE, L.; CONNER, S. J.; SALPEKAR, A.; OLUFOWOBI, O.; ASHTON, P.;

PAVLOVIC, B.; LENTON, W.; AFNAN, M.; BREWIS, I. A.; MONK, M.; HUGHES, D. C.;

188

BARRATT, C. L. Four zona pellucida glycoproteins are expressed in the human. Human

Reproduction, v. 19, p. 1580-1586, 2004.

LEVINE, A.; BRIVANLOU, A. GDF3 at the crossroads of TGF-beta signaling. Cell Cycle, v. 5,

n. 10, p. 1069-1073, 2006.

LEYDON, C.; SELEKMAN, J. A.; PALECEK, S.; THIBEAULT, S. L. Human embryonic stem

cell-derived epithelial cells in a novel in vitro model of vocal mucosa. Tissue Engineering Part

A, v. 19, p. 2233-2241, 2013.

LEWITZKY, M.; YAMANAKA, S. Reprogramming somatic cells towards pluripotency by

defined factors. Current Opinion in Biotechnology, v. 18, n. 5, p. 467-473, 2007.

LIANG, L.; SOYAL, S.M.; DEAN, J. FIGalpha, a germ cell specific transcription factor involved

in the coordinate expression of the zona pellucida genes. Development, v. 124, n. 24, p. 4939-

4947, 1997.

LI, E. Chromatin modification and epigenetic reprogramming in mammalian development.

Nature Reviews Genetics, v. 3, n. 9, p. 662-673, 2002.

LI, J.; WANG, G.; WANG, C.; ZHAO, Y.; ZHANG, H.; TAN, Z.; SONG, Z.; DING, M.;

DENG, H. MEK/ERK signaling contributes to the maintenance of human embryonic stem cell

self-renewal. Differentiation, v. 75, n. 4, p. 299-307, 2007.

LIN, Y.; PAGE, D. C. Dazl deficiency leads to embryonic arrest of germ cell development in XY

C57BL/6 mice. Developmental Biology, v. 288, n. 2, p. 309-316, 2005.

LIN, C. Y.; CHEN, C. Y.; YU, C. H.; YU, I. S.; LIN, S. R.; WU, J. T.; LIN, Y. H.; KUO, P. L.;

WU, J. C.; LIN, S. W. Human X-linked Intellectual Disability Factor CUL4B Is Required for

Post-meiotic Sperm Development and Male Fertility. Scientific Reports, v. 6, n. 20227, 2016.

LIN, T.; WU, S. Reprogramming with Small Molecules instead of Exogenous Transcription

Factors. Stem Cells International, v. 2015, 794632, 2015.

LIN, Y.; GILL, M. E.; KOUBOVA, J.; PAGE, D. C. Germ cell-intrinsic and-extrinsic factors

govern meiotic initiation in mouse embryos. Science, v. 322, p. 1685-1687, 2008.

LINHER, K.; DYCE, P.; LI, J. Primordial germ cell-like cells differentiated in vitro from skin-

derived stem cells. PLoS ONE, v. 4, n. 12, e8263, 2009.

LIVAK, J. K.; SCHMITTGEN, T. D. Analysis of relative gene expression data using real-time

quantitative PCR and the 2-ΔΔCt method. Methods, v. 25, p. 402-408, 2001.

MACGREGOR, G. R; ZAMBROWICZ, B. P; SORIANO, P. Tissue non-specific alkaline

phosphatase is expressed in both embryonic and extraembryonic lineages during mouse

embryogenesis but is not required for migration of primordial germ cells. Development, v. 121,

n. 5, p. 1487-1496, 1995.

189

MAHERALI, N.; SRIDHARAN, R.; XIE, W.; UTIKAL, J.; EMINLI, S.; ARNOLD, K.;

STADTFELD, M.; YACHECHKO, R.; TCHIEU, J.; JAENISCH, R.; PLATH, K. Directly

reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.


MALI, P.; CHOU, B. K.; YEN, J; YE, Z.; ZOU, J.; DOWEY, S.; BRODSKY, R. A.; OHM, J. E.;

YU, W.; BAYLIN, S. B.; YUSA, K.; BRADLEY, A.; MEYERS, D. J.; MUKHERJEE, C.;

COLE, P. A.; CHENG, L. Butyrate greatly enhances derivation of human induced pluripotent

stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated

genes. Stem Cells, v. 28, n. 4, p. 713-720, 2010.

MALTSEV, V. A.; ROHWEDEL, J.; HESCHELER, J.; WOBUS, A. M. Embryonic stem cells

differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell

types. Mechanisms of Development, v. 44, n. 1, p. 41-50, 1993.

MARCHAND, M.; HORCAJADAS, J. A.; ESTEBAN, F. J.; MCELROY, S. L.; FISHER, S. J.;

GIUDICE, L. C. Transcriptomic signature of trophoblast differentiation in a human embryonic

stem cell model. Biology of Reproduction, v. 84, p. 1258-1271, 2011.

MARQUES-MARI, A. I; LACHAM-KAPLAN, O; MEDRANO, J. V; PELLICER, A; SIMÓN,

C. Differentiation of germ cells and gametes from stem cells. Human Reproduction Update,

v. 15, n. 3, p. 379-390, 2009.

MARTHALER, A. G.; TUBSUWAN, A.; SCHMID, B.; POULSEN, U. B.; HYTTEL, P.;

NIELSEN, J. E.; NIELSEN, T. T.; HOLST, B. Generation of spinocerebellar ataxia type 2

patient-derived iPSC line H271. Stem Cell Research, v. 16, n. 1, p. 159-161, 2016.

MARTIN, G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium

conditioned by teratocarcinoma stem cells. Proceedings of the National Academy of Sciences,

v. 78, n. 12, p. 7634-7638, 1981.

MASUI, S.; NAKATAKE, Y.; TOYOOKA, Y.; SHIMOSATO, D.; YAGI, R.; TAKAHASHI,

K.; OKOCHI, H.; OKUDA, A.; MATOBA, R.; SHAROV, A. A.; KO, M. S.; NIWA, H.

Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem

cells. Nature Cell Biology, v. 9, p. 625-635, 2007.

MCCONNELL, N. A.; YUNUS, R. S.; GROSS, S. A.; BOST, K. L.; CLEMENS, M. G.;

HUGHES JR, F. M. Water permeability of an ovarian antral follicle is predominantly

transcellular and mediated by aquaporins. Endocrinology, v. 143, n. 8, p. 2905-2912, 2002.

MCLAREN, A. Primordial germcells in the mouse. Developmental Biology, v. 262, n. 1, p. 1-

15, 2003.

MCLAREN, A. Signalling for germ cells. Genes & Development, v. 13, p. 373-376, 1999.

190

MCLAUGHLIN, M.; KELSEY, T.W.; WALLACE, W.H.B.; ANDERSON, R.A.; TELFER, E.E.

An externally validated age-related model of mean follicle density in the cortex of the human

ovary.Journal of Assisted Reproduction and Genetics, v. 32, n. 7, p. 1089-1095, 2015.

MENKE, D. B.; KOUBOVA, J.; PAGE, D. C. Sexual differentiation of germ cells in XX mouse

gonads occurs in an anterior-toposterior wave. Developmental Biology, v. 262, n. 2, p. 303-312,

2003.

MEDRANO, J.V.; RAMATHAL, C.; NGUYEN, H.N.; SIMON, C.; REIJO PERA, R.A.

Divergent RNA-binding proteins, DAZL and VASA, induce meiotic progression in human germ

cells derived in vitro. Stem Cells, v. 30, n. 3, p. 441-451, 2012.

MEDRANO, J.V.; MARQUÉS-MARÍ, A.I.; AGUILAR, C.E.; RIBOLDI, M.; GARRIDO, N.;

MARTÍNEZ-ROMERO, A.; O'CONNOR, E.; GIL-SALOM, M.; SIMÓN C. Comparative

analysis of the germ cell markers c-KIT, SSEA-1 and VASA in testicular biopsies from secretory

and obstructive azoospermias. Molecular human reproduction, v. 16, n. 11, p. 811-817, 2010.

MIKKELSEN, T. S.; HANNA, J.; ZHANG, X.; KU, M.; WERNIG, M.; SCHORDERET, P.;

BERNSTEIN, B. E.; JAENISCH, R.; LANDER, E. S.; MEISSNER, A. Dissecting direct

reprogramming through integrative genomic analysis. Nature, v. 454, p. 49-55, 2008.

MISHRA, L.; DERYNCK, R.; MISHRA, B. Transforming growth factor-beta signaling in stem

cells and cancer. Science, v. 310, p. 68-71, 2005.

MIYOSHI, N.; ISHII, H., NAGANO, H.; HARAGUCHI, N.; DEWI, D. L.; KANO, Y.;

NISHIKAWA, S.; TANEMURA, M.; MIMORI, K.; TANAKA, F.; SAITO, T.; NISHIMURA,

J.; TAKEMASA, I.; MIZUSHIMA, T.; IKEDA, M.; YAMAMOTO, H.; SEKIMOTO, M.;

DOKI, Y.; MORI, M. Reprogramming of mouse and human cells to pluripotency using mature

microRNAs. Cell Stem Cell, v. 8, n. 6, p. 633-638, 2011.

MOLYNEAUX, K. A.; STALLOCK, J.; SCHAIBLE, K.; WYLIE, C. Timelapse analysis of

living mouse germ cell migration. Developmental Biology, v. 240, n. 2, p. 488-498, 2001.

MONTI, B.; POLAZZI, E.; CONTESTABILE, A. Biochemical, molecular and epigenetic

mechanisms of valproic acid neuroprotection. Current Molecular Pharmacology, v. 2, n. 1, p.

95-109, 2009.

MOHAMED, J. S.; GAUGHWIN, P. M.; LIM, B.; ROBSON, P.; IPOVICH, L. Conserved long

noncoding RNAs transcriptionally regulated by Oct4 and Nanog modulate pluripotency in mouse

embryonic stem cells. RNA, v. 16, n. 2, p. 324–337, 2010.

MOHANA KUMAR, B.; JIN, H. F.; KIM, J. G.; SONG, H. J.; HONG, Y.;

BALASUBRAMANIAN, S.; CHOE, S. Y.; RHO, G. J. DNA methylation levels in porcine fetal

fibroblasts induced by an inhibitor of methylation, 5-azacytidine. Cell and Tissue Research, v.

325, n. 3, p. 445-454, 2006.

191

MOOYOTTU, S.; ANEES, S.; CHERIAN, S. Ovarian stem cells and neo-oogenesis: A

breakthrough in reproductive biology research. Veterinary World, v. 4, n. 2, p. 89-91, 2011.

NAGANO, M., PATRIZIO, P.; BRINSTER, R.L. Long-term survival of human spermatogonial

stem cells in mouse testes. Fertility and Sterility, v. 78, n. 6, p. 1225-1233, 2002.

NAKAGAWA, M.; KOYANAGI, M.; TANABE, K.; TAKAHASHI, K.; ICHISAKAM T.; AOI,

T.; OKITA, K.; MOCHIDUKI, Y.; TAKIZAWA, N.; YAMANAKA, S. Generation of induced

pluripotent stem cells without Myc from mouse and human fibroblasts. Nature Biotechnology,

v. 26, p. 101-106, 2008.

NAYERNIA, K.; NOLTE, J.; MICHELMANN, H. W.; LEE, J. H.; RATHSACK, K.;

DRUSENHEIMER, N.; DEV, A.; WULF, G.; EHRMANN, I. E.; ELLIOTT, D. J.; OKPANYI,

V.; ZECHNER, U.; HAAF, T.; MEINHARDT, A.; ENGEL, W. In vitro-differentiated embryonic

stem cells give rise to male gametes that can generate offspring mice. Developmental Cell, v. 11,

p. 125-132, 2006.

NICHOLAS, C. R.; CHAVEZ, S. H.; BAKER, V. L.; REIJO PERA, R. A. Instructing an

embryonic stem cell-derived oocyte fate: lessons from endogenous oogenesis. Endocrine

Reviews, v. 30, n. 3, p. 264–283, 2009a.

NICHOLAS, C. R.; HASTON, K. M.; GREWALL, A. K.; LONGACRE, T. A.; REIJO PERA,

R. A. Transplantation directs oocyte maturation from embryonic stem cells and provides a

therapeutic strategy for female infertility. Human Molecular Genetics, v. 18, n. 22, p. 4376-

4389, 2009b.

NICHOLS, J.; ZEVNIK, B.; ANASTASSIADIS, K.; NIWA, H.; KLEWE-NEBENIUS, D.;

CHAMBERS, I.; SCHÖLER, H.; SMITH, A. Formation of pluripotent stem cells in the

mammalian embryo depends on the POU transcription factor Oct4. Cell, v. 95, n. 3, p. 379-391,

1998.

NIE, B.; WANG, H.; LAURENT, T.; DING, S. Cellular reprogramming: a small molecule

perspective. Current Opinion in Cell Biology, v. 24, n. 6, p. 784-792, 2012.

NIIKURA, Y.; NIIKURA, T.; TILLY, J.L. Aged mouse ovaries possess rare premeiotic germ

cells that can generate oocytes following transplantation into a young host environment. Aging,

v. 1, n. 12, p. 971-978, 2009.

NIKOLIC, A.; VOLAREVIC, V.; ARMSTRONG, L.; LAKO, M.; STOJKOVIC, M. Primordial

Germ Cells: Current Knowledge and Perspectives. Stem Cells International, v. 2016, 1741072.

2016.

NIWA, H. How is pluripotency determined and maintained? Development, v. 134, p. 635-646,

2007.

192

NIWA, H.; MIYAZAKI, J.; SMITH, A. G. Quantitative expression of Oct-3/4 defines

differentiation, dedifferentiation or self-renewal of ES cells. Nature Genetics, 24, 372-376,

2000.

NIWA, H.; OGAWA, K.; SHIMOSATO, D.; ADACHI, K. A parallel circuit of LIF signalling

pathways maintains pluripotency of mouse ES cells. Nature, v. 460, n. 7251, p. 118-122, 2009.

NÖR, C.; SASSI, F. A.; DE FARIAS, C. B.; SCHWARTSMANN, G.; ABUJAMRA, A. L.;

LENZ, G.; BRUNETTO, A. L.; ROESLER, R. The histone deacetylase inhibitor sodium butyrate

promotes cell death and differentiation and reduces neurosphere formation in human

medulloblastoma cells. Molecular Neurobiology, v. 48, n. 3, p. 533-543, 2013.

NOVAK, I.; LIGHTFOOT, D.A.; WANG, H.; ERIKSSON, A.; MAHDY, E.; HÖÖG C. Mouse

embryonic stem cells form follicle-like ovarian structures but do not progress through meiosis.

Stem Cells, v. 24, n. 8, p. 1931-1936, 2006.

OHINATA, Y.; OHTA, H.; SHIGETA, M.; YAMANAKA, K.; WAKAYAMA, T.; SAITOU, M.

A signaling principle for the specification of the germ cell lineage in mice. Cell, v. 137, p. 571-

584, 2009.

OHINATA, Y.; PAYER, B.; O'CARROLL, D.; ANCELIN, K.; ONO, Y.; SANO, M.; BARTON,

S. C.; OBUKHANYCH, T.; NUSSENZWEIG, M.; TARAKHOVSKY, A.; SAITOU, M.;

SURANI, M. A. Blimp1 is a critical determinant of the germ cell lineage in mice. Nature, v. 436,

p. 207-213, 2005.

OKAMURA, D.; HAYASHI, K.; MATSUI, Y. Mouse epiblasts change responsiveness to BMP4

signal required for PGC formation through functions of extraembryonic ectoderm. Molecular

Reproduction and Development, v. 70, p. 20-29, 2005.

OKITA, K.; YAMANAKA, S. Intracellular signaling pathways regulating pluripotency of

embryonic stem cells. Current Stem Cell Research & Therapy, v. 1, n. 1, p. 103-111, 2006.

OKITA, K.; ICHISAKA, T.; YAMANAKA, S. Generation of germline-competent induced

pluripotent stem cells. Nature, v. 448, n. 7151, p. 313-317, 2007.

OKITA, K.; YAMANAKA, S. Induced pluripotent stem cells: opportunities and challenges.

Philos. Trans. R. Philosophical Transactions of the Royal Society of London B: Biological

Sciences, v. 366, n. 1575, p. 2198-2207, 2011.

OMBELET, W.; COOKE, I.; DYER, S.; SEROUR, G.; DEVROEY, P. Infertility and the

provision of infertility medical services in developing countries. Human Reproduction Update,

v. 14, n. 6, p. 605-621, 2008.

PACCHIAROTTI, J.; MAKI, C.; RAMOS, T.; MARH, J.; HOWERTON, K.; WONG, J.;

PHAM, J.; ANORVE, S.; CHOW, Y. C.; IZADYAR, F. Differentiation potential of germ line

stem cells derived from the postnatal mouse ovary. Differentiation, v. 79, p. 159-170, 2010.

193

PAN, Z.; SUN, M.; LI, J.; ZHOU, F.; LIANG, X.; HUANG, J.; ZHENG, T.; ZHENG, L.;

ZHENG, Y. The Expression of Markers Related to Ovarian Germline Stem Cells in the Mouse

Ovarian Surface Epithelium and the Correlation with Notch Signaling Pathway. Cell Physiology

and Biochemistry, v. 37, n. 6, p. 2311-2322, 2015.

PANULA, S.; MEDRANO, J. V.; KEE, K.; BERGSTRÖM, R.; NGUYEN, H. N.; BYERS, B.;

WILSON, K. D.; WU, J. C.; SIMON, C.; HOVATTA, O.; REIJO PERA, R. A. Human germ cell

differentiation from fetal- and adult-derived induced pluripotent stem cells. Human Molecular

Genetics, v. 20, n. 4, p. 752 – 762, 2011.

PARK, E. S; WOODS, D. C; TILLY, J. L. Bone morphogenetic protein 4 promotes mammalian

oogonial stem cell differentiation via Smad1/5/8 signaling. Fertility and Sterility, v. 100, n. 5, p.

1468-1475, 2013.

PARK, T. S.; GALIC, Z.; CONWAY, A. E.; LINDGREN, A.; VAN HANDEL, B. J.;

MAGNUSSON, M.; RICHTER, L.; TEITELL, M. A.; MIKKOLA, H. K.; LOWRY, W. E.;

PLATH, K.; CLARK, A. T. Derivation of primordial germ cells from human embryonic and

induced pluripotent stem cells is significantly improved by coculture with human fetal gonadal

cells. Stem Cells, v. 27, n. 4, p. 783-795, 2009.

PARTE, S.; BHARTIYA, D.; PATEL, H.; DAITHANKAR, V.; CHAUHAN, A.; ZAVERI, K.;

HINDUJA, I. Dynamics associated with spontaneous differentiation of ovarian stem cells in

vitro. Journal of Ovarian Research, v. 7, p.25, 2014.

PARTE, S.; BHARTIYA, D.; TELANG, J.; DAITHANKAR, V.; SALVI, V.; ZAVERI, K.;

HINDUJA, I. Detection, characterization, and spontaneous differentiation in vitro of very small

embryonic-like putative stem cells in adult mammalian ovary. Stem Cells and Development, v.

20, n. 8, p. 1451-1464, 2011.

PATEL, D.M.; SHAH, J.; SRIVASTAVA, A.S. Therapeutic Potential of Mesenchymal Stem

Cells in Regenerative Medicine. Stem Cells International. v. 2013, n. 496218, p. 15, 2013.

PAYER, B.; SAITOU, M.; BARTON, S. C.; THRESHER, R.; DIXON, J. P.; ZAHN, D.;

COLLEDGE, W. H.; CARLTON, M. B.; NAKANO, T.; SURANI, M. A. Stella is a maternal

effect gene required for normal early development in mice. Current Biology, v. 13, p. 2110-

2117, 2003.

PENNAROSSA, G.; MAFFEI, S.; CAMPAGNOL, M.; RAHMAN, M.M.; BREVINI, T.A.L.;

GANDOLFI, F. Reprogramming of Pig Dermal Fibroblast into Insulin Secreting Cells by a Brief

Exposure to 5-aza-cytidine. Stem Cell Reviews and Reports, v. 10, n. 1, p. 31-43, 2014.

PENNAROSSA, G.; MAFFEI, S.; CAMPAGNOL, M.; TARANTINI, L.; GANDOLFI, F.;

BREVINIA, T. A. L. Brief demethylation step allows the conversion of adult human skin

fibroblasts into insulin-secreting cells. Proceedings of the National Academy of Sciences of the

United State of America, v. 110, n. 22, p. 8948–8953, 2013.

194

PERA, M. F.; ANDRADE, J.; HOUSSAMI, S.; REUBINOFF, B.; TROUNSON, A.; STANLEY,

E. G.; WARD-VAN OOSTWAARD, D.; MUMMERY, C. Regulation of human embryonic stem

cell differentiation by BMP-2 and its antagonist noggin. Journal of Cell Science, v. 117, p.

1269-1280, 2004.

PERA, M. F.; TROUNSON, A. O. Human embryonic stem cells: prospects for development.

Development, v. 131, n. 22, p. 5515-5525, 2004.

PESCE, M.; GIOIA KLINGER, F.; DE FELICI, M. Derivation in culture of primordial germ

cells from cells of the mouse epiblast: Phenotypic induction and growth control by Bmp4

signalling. Mechanisms of Development, v. 112, p. 15-24, 2002.

PESCE, M.; GROSS, M.K.; SCHOLER, H.R. In line with our ancestors: Oct-4 and the

mammalian germ. Bioessays, v. 20, n. 9, p. 722-732, 1998.

PHIEL, C.J.; ZHANG, F.; HUANG, E.Y.; GUENTHER, M.G.; LAZAR, M.A.; KLEIN, P.S.

Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer,

and teratogen. Journal of Biological Chemistry, v. 276, n. 39, p. 36734-36741, 2001.

PFANNKUCHE, K.; FATIMA, A.; GUPTA, M. K.; DIETERICH, R.; HESCHELER, J. Initial

colony morphology-based selection for iPS cells derived from adult fibroblasts is substantially

improved by temporary UTF1-based selection. PLoS One, v. 5, e9580, 2010.

PICANÇO-CASTRO, V.; RUSSO-CARBOLANTE, E.; REIS, L. C.; FRAGA, A. M.;

MAGALHÃES, D. A.; ORELLANA, M. D.; PANEPUCCI, R. A.; PEREIRA, L. V.; COVAS, D.

T. Pluripotent reprogramming of fibroblasts by lentiviral mediated insertion of SOX2, C-MYC,

and TCL-1A. Stem Cells and Development, v. 20, n. 1, p. 169-180, 2011.

PICTON, H. M. Activation of follicle development: the primordial follicle. Theriogenology, v.

55, n. 6, p. 1193-1210, 2001.

PICTON, H. M.; BRIGGS, D.; GOSDEN, R. The molecular basis of oocyte growth and

development. Molecular and Cellular Endocrinology, v. 145, n. 1-2, p. 27-37, 1998.

PLOCK, J. A.; SCHNIDER, J. T.; SOLARI, M. G.; ZHENG, X. X.; GORANTLA, V. S.

Perspectives on the use of mesenchymal stem cells in vascularized composite allotransplantation.

Frontiers in Immunology, v. 4:175, 2013.

PROCHAZKA, R; NEMCOVA, L; NAGYOVA, E; KANKA, J. Expression of growth

differentiation factor 9 messenger RNA in porcine growing and preovulatory ovarian follicles.

Biology of Reproduction, v. 71, n. 4, p. 1290–1295, 2004.

PSATHAKI, O. E.; HÜBNER, K.; SABOUR, D.; SEBASTIANO, V.; WU, G.; SUGAWA, F.;

WIEACKER, P.; PENNEKAMP, P.; SCHÖLER, H. R. Ultrastructural characterization of mouse

embryonic stem cell-derived oocytes and granulosa cells. Stem Cells Development, v. 20, p.

2205-2215, 2011.

195

QING, T.; SHI ,Y.; QIN, H.; YE, X.; WEI, W.; LIU, H.; DING, M.; DENG, H. Induction of

oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells.

Differentiation, v. 75, n. 10, p. 902–911, 2007.

RATAJCZAK, M. Z.; ZUBA-SURMA, E.; KUCIA, M.; PONIEWIERSKA, A.; SUSZYNSKA,

M.; RATAJCZAK, J. Pluripotent and multipotent stem cells in adult tissues. Advances in

Medical Sciences, v. 57, n. 1, p. 1-17, 2012.

REIJO PERA, R. A. Status of human germ cell differentiation from pluripotent stem cells.

Reproduction, Fertility and Development, v. 25, n. 2, p. 396-404, 2013.

REMENYI, A.; LINS, K.; NISSEN, L. J.; REINBOLD, R.; SCHÖLER, H. R.; WILMANNS, M.

Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4

and Sox2 on two enhancers. Genes & Development, v. 17, p. 2048-2059, 2003.

REUBINOFF, B. E.; PERA, M. F.; FONG, C. Y.; TROUNSON, A.; BONGSO, A. Embryonic

stem cell lines from human blastocysts: somatic differentiation in vitro. Nature Biotechnology,

v. 18, p. 399-404, 2000.

REYNOLDS, N.; COLLIER, B.; BINGHAM, V.; GRAY, N. K.; COOKE, H. J. Translation of

the synaptonemal complex component Sycp3 is enhanced in vivo by the germcell specific

regulator Dazl. RNA, v. 13, n. 7, p. 974-981, 2007.

REYNOLDS, N.; COLLIER, B.; MARATOU, K.; BINGHAM, V.; SPEED, R. M.; TAGGART,

M.; SEMPLE, C. A.; GRAY, N. K.; COOKE, H. J. Dazl binds in vivo to specific transcripts and

can regulate the pre-meiotic translation of Mvh in germ cells. Human Molecular Genetics, v.

14, n. 24, p. 3899-3909, 2005.

RICHARDS, M.; FONG, C.Y.; BONGSO A. Comparative evaluation of different in vitro

systems that stimulate germ cell differentiation in human embryonic stem cells. Fertility and

Sterility, v. 93, n. 3, p. 986-994, 2010.

RODDA D. J.; CHEW J. L.; LIM, L. H.; LOH, Y. H.; WANG B.; NG H. H.; ROBSON P.

Transcriptional regulation of nanog by OCT4 and SOX2. The Journal of Biology Chemistry, v.

280, n. 26, p. 24731-24737, 2005.

ROHANI, L.; JOHNSON, A.A.; ARNOLD, A.; STOLZING, A. The aging signature: a hallmark

of induced pluripotent stem cells? Aging Cell, v. 13, n. 1, p. 2-7, 2014.

RODGERS, R. J.; IRVING-RODGERS, H. F. Formation of the ovarian follicular antrum and

follicular fluid. Biology of Reproduction, v. 82, n. 6, p. 1021-1029, 2010.

ROHWEDEL, J.; MALTSEV, V.; BOBER, E. Muscle cell differentiation of embryonic stem

cells reflects myogenesis in vivo: developmentally regulated expression of myogenic

determination genes and functional expression of ionic currents. Developmental Biology, 164, n.

1, p. 87-101, 1994.

196

ROSS, A. J.; TILMAN, C.; YAO, H.; YAO, H.; MACLAUGHLIN, D.; CAPEL, B. AMH

induces mesonephric cell migration in XX gonads. Molecular and Cellular Endocrinology, v.

211, p. 1-7, 2003.

ROSSANT, J.; TAM, P. P. Blastocyst lineage formation, early embryonic asymmetries and axis

patterning in the mouse. Development, v. 136, n. 5, p. 701-713, 2009.

ROSSI, R. O. D. S.; CUNHA, E. V.; PORTELA, A. M. L. R.; PASSOS, J. R. S.; COSTA, J. J.

N.; SILVA, A. W. B.; SARAIVA, M. V. A.; PEIXOTO, C. A.; DONATO, M. A. M.; HURK, R

VAN DEN; SILVA J. R.V. Influence of BMP-2 on early follicular development and mRNA

expression of oocyte specific genes in bovine preantral follicles cultured in vitro. Histology and

Histopathology, v. 31, p. 339-348, 2016.

RUGGIU, M.; SPEED, R.; TAGGART, M.; MCKAY, S. J.; KILANOWSKI, F.; SAUNDERS,

P.; DORIN, J.; COOKE, H. J. The mouse Dazla gene encodes a cytoplasmic protein essential for

gametogenesis. Nature, v. 389, n. 6646, p. 73-77, 1997.

RÜSSE, I. Oogenesis in cattle and sheep. Bibliotheca Anatômica, v. 24, p. 77-92, 1983.

SACHS, P. C.; FRANCIS, M. P.; ZHAO, M.; BRUMELLE, J.; RAO, R. R.; ELMORE, L. W.;

HOLT, S. E. Defining essential stem cell characteristics in adipose-derived stromal cells

extracted from distinct anatomical sites. Cell and Tissue Research, v. 349, n. 2, p. 505-515,

2012.

SAFFMAN, E. E.; LASKO, P. Germline development in vertebrates and invertebrates. Cellular

and Molecular Life Sciences, v. 55, n. 8-9, p. 1141-1163, 1999.

SAITOU, M.; BARTON, S. C.; SURANI, M. A. A molecular programme for the specification of

germ cell fate in mice. Nature, v. 418, p. 293-300, 2002.

SAITOU, M.; KAGIWADA, S.; KURIMOTO, K. Epigenetic reprogramming in mouse pre-

implantation development and primordial germ cells. Development, v. 139, p. 15-31, 2012.

SAITOU, M.; YAMAJI, M. Germ cell specification in mice: Signaling, transcription regulation,

and epigenetic consequences. Reproduction, v. 139, p. 931-942, 2010.

SAITOU, M.; YAMAJI, M. Primordial germ cells in mice. Cold Spring Harbor Perspectives in

Biology, v. 4, n. 11, 2012.

SALVADOR, L. M.; SILVA, C. P.; KOSTETSKII, I.; RADICE, G. L.; STRAUSS, J. F. The

promoter of the oocyte-specific gene, Gdf9, is active in population of cultured mouse embryonic

stem cells with an oocyte-like phenotype. Methods, v. 45, n. 2, p. 172-181, 2008.

SATO, M.; KIMURA, T.; KUROKAWA, K.; FUJITA, Y.; ABE, K.; MASUHARA, M.;

YASUNAGA, T.; RYO, A.; YAMAMOTO, M.; NAKANO, T. Identification of PGC7, a new

gene expressed specifically in preimplantation embryos and germ cells. Mechanisms of

Development, v. 113, p. 91-94, 2002.

197

SAWYER, H. R.; SMITH, P.; HEATH, D. A.; JUENGEL, J. L.; WAKEFIELD, S. J.;

MCNATTY, K. P. Formation of ovarian follicles during fetal development in sheep. Biology of

Reproduction, v. 66, p. 1134-1150, 2002.

SCHENKE-LAYLAND, K.; BRUCKER, S.Y. Prospects for regenerative medicine approaches in

women's health. Journal of Anatomy, v. 227, n. 6, p. 781-785, 2015.

SCHEPER, W.; COPRAY, S. The molecular mechanism of induced pluripotency: a two-stage

switch. Stem Cell Reviews, v. 5, n. 3, p. 204-223, 2009.

SCHMIDT, L.; SOBOTKA, T.; BENTZEN, J. G.; NYBOE ANDERSEN, A. Demographic and

medical consequences of the postponement of parenthood. Human Reproduction Update, v. 18,

p. 29-43, 2012.

SCHOENFELDER, M.; EINSPANIER, R. Expression of hyaluronan synthases and

corresponding hyaluronan receptors is differentially regulated during oocyte maturation in cattle.

Biology of Reproduction, v. 69, n. 1, p. 269-277, 2003.

SELESNIEMI, K.; LEE, H-J.; NIIKURA, T.; TILLY J.L. Young adult donor bone marrow

infusions into female mice postpone age-related reproductive failure and improve offspring

survival. Aging, v. 1, n. 1, p. 49-57, 2009.

SHAH, S. M; SAINI, N; ASHRAF, S; SINGH, M. K; MANIK, R. S; SINGLA, S. K; PALTA, P;

CHAUHAN, M. S. Bone morphogenetic protein 4 (BMP4) induces buffalo (Bubalus bubalis)

embryonic stem cell differentiation into germ cells. Biochimie, v. 119, p. 113-124, 2015a.

SHAH, S. M; SAINI, N; ASHRAF, S; SINGH, M. K; MANIK, R; SINGLA, S. K; PALTA, P;

CHAUHAN, M. S. Development of buffalo (Bubalus bubalis) embryonic stem cell lines from

somatic cell nuclear transferred blastocysts. Stem Cell Research, v. 15, n. 3, p. 633-639, 2015b.

SHALGI, R.; KRAICER, P.; RIMON, A.; PINTO, M.; SOFERMAN, N. Proteins of human

follicular fluid: the blood-follicle barrier. Fertility and Sterility, v. 24, n. 6, p. 429-434, 1973.

SHI, Y.; DESPONTS, C.; DO, J.T.; HAHM, H.S.; SCHOLER, H.R.; DING, S. Induction of

pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule

compounds. Cell stem cell, v. 3, n. 5, p. 568-574, 2008.

SHI, G.; JIN, Y. Role of Oct4 in maintaining and regaining stem cell pluripotency. Stem Cell

Research & Therapy, v. 1, n. 39, 2010.

SHI, W.; WANG, H.; PAN, G.; GENG, Y.; GUO, Y.; PEI, D. Regulation of the pluripotency

marker Rex-1 by Nanog and Sox2. The Journal of Biological Chemistry, v. 281, p. 23319-

23325, 2006.

SHU, J.; WU, C.; WU, Y.; LI, Z.; SHAO, S.; ZHAO, W.; TANG, X.; YANG, H.; SHEN, L.;

ZUO, X.; YANG, W.; SHI, Y.; CHI, X.; ZHANG, H.; GAO, G.; SHU, Y.; YUAN, K.; HE, W.;

198

TANG, C.; ZHAO, Y.; DENG, H. Induction of pluripotency in mouse somatic cells with lineage

specifiers. Cell, v. 153, n. 5, p. 963-975, 2013.

SILVESTRIS, E.; D’ORONZO, S.; CAFFORIO, P.; D’AMATO, G.; LOVERRO, G. Perspective

in infertility: the ovarian stem cells. Journal of Ovarian Research, n. 8:55, 2015.

SINGH, V. K.; KALSAN, M.; KUMAR, N.; SAINI, A.; CHANDRA, R. Induced pluripotent

stem cells: applications in regenerative medicine, disease modeling, and drug discovery.

Frontiers in Cell and Developmental Biology, v. 3, n. 2, 2015.

SINGHAL, D. K.; MALIK, H. N.; SINGHAL, R.; SAUGANDHIKA, S.; DUBEY, A.;

BOATENG, S.; KUMAR, S.; KAUSHIK, J. K.; MOHANTY, A. K.; MALAKAR, D. 199 germ-

cell-like cells generation from goat induced pluripotent stem cells. Reproduction, Fertility and

Development, v. 26, n. 1, p. 214-214, 2013.

SINGHAL, D. K.; SINGHAL, R.; MALIK, H. N.; SINGH, S.; KUMAR, S.; KAUSHIK, J. K.;

MOHANTY, A. K.; DHRUBA, M. Generation of Germ Cell-Like Cells and Oocyte-Like Cells

from Goat Induced Pluripotent Stem Cells. Journal Stem Cell Research and Therapy, v. 5, n.

279, p. 2, 2015.

SINGHAL, N.; ESCH, D.; STEHLING, M.; SCHÖLER, H.R. BRG1 Is Required to Maintain

Pluripotency of Murine Embryonic Stem Cells. BioResearch open access, v. 3, n. 1, p. 1-8, 2014

SINGHAL, V. K.; KALSAN, M.; KUMAR, N.; SAINI, A.; CHANDRA, R. Induced pluripotent

stem cells: applications in regenerative medicine, disease modeling, and drug discovery.

Frontiers in Cell and Developmental Biology, v. 3:2, 2015.

SKOWRONSKI, M. T.; KWON, T. H.; NIELSEN, S. Immunolocalization of aquaporin 1, 5, and

9 in the female pig reproductive system. Journal of Histochemistry & Cytochemistry, v. 57, n.

1, p. 61-67, 2009.

SOLDNER, F.; HOCKEMEYER, D.; BEARD, C.; GAO, Q.; BELL, G.W.; COOK, E.G.;

HARGUS, G.; BLAK, A.; COOPER, O.; MITALIPOVA, M.; ISACSON, O.; JAENISC, R.

Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming

factors. Cell, v. 136, n. 5, p. 964-977, 2009.

SON, M.-Y.; CHOI, H.; HAN, Y.-M.; CHO, Y. S. Unveiling the critical role of REX1 in the

regulation of human stem cell pluripotency. Stem Cells, v. 31, n. 11, p. 2374-2387, 2013.

SONG, S-H.; KUMAR, B.M.; KANG, E-J.; LEE, Y-M.; KIM, T-H.; OCK, S-A.; LEE, S.L.;

JEON, B.G.; RHO, G.J. Characterization of Porcine Multipotent Stem/Stromal Cells Derived

from Skin, Adipose, and Ovarian Tissues and Their Differentiation In Vitro into Putative Oocyte-

Like Cells. Stem cells and development, v. 20, n. 8, p. 1359-1370, 2011.

SOTO-SUAZO, M.; ZORN, T. M. Primordial germ cells migration: morphological and

molecular aspects. Animal Reproduction, v. 2, p. 147-160, 2005.

199

SPEED, R. M. Meiosis in the foetal mouse ovary. I. An analysis at the light microscope level

using surface-spreading. Chromosoma, v. 85, p. 427-437, 1982.

SRIRAMAN, K.; BHARTIYA, D.; ANAND, S.; BHUTDA, S. Mouse Ovarian Very Small

Embryonic-Like Stem Cells Resist Chemotherapy and Retain Ability to Initiate Oocyte-Specific

Differentiation. Reproductive Sciences, v.22, n. 7, p.884-903, 2015.

STADTFELD, M.; NAGAYA, M.; UTIKAL, J.; WEIR, G.; HOCHEDLINGER, K. Induced

pluripotent stem cells generated without viral integration. Science, v. 322, n. 5903, p. 945-949,

2008.

ŠTEFKOVÁ, K.; PROCHÁZKOVÁ, J.; PACHERNÍK, J. Alkaline Phosphatase in Stem Cells.

Stem Cells International, v. 2015, Article ID 628368, 2015.

STERNECKERT, J.; HOING, S.; SCHOLER, H. R. Concise review: Oct4 and more: the

reprogramming expressway. Stem Cells, v. 30, p. 15-21, 2012.

STIMPFEL, M.; SKUTELLA, T.; CVJETICANIN, B.; MEZNARIC, M.; DOVC, P.;

NOVAKOVIC, S.; CERKOVNIK, P.; VRTACNIK-BOKAL, E.; VIRANT-KLUN, I. Isolation,

characterization and differentiation of cells expressing pluripotent/multipotent markers from adult

human ovaries. Cell and Tissue Research, v. 354, p. 593-607, 2013.

STRIOGA, M.; VISWANATHAN, S.; DARINSKAS, A.; SLABY, O.; MICHALEK, J. Same or

not the same? Comparison of adipose tissue-derived versus bone marrow-derived mesenchymal

stem and stromal cells. Stem Cells and Development, v. 21, n. 14, p. 2724-2752, 2012.

SUH, C. S.; SONNTAG, B.; ERICKSON, G. F. The ovarian life cycle: a contemporary view.

Reviews in Endocrine & Metabolic Disorders, v. 3, n. 1, p. 5-12, 2002.

TADDEI, A.; ROCHE, D.; BICKMORE, W. A.; ALMOUZNI, G. The effects of histone

deacetylase inhibitors on heterochromatin: implications for anticancer therapy? EMBO Reports,

v. 6, n. 6, p. 520-524, 2005.

TAKAHASHI, K.; TANABE, K.; OHNUKI, M.; NARITA, M.; ICHISAKA, T.; TOMODA, K.;

YAMANAKA, S. Induction of pluripotent stem cells from adult human fibroblasts by defined

factors. Cell, v. 131, p. 861-872, 2007.

TAKAHASHI, K.; YAMANAKA, S. Induction of pluripotent stem cells from mouse embryonic

and adult fibroblast cultures by defined factors. Cell, v. 126, p. 663-676, 2006.

TAKEMITSU, H.; ZHAO, D.; YAMAMOTO, I.; HARADA, Y.; MICHISHITA, M.; ARAI, T.

Comparison of bone marrow and adipose tissue-derived canine mesenchymal stem cells. BMC

Veterinary Research, v. 8:150, 2012.

TAM, P. P. L.; SNOW, M. H. L. Proliferation and migration of primordial germ cells during

compensatory growth in mouse embryos. Journal of Embryology and Experimental

Morphology, v. 64, p. 133-147, 1981.

200

TANABE, K.; NAKAMURA, M.; NARITA, M.; TAKAHASHI, K.; YAMANAKA, S.

Maturation, not initiation, is the major roadblock during reprogramming toward pluripotency

from human fibroblasts. Proceedings of the National Academy of Sciences, v. 110, n. 30, p.

12172-12179, 2013.

TANABE, K.; TAKAHASHI, K.; YAMANAKA, S. Induction of pluripotency by defined

factors. Proceedings of the Japan Academy, Series B, v. 90, n. 3, p. 83-96, 2014.

TAKAI, H.; SMOGORZEWSKA, A.; DE LANGE, T. DNA damage foci at dysfunctional

telomeres. Current Biology, v. 13, n. 17, p. 1549–1556, 2003.

TAKEBE, T.; SEKINE, K.; ENOMURA, M.; KOIKE, H.; KIMURA, M.; OGAERI, T.;

ZHANG, R. R.; UENO, Y.; ZHENG, Y. W.; KOIKE, N.; AOYAMA, S.; ADACHI, Y.;

TANIGUCHI, H. Vascularized and functional human liver from an iPSC-derived organ bud

transplant. Nature, v. 499, n. 7459, p. 481-484, 2013.

TANAKA, S. S.; MATSUI, Y. Developmentally regulated expression of mil-1 and mil-2, mouse

interferon-induced transmembrane protein like genes, during formation and differentiation of

primordial germ cells. Mechanisms of Development, v. 119, p. S261-S267, 2002.

TAYLOR, S. M.; JONES, P. A. Multiple new phenotypes induced in 10T1/2 and 3T3 cells

treated with 5-azacytidine. Cell, v. 17, n. 4, p. 771-779, 1979.

TAYLOR, S. M.; JONES, P. A. Changes in phenotypic expression in embryonic and adult cells

treated with 5-azacytidine. Journal of Cellular Physiology, v. 111, n. 2, p. 187-194, 1982.

TELFER, E. E.; ALBERTINI, D. F. The quest for human ovarian stem cells. Nature medicine,

v. 18, n. 3, p. 413-421, 2012.

TILLY, J. L.; SINCLAIR, D. A. Germline energetics, aging, and female infertility.

Cell metabolism, v. 17, n. 6, p. 838-850, 2013.

TERADA, Y.; NAKASHIMA, O.; INOSHITA, S.; KUWAHARA, M.; SASAKI, S.; MARUMO,

F. Mitogen-activated protein kinase cascade and transcription factors: the opposite role of

MKK3/6-p38K and MKK1-MAPK. Nephrology Dialysis Transplantation, v. 14, p. 45-47,

1999.

THOMSON, J. A.; ITSKOVITZ-ELDOR, J.; SHAPIRO, S. S.; WAKNITZ, M. A.;

SWIERGIEL, J. J.; MARSHALL, V. S.; JONES, J. M. Embryonic stem cell lines derived from

human blastocysts. Science, v. 282, p. 1145-1147, 1998.

TILGNER, K.; ATKINSON, S. P.; GOLEBIEWSKA, A.; STOJKOVIC, M.; LAKO, M.;

ARMSTRONG, L. Isolation of primordial germ cells from differentiating human embryonic stem

cells. Stem Cells, v. 26, p. 3075-3085, 2008.

201

TILGNER, K.; ATKINSON, S. P.; YUNG, S.; GOLEBIEWSKA, A.; STOJKOVIC, M.;

MORENO, R.; LAKO, M.; ARMSTRONG, L. Expression of GFP under the control of the RNA

helicase VASA permits fluorescence-activated cell sorting isolation of human primordial

germ cells. Stem Cells, v. 28, n. 1, p. 84-92, 2010.

TILL, J. E.; MCCULLOCH, E. A.; SIMINOVITCH, L. A stochastic model of stem cell

proliferation, based on the growth of spleen colony-forming cells. Proceedings of the National

Academy of Sciences of the United State of America, v. 51, n. 1, p. 29-36, 1964.

TILLY, J. L.; JOHNSON, J. Recent arguments against germ cell renewal in the adult human

ovary. Cell cycle, v. 8, p. 879-883, 2007.

TINGEN, C.; KIM, A.; WOODRUFF, T. K. The primordial pool of follicles and nest breakdown

in mammalian ovaries. Molecular Human Reproduction, v. 15, n. 12, p. 795-803, 2009.

TOYOOKA, Y.; TSUNEKAWA, N.; AKASU, R.; NOCE, T. Embryonic stem cells can form

germ cells in vitro. Proceedings of the National Academy of Sciences of the United States of

America, v. 100, p. 11457-11462, 2003.

TOYOOKA, Y.; TSUNEKAWA, N.; TAKAHASHI, Y.; MATSUI, Y.; SATOH, M.; NOCE, T.

Expression and intracellular localization of mouse Vasa-homologue protein during germ cell

development. Mechanisms of development, v. 93, n. 1, p. 139-149, 2000.

TSUI, S.; DAI, T.; WARREN, S. T.; SALIDO, E. C.; YEN, P. H. Association of the mouse

infertility factor DAZL1 with actively translating polyribosomes. Biology of Reproduction, v.

62, n. 6, p. 1655-1660, 2000.

VAYENA, E.; ROWE, P. J.; GRIFFIN, P. D. (eds). Medical, ethical & social aspects of assisted

reproduction. Current practices & controversies in assisted reproduction: Report of a WHO

meeting, 2001: Geneva, Switzerland.

VAN DEN HURK R., ZHAO J. Formation of mammalian oocytes and their growth,

differentiation and maturation within ovarian follicles. Theriogenology, v. 63, p. 1717–1751,

2005.

VASCONCELOS, G. L.; SARAIVA, M. V.; COSTA, J. J.; PASSOS, M. J.; SILVA, A. W.;

ROSSI, R. O.; PORTELA, A. M.; DUARTE, A. B.; MAGALHÃES-PADILHA D. M.;

CAMPELO, C. C.; FIGUEIREDO, J. R.; VAN DEN HURK, R.; SILVA, J. R.V. Effects of

growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression

in bovine preantral follicles. Reproduction, Fertility and Development, v. 25, n. 8, p. 1194-

1203, 2013.

VIDANE, A. S.; ZOMER, H. D.; OLIVEIRA, B. M.; GUIMARÃES, C. F.; FERNANDES, C.

B.; PERECIN, F.; SILVA, L. A.; MIGLINO, M. A.; MEIRELLES, F. V.; AMBRÓSIO, C. E.

Reproductive stem cell differentiation: extracellular matrix, tissue microenvironment, and growth

factors direct the mesenchymal stem cell lineage commitment. Reproductive Sciences, v. 20, n.

10, p. 1137-1143, 2013.

202

VINCENT, S. D.; DUNN, N. R.; SCIAMMAS, R.; SHAPIRO-SHALEF, M.; DAVIS, M. M.;

CALAME, K.; BIKOFF, E. K.; ROBERTSON, E. J. The zinc finger transcriptional repressor

Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of

primordial germ cells in the mouse. Development, v. 132, p. 1315-1325, 2005.

VIRANT-KLUN I.; STIMPFEL, M.; CVJETICANIN, B.; VRTACNIK-BOKAL, E.;

SKUTELLA, T. Small SSEA-4-positive cells from human ovarian cell cultures: related to

embryonic stem cells and germinal lineage? Journal of ovarian research, v. 6, n. 1, p. 1, 2013.

VIRANT-KLUN, I.; ROZMAN, P.; CVJETICANIN, B.; VRTACNIK-BOKAL, E.;

NOVAKOVIC, S.; RÜLICKE, T.; DOVC, P.; MEDEN-VRTOVEC, H. Parthenogenetic

embryo-like structures in the human ovarian surface epithelium cell culture in postmenopausal

women with no naturally present follicles and oocytes. Stem Cells and Development, v. 18, p.

137-150, 2009.

VIRANT-KLUN, I.; SKUTELLA, T. Stem cells in aged mammalian ovaries. Aging, v. 2, n. 1, p.

3-6, 2010.

VIRANT-KLUN, I.; SKUTELLA, T.; KUBISTA, M.; VOGLER, A.; SINKOVEC, J.; MEDEN-

VRTOVEC, H. Expression of pluripotency and oocyte-related genes in single putative stem cells

from human adult ovarian surface epithelium cultured in vitro in the presence of follicular fluid.

BioMed Research International, 2013, 861460, 2013.

VIRANT-KLUN, I.; STIMPFEL, M.; SKUTELLA, T. Ovarian pluripotent/multipotent stem cells

and in vitro oogenesis in mammals. Histology and histopathology, v. 26, n. 8, p. 1071-1082,

2011.

VIRANT-KLUN, I.; ZECH, N.; ROZMAN, P.; VOGLER, A.; CVJETICANIN, B.; KLEMENC

P.; MALICEV, E.; MEDEN-VRTOVEC, H. Putative stem cells with an embryonic character

isolated from the ovarian surface epithelium of women with no naturally present follicles and

oocytes. Differentiation, v. 76, n. 8, p. 843-856, 2008.

VOLAREVIC, V.; BOJIC, S.; NURKOVIC, J.; VOLAREVIC, A.; LJUJIC, B.; ARSENIJEVIC,

N.; LAKO, M.; STOJKOVIC, M. Stem Cells as New Agents for the Treatment of Infertility:

Current and Future Perspectives and Challenges. BioMed research international, v. 2014, 2014.

WAKITANI, S.; TAKAOKA, K.; HATTORI, T.; MIYAZAWA, N.; IWANAGA, T.; TAKEDA,

S.; WATANABE, T. K.; TANIGAMI, A. Embryonic stem cells injected into the mouse knee

joint form teratomas and subsequently destroy the joint. Rheumatology, v. 42, p. 162-165, 2003.

WANG, J, ROY, S. K. Growth differentiation factor-9 and stem cell factor promote primordial

follicle formation in the hamster, p. modulation by follicle-stimulating hormone. Biology of

Reproduction, v.70, n. 3, p. 577–585, 2004.

203

WANG, Y.; HU, J.; JIAO, J.; LIU, Z.; ZHOU, Z.; ZHAO, C.; CHANG, L. J.; CHEN, Y. E.; MA,

P. X.; YANG, B. Engineering vascular tissue with functional smooth muscle cells derived from

human iPS cells and nanofibrous scaffolds. Biomaterials, v. 35, n. 32, p. 8960-8969, 2014.

WANG, Y.; MAH, N.; PRIGIONE, A.; WOLFRUM, K.; ANDRADE-NAVARRO, M. A.;

ADJAYE, J. A transcriptional roadmap to the induction of pluripotency in somatic cells. Stem

Cell Reviews, v. 6, n. 2, p. 282-296, 2010.

WANG, Z.; ORON, E.; NELSON, B.; RAZIS, S.; IVANOVA, N. Distinct lineage specification

roles for NANOG, OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell, v. 10, p.

440-454, 2012.

WARREN, L.; MANOS, P. D.; AHFELDT, T.; LOH, Y. H.; LI, H.; LAU, F.; EBINA, W.;

MANDAL, P. K.; SMITH, Z. D.; MEISSNER, A.; DALEY, G. Q.; BRACK, A. S.; COLLINS, J.

J.; COWAN, C.; SCHLAEGER, T. M.; ROSSI, D. J. Highly efficient reprogramming to

pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell

Stem Cell, v. 7, n. 5, p. 618-630, 2010.

WATT, F. M.; HOGAN, B. L. M. Out of Eden: stem cells and their niches. Science, v. 287, p.

1427-1430, 2000.

WEBB, R.; NICHOLAS, B.; GONG, J. G.; CAMPBELL, B. K.; GUTIERREZ, C. G.;

GARVERICK; H. A.; ARMSTRONG, D. G. Mechanisms regulating follicular development and

selection of the dominant follicle. Reproduction Supplement, n. 61, p.71-90, 2003.

WEBER, S.; ECKERT, D.; NETTERSHEIM, D.; GILLIS, A. J.; SCHÄFER, S.;

KUCKENBERG, P.; EHLERMANN, J.; WERLING, U.; BIERMANN, K.; LOOIJENGA, L. H.;

SCHORLE, H. Critical function of AP-2g/TCFAP2C in mouse embryonic germ cell

maintenance. Biology of Reproduction, v. 82, p. 214-223, 2010.

WEBSTER, R. A.; BLABER, S. P.; HERBERT, B. R.; WILKINS, M. R.; VESEY, G. The role

of mesenchymal stem cells in veterinary therapeutics – a review. New Zealand Veterinary

Journal, v. 60, n. 5, p. 265-272, 2012.

WEI, W; QING, T; YE, X; LIU, H; ZHANG, D; YANG, W; DENG, H. Primordial germcell

specification from embryonic stem cells. PLoS One, v. 3, n. 12, 2008.

WERNIG, M.; MEISSNER, A.; FOREMAN, R.; BRAMBRINK, T.; KU, M.;

HOCHEDLINGER, K.; BERNSTEIN, B. E.; JAENISCH, R. In vitro reprogramming of

fibroblasts into a pluripotent ES cell-like state. Nature, v. 448, p. 318-324, 2007.b

WEST, J.A.; PARK, I.H.; DALEY, G.Q.; GEIJSEN, N. In vitro generation of germ cells from

murine embryonic stem cells. Nature protocols, v. 1, n. 4, p. 2026-2036, 2006.

WEST, J. A.; VISWANATHAN, S. R.; YABUUCHI, A.; CUNNIFF, K.; TAKEUCHI, A.;

PARK, I. H.; SERO, J. E.; ZHU, H.; PEREZ-ATAYDE, A.; FRAZIER, A. L.; SURANI, M. A.;

204

DALEY, G. Q. A role for Lin28 in primordial germ-cell development and germ-cell malignancy.

Nature, v. 460, p. 909-913, 2009.

WEST, F. D.; ROCHE-RIOS, M. I.; ABRAHAM, S.; RAO, R. R.; NATRAJAN, M. S.;

BACANAMWO, M.; STICE, S. L. KIT ligand and bone morphogenetic protein signaling

enhances human embryonic stem cell to germ-like cell differentiation. Human Reproduction, v.

25, n. 1, p. 168-178, 2010.

WEST, F. D.; MACHACEK, D. W.; BOYD, N. L.; PANDIYAN, K.; ROBBINS, K. R.; STICE,

S. L. Enrichment and differentiation of human germ-like cells mediated by feeder cells and basic

fibroblast growth factor signaling. Stem Cells, v. 26, p. 2768-2776, 2008.

WHITE, Y. A.; WOODS, D. C.; TAKAI, Y.; ISHIHARA, O.; SEKI, H.; TILLY, J. L. Oocyte

formation by mitotically active germ cells purified from ovaries of reproductive age women.

Nature Medicine v. 18, p. 413-21, 2012.

WOODS, D.C.; TELFER, E.E.; TILLY, J.L. Oocyte Family Trees: Old Branches or New Stems?

PLoS Genet, v. 8, n. 7, p. e1002848, 2012.

WOODS, D.C.; WHITE, Y.A.; NIIKURA ,Y.; KIATPONGSAN, S.; LEE, H.J.; TILLY, J.L.

Embryonic stem cell-derived granulosa cells participate in ovarian follicle formation in vitro and

in vivo. Reproductive Sciences, v. 20, p. 524-535, 2013.

WU, G.; SCHÖLER, H. R. Role of Oct4 in the early embryo development. Cell Regeneration, v.

3, n. 1, p. 1, 2014.

WU, Y. T.; TANG, L.; CAI, J.; LU, X. E.; XU, J.; ZHU, X. M.; LUO, Q.; HUANG, H. F. High

bone morphogenetic protein-15 level in follicular fluid is associated with high quality oocyte and

subsequent embryonic development. Human Reproduction, v. 22, n. 6, p. 1526-1531, 2007.

XIAO, L.; TSUTSUI, T. Characterization of human dental pulp cells-derived spheroids in serum-

free medium: Stem cells in the core. Journal of Cellular Biochemistry, v. 114, n. 11, p. 2624-

2636, 2013.

XU, R. H.; CHEN, X.; LI, D. S.; LI, R.; ADDICKS, G. C.; GLENNON, C.; ZWAKA, T. P.;

THOMSON, J. A. BMP4 initiates human embryonic stem cell differentiation to trophoblast.

Nature Biotechnology, v. 20, p. 1261-1264, 2002.

XU, Y.; WEI, X.; WANG, M.; ZHANG, R.; FU, Y.; XING, M.; HUA, Q.; XIE, X. Proliferation

rate of somatic cells affects reprogramming efficiency. The Journal of Biological Chemistry, v.

288, n. 14, p. 9767-9778, 2013.

YABUTA, Y.; KURIMOTO, K.; OHINATA, Y.; SEKI, Y.; SAITOU, M. Gene expression

dynamics during germline specification in mice identified by quantitative single-cell gene

expression profiling. Biology of Reproduction, v. 75, p, 705-716, 2006.

205

YAKUBOV, E.; RECHAVI, G.; ROZENBLATT, S.; GIVOL, D. Reprogramming of human

fibroblasts to pluripotent stem cells using mRNA of four transcription factors. Biochemical and

biophysical research communications, v. 394, n. 1, p. 189-193, 2010.

YAMAGUCHI, S.; KIMURA, H.; TADA, M.; NAKATSUJI, N.; TADA, T. Nanog expression in

mouse germ cell development. Gene Expression Patterns, v. 5, p. 639-646, 2005.

YAMAJI, M.; SEKI, Y.; KURIMOTO, K.; YABUTA,Y.; YUASA, M.; SHIGETA, M.;

YAMANAKA, K.; OHINATA, Y.; SAITOU, M. Critical function of Prdm14 for the

establishment of the germ cell lineage in mice. Nature Genetics, v. 40, p. 1016-1022, 2008.

YAMANAKA, S. Strategies and new developments in the generation of patient-specific

pluripotent stem cells. Cell stem cell, v. 1, n. 1, p. 39-49, 2007.

YAMANAKA, S. Pluripotency and nuclear reprogramming. Philosophical transactions of the

Royal Society of London. Series B, Biological Sciences, v. 363, n. 1500, p. 2079-2087, 2008.

YAMANAKA, Y.; RALSTON, A.; STEPHENSON, R. O.; ROSSANT, J. Cell and molecular

regulation of the mouse blastocyst. Developmental Dynamics, v. 235, p. 2301-2314, 2006.

YEOM, Y. I.; FUHRMANN, G.; OVITT, C. E.; BREHM, A.; OHBO, K.; GROSS, M.;

HÜBNER, K.; SCHÖLER, H. R. Germline regulatory element of Oct-4 specific for the totipotent

cycle of embryonal cells. Development, v. 122, n. 3, p. 881-894, 1996.

YING, Y. L.; MARBLE, X. M.; LAWSON, A.; ZHAO, G. Q. Requirement of Bmp8b for the

generation of primordial germ cells in the mouse. Molecular Endocrinology, v. 14, p. 1053-

1063, 2000.

YING, Y.; QI, X.; ZHAO, G-Q. Induction of primordial germ cells from murine epiblasts by

synergistic action of BMP4 and BMP8B signaling pathways. Proceedings of the National

Academy of Sciences of the United States of America, v. 98, p. 7858-7862, 2001.

YING, Y.; ZHAO, G. Q. Cooperation of endoderm-derived BMP2 and extraembryonic

ectoderm-derived BMP4 in primordial germ cell generation in the mouse. Developmental

Biology, v. 232, p. 484-492, 2001.

YING, Q-L.; NICHOLS, J.; CHAMBERS, I.; SMITH, A. BMP Induction of Id Proteins

Suppresses Differentiation and Sustains Embryonic Stem Cell Self-Renewal in Collaboration

with STAT3. Cell, v. 115, p. 281-292, 2003.

YOUNG, J. C.; DIAS, V. L.; LOVELAND, K. L. Defining the window of germline genesis in

vitro from murine embryonic stem cells. Biology of Reproduction, v. 82, p. 390-401, 2010.

YUAN, L.; LIU, J.G.; ZHAO, J.; BRUNDELL, E.; DANEHOLT, B.; HOOG, C. The murine

SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male

fertility. Molecular cell, v. 5, n. 1, p. 73-83, 2000.

206

YU, H.; VU, T.H.; CHO, K.S.; GUO, C.; CHEN, D.F. Mobilizing endogenous stem cells for

retinal repair. Translational Research, v. 163, n. 4, p. 387-398, 2014.

YU, J.; HU, K.; SMUGA-OTTO, K.; TIAN, S.; STEWART, R.; SLUKVIN, I. I.; THOMSON, J.

A. Human induced pluripotent stem cells free of vector and transgene sequences. Science, v. 324,

n. 5928, p. 797-801, 2009.

YU, J.; VODYANIK, M. A.; SMUGA-OTTO, K.; ANTOSIEWICZ-BOURGET, J.; FRANE, J.

L.; TIAN, S.; NIE, J.; JONSDOTTIR, G. A.; RUOTTI, V.; STEWART, R.; SLUKVIN, I. I.;

THOMSON, J. A. Induced pluripotent stem cell lines derived from human somatic cells. Science,

v. 318, p. 1917-1920, 2007.

YU, Z.; JI, P.; CAO, J.; ZHU S.; LI, Y.; ZHENG, L.; CHEN, X.; FENG, L. Dazl Promotes Germ

Cell Differentiation from Embryonic Stem Cells. Journal of molecular cell biology, v. 1, p. 93-

103, 2009.

ZAGO, M.A.; COVAS, D.T. Células-tronco: a nova fronteira da medicina. São Paulo: Atheneu,

2006. p. 35-48.

ZARZECZNY, A.; CAULFIELD, T. Emerging ethical, legal and social issues associated with

stem cell research and the current role of the moral status of the embryo. Stem Cell Reviews and

Reports, v. 5, p. 96-101, 2009.

ZENG, M.; LU, Y.; LIAO, X.; LI, D.; SUN, H.; LIANG, S.; ZHANG, S.; MA, Y.; YANG, Z.

DAZL binds to 3’UTR of Tex19.1 mRNAs and regulates Tex19.1 expression. Molecular

Biology Reports, v. 36, n. 8, p. 2399-2403, 2009.

ZHANG, J.; LI, L. BMP signaling and stem cell regulation. Developmental Biology, 284(1), 1-

11, 2005.

ZHANG, H.; ZHENG, W.; SHEN, Y.; ADHIKARI, D.; UENO, H.; LIU, K. Experimental

evidence showing that no mitotically active female germline progenitors exist in postnatal mouse

ovaries. Proceedings of the National Academy of Sciences of the United States of America, v.

109, p. 12580-12585, 2012.

ZHANG, Y.; LI, W.; LAURENT, T.; DING, S. Small molecules, big roles – the chemical

manipulation of stem cell fate and somatic cell reprogramming. Journal of Cell Science, v. 125,

n. 23, p. 5609-5620, 2012.

ZHAO, G. Q. Consequences of knocking out BMP signaling in the mouse. Genesis, v. 35, p. 43-

56, 2003.

ZHAO, G. Q.; CHEN, Y. X.; LIU, X. M.; XU Z.; QI, X. Mutation in Bmp7 exacerbates the

phenotype of Bmp8a mutants in spermatogenesis and epididymis. Developmental Biology, v.

240, p. 212-222, 2001.

207

ZHAO, G.; DENG, K.; LABOSKY, P. A.; LIAW, L.; HOGAN, B. L. M. The gene encoding

bone morphogenetic protein 8B is required for the initiation and maintenance of spermatogenesis

in the mouse. Genes & Development, v. 10, p. 1657-1669, 1996.

ZHANG, J.; NUEBEL, E.; DALEY, G.Q.; KOEHLER, C.M.; TEITELL, M.A. Metabolic

Regulation in Pluripotent Stem Cells during Reprogramming and Self-Renewal. Cell stem cell, v.

11, n. 5, p. 589-595, 2012.

ZHAO, W.; JI, X.; ZHANG, F.; LI, L.; MA, L. Embryonic Stem Cell Markers. Molecules, v. 17,

n. 6, p. 6196-6236, 2012.

ZHOU, H.; WU, S.; JOO, J. Y.; ZHU, S.; HAN, D. W.; LIN, T.; TRAUGER, S.; BIEN, G.;

YAO, S.; ZHU, Y.; SIUZDAK, G.; SCHÖLER, H. R.; DUAN, L.; DING, S. Generation of

induced pluripotent stem cells using recombinant proteins. Cell Stem Cell, v. 4, n. 5, p. 381-384,

2009.

ZHU, J.; ZHANG, K.; SUN, Y.; GAO, X.; LI, Y.; CHEN, Z.; WU, X. Reconstruction of

functional ocular surface by acellular porcine cornea matrix scaffold and limbal stem cells

derived from human embryonic stem cells. Tissue Engineering Part A, v. 19, p. 2412-2425,

2013.

ZHU, S.; LI, W.; ZHOU, H.; WEI, W.; AMBASUDHAN, R.; LIN, T.; KIM, J.; ZHANG, K.;

DING, S. Reprogramming of human primary somatic cells by OCT4 and chemical compounds.


ZHU, Y.; HU, H. L.; LI, P.; YANG, S.; ZHANG, W.; DING, H.; TIAN, R. H.; NING, Y.;

ZHANG, L. L.; GUO, X. Z.; SHI, Z. P.; LI, Z.; HE, Z. Generation of male germ cells from

induced pluripotent stem cells (iPS cells): an in vitro and in vivo study. Asian Journal of

Andrology, v. 14, n. 4, p. 574-579, 2012.

ZOMER, H. D.; VIDANE, A. S.; GONÇALVES, N. N.; AMBRÓSIO, C. E. Mesenchymal and

induced pluripotent stem cells: general insights and clinical perspectives. Stem Cells and

Cloning: Advances and Applications, v. 8, p. 125-134, 2015.

ZOU, K.; YUAN, Z.; YANG, Z.; LUO, H.; SUN, K.; ZHOU, L.; XIANG, J.; SHI, L.; YU, Q.;

ZHANG, Y.; HOU, R.; WU, J. Production of offspring from a germline stem cell line derived

from neonatal ovaries. Nature Cell Biology, v. 11, p. 631-636, 2009.

ZUCKERMAN, S. The number of oocytes in the mature ovary. Recent Progress in Hormone

Research, v. 6, p. 63-108, 1951.

ZWAKA, T. P.; THOMSON, J. A. A germ cell origin of embryonic stem cells? Development, v.

132, p. 227-233, 2005.

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