UNIVERSITI PUTRA MALAYSIA

PARVIN SALEHINEJAD

FBSB 2012 16

IN VITRO STUDY OF VARIOUS FACTORS IN ISOLATION, EXPANSION AND DIFFERENTIATION OF MESENCHYMAL STEM CELLS FROM

HUMAN UMBILICAL CORD

© COPYRIG

HT UPM

IN VITRO STUDY OF VARIOUS FACTORS IN ISOLATION, EXPANSION AND DIFFERENTIATION OF MESENCHYMAL STEM CELLS FROM

HUMAN UMBILICAL CORD

By

PARVIN SALEHINEJAD

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirement for the Degree of Doctor of Philosophy

June 2012

© COPYRIG

HT UPM

ii

Abstract of Thesis Presented to the Senate of Universiti Putra Malaysia, in Fulfillment of the Requirement for the Degree of Doctor of Philosophy

IN VITRO STUDY OF VARIOUS FACTORS IN ISOLATION, EXPANSION AND DIFFERENTIATION OF MESENCHYMAL STEM CELLS FROM

HUMAN UMBILICAL CORD

By

PARVIN SALEHINEJAD

June 2012

Chairman: Noorjahan Banu Binti Mohamed Alitheen, PhD

Faculty: Biotechnology and Biomolecular Sciences

The Wharton’s Jelly (WJ) of the umbilical cord is a rich source of mesenchymal

stem cells (MSCs) with a various potential therapeutic applications. Human

umbilical cord mesenchymal stem cells (hUCMCs) can be isolated with different

methods, expanded and differentiated into neuron, astrocyte and oligodendrocyte in

vitro.

In the first study, four methods to isolate hUCMCs were compared. Three of them

were based on using an enzyme cocktail containing collagenase, hyaluronidase and

trypsin (CHT), collagenase /trypsin (CT) or trypsin alone (TRP) and the other was

based on the explant culture (Exp) method. In the second experiment, the

competence of DMEM/ F12 and alpha-MEM/Glutamax (α-MEM/GL), also the

epidermal growth factor (EGF) and fibroblast growth factor (FGF) role to expand

hUCMCs were evaluated. Finally, hUCMCs were examined for their differentiation

rate into neural lineage cells by six various cocktails made of a base media

© COPYRIG

HT UPM

iii

(DMEM/LG), 10 % FBS, retinoic acid (RA), dimethyl sulfoxide (DMSO), EGF and

FGF.

Comparison of the four isolation methods of the hUCMCs showed that more cells

were isolated in the CT, CHT and TRP groups, respectively. Cells were successfully

isolated in TRP group but they did not propagate normally. Flow cytometry analysis

revealed that CD44, CD73, CD90 and CD105 were expressed on the cell surface in

all groups, but there was no expression of hematopoietic lineage markers, CD34 and

CD45, in any of the groups. In addition, the expression of C-kit and OCT-4 in

enzymatic (CT and CHT) groups was greater than Exp group. One-way ANOVA

showed that cell activity rate in the Exp group was significantly higher (P<0.001)

than the other groups. A positive alkaline phosphatase activity and differentiation to

adipogenic and osteogenic lineage was detected in the hUCMCs of all groups.

The ratio of CD44 and CD105 expression cells were similar in DMEM/F12 and α-

MEM/GL groups, while in both of the groups expression of the hematopoietic cells

surface marker CD34 were negative. Differentiation potential into adipogenic and

osteogenic cells were similar in both groups. Student`s t-test for evaluation of cell

metabolism with WST-1 showed that the quality of the expanded cells in α-

MEM/GL group was significantly higher (P<0.001) than DMEM/F12

group. Population doubling time (PDT) was calculated at 60 hours for DMEM/F12

group, while it was 47 hours in α-MEM/GL group that this difference was

statistically significant (P<0.001). Data sets were compared using two- way

ANOVA.

© COPYRIG

HT UPM

iv

Semi-quantitative RT-PCR revealed that the TERT in hUCMs/EGF was 0.26 ± 0.06.

Cell cycle analysis showed that EGF has caused 37.34± 9.8% and 7.71± 0.53% of the

hUCMs to move in the S and G2/M phases, respectively. While, after exposure to

FGF 29.92± 2.55% and 9.92± 0.24% of the cells progressed to S and G2/M phases,

respectively.

Induction of neuronal differentiation with various cocktails showed that there was no

statistically significant difference (one-way ANOVA with a post hoc Tukey’s test)

among the groups. Despite, the cocktail consist of DMEM/LG, FBS, RA, FGF, and

EGF (DF/R/Fg/E group) resulted in the expression of the highest percentage of

nestin, ß-tubulin III, neurofilament, and CNPase 69.7 ± 6%, 76 ±1.7%, 89 ±9.5%,

and 16.3 ±5.2%, respectively. Also, in this group and the DF/Ds/Fg/E group, the

highest cell proliferation was observed. On the other hand, DF/Ds/Fg/E group had

the highest percentage of GFAP expression (36 ±12.5%). While, the expression

level of NF, GFAP, and CNPase was at least in the DF group. The least percentage

of nestin and ß-tubulin III expression was observed in the DF/Ds group.

It may be concluded that isolation technique affects both the amount and the quality

of the isolated hUCMCs. When collagenase/trypsin enzymes are used for isolation of

hUCMCs, the waiting time for primary culture is shorter, the number of isolated cells

is higher and pluripotency properties of them were greater. The type of culture media

also affects the quality of the harvested cells. α-MEM/GL supports hUCMCs growth

more strongly than DMEM/F12. Furthermore, growth factors also affect the TERT

expression and cell cycle phases. In this case, EGF increases TERT expression and

the number of cells that progress towards S/G2/M phases. In addition, hUCMCs

© COPYRIG

HT UPM

v

differentiate into various neuronal cell types successfully; the amount of different

neuronal cell types is influenced by inducing agents. FGF and EGF are important

inducers for differentiation of hUCMCs into neuron, astrocyte and oligodendrocyte.

RA can induce hUCMCs to differentiate into neuron and oligodendrocyte; while in

contribution to astrocyte differentiation, DMSO had a pivotal role.

From this study, it can be concluded that different isolation methods can effect on

characteristics of the mesenchymal cells from human Wharton’s Jelly. These cells

are able to expand in culture with various media and growth factors and differentiate

into neural cells.

© COPYRIG

HT UPM

vi

Abstrak Tesis Untuk Dikemukakan Kepada Senat Universiti Putra Malaysia Sebagai Memenuhi Keperluan Untuk Ijazah Doktor Falsafah

KAJIAN IN VITRO MENGENAI PERBAGAI FAKTOR DALAM PEMENCILAN, PERKEMBANGAN, DAN PEMBEZAAN SEL INDUK

MESENKIMAL DARI TALI PUSAT MANUSIA

Oleh

PARVIN SALEHINEJAD

June 2012

Pengerusi: Noorjahan Banu Binti Mohammed Alitheen, PhD Fakulti: Bioteknologi dan Sains Biomolekul Whaton Jeli (WJ) dari tali pusat adalah sumber yang kaya dengan sel induk

mesenkimal (MSCs) dan mempunyai pelbagai potensi terapeutik. Sel induk

mesenkimal dari tali pusat manusia (hUCMCs) boleh diasingkan dengan pelbagai

kaedah, dan boleh berkembang dan dibezakan menjadi neuron, astrocyte dan

oligodendrocyte secara in vitro.

Dalam kajian pertama, empat kaedah untuk mengasingkan hUCMCs telah

dibandingkan. Tiga daripada kaedah itu berdasarkan penggunaan koktel enzim yang

mengandungi kolagenase, hyaluronidase dan trypsin (CHT), kolagenase / trypsin

(CT) atau trypsin sahaja (TRP) dan selebihnya adalah berdasarkan kaedah kultur

eksplan (Exp). Dalam kajian kedua, kemampuan media DMEM / F12, α-

MEM/Glutamax (α -MEM/GL dan juga peranan faktor pertumbuhan dalam

perkembangan hUCMCs dikaji. Akhir sekali, pemeriksaan dijalankan ke atas sel

hUCMCs untuk mengenal pasti kadar perkembangan mereka menjadi sel keturunan

© COPYRIG

HT UPM

vii

neural menggunakan kombinasi enam koktail yang dibuat daripada media (DMEM /

LG), 10% FBS, Retinoic asid (RA),sulfoxide dimetil (DMSO), EGF dan FGF.

Perbandingan antara empat kaedah pengasingan sel hUCMCs menunjukkan bahawa

lebih banyak sel dapat diasingkan dalam kumpulan CT, CHT dan TRP. Sel-sel telah

berjaya diasingkan di kumpulan TRP tetapi tidak dapat berkembang secara normal.

Analisis aliran sitometri menunjukkan bahawa CD44, CD73, CD90 dan CD105 telah

diekspreskan di atas permukaan sel dalam semua kumpulan, tetapi CD34 dan CD45

tidak diekspreskan pada keturunan hematopoietik, dalam mana-mana kumpulan. Di

samping itu, ekspresi C-kit dan OCT- 4 pada kumpulan enzim (CT dan CHT) adalah

lebih baik daripada kumpulan Exp. Kadar perkembangan sel dalam kumpulan Exp

adalah jauh lebih tinggi (P <0.001) berbanding kumpulan lain. Aktiviti fosfatase alkali

yang positif dan pembezaan kepada keturunan adipogenik dan osteogenik dapat dikesan

pada sel hUCMCs dari mana-mana kumpulan.

Ekspresi stem sel mesenkimal penanda termasuklah CD44 dan CD105 adalah sama

dalam kumpulan media DMEM/F12 dan α-MEM/GL, manakala dalam kedua-dua

kumpulan ekspresi penanda CD34 pada permukaan sel-sel hematopoietic adalah negatif.

Potensi perkembangan sel kepada sel adipogenik dan osteogenik adalah sama dalam

kedua-dua kumpulan. Penilaian kadar metabolisme sel dengan WST-1 menunjukkan

bahawa kualiti sel-sel yang berkembang dalam kumpulan media α-MEM/GL adalah

lebih tinggi (P <0.001) berbanding kumpulan DMEM/F12. Populasi masa dua kali ganda

(PDT) telah dikira pada 60 jam untuk kumpulan DMEM/F12, sementara pada kumpulan

α-MEM/GL adalah 47 jam. Perbezaan ini adalah signifikan secara statistik (P <0.001).

© COPYRIG

HT UPM

viii

Separa-kuantitatif RT-PCR telah menunjukkan bahawa TERT di dalam hUCMs/EGF

adalah 0.26 ± 0.06. Analisis kitaran sel juga menunjukkan yang EGF adalah punca

kepada pergerakan 37.34± 9.8% dan 7.71± 0.53% hUCMs kepada fasa S dan G2/M.

Selepas pendedahan FGF, 29.92± 2.55% dan 9.92± 0.24% sel telah memasuki fasa S

dan G2/M.

Induksi pembezaan neuron dengan pelbagai koktel menunjukkan bahawa koktel

mengandungi kumpulan DMEM/LG, FBS, RA, FGF dan EGF (DF/R/Fg/E)

menghasilkan ekspresi dengan peratusan yang tertinggi untuk nestin, ß-tubulin III,

neurofilament, and CNPase 69.7 ± 6%, 76 ±1.7%, 89 ±9.5%, and 16.3 ±5.2%,

masing-masing. Juga di dalam kumpulan ini dan kumpulan DF/Ds/Fg/E,

perkembangan sel yang paling tinggi diperhatikan. Selain itu, kumpulan DF/Ds/Fg/E

mempunyai peratusan ekspresi GFAP yang tertinggi ((36 ±12.5%). Sementara itu,

peratusan ekspresi NF, GFAP, dan CNPase adalah yang terendah di dalam kumpulan

DF. Peratusan ekspresi terendah nestin dan ß-tubulin III diperhatikan di dalam

kumpulan DF/Ds.

MEM/GL menyokong penumbuhan hUCMCs lebih baik daripada DMEM/F12. Di

samping itu, faktor penumbuhan juga mempengaruhi ekspresi TERT dan fasa kitaran

sel. Di dalam kes ini, EGF meningkatkan ekspresi TERT dan jumlah sel yang

memasuki fasa S/G2/M. Selain itu, kejayaan hUCMCs untuk membezak kepada

pelbagai sel neuron dan jumlah pelbagai sel neuron adalah dipengaruhi oleh agen-

agen induksi. FGF dan EGF adalah ejen pendorong yang penting untuk pembezaan

hUCMCs kepada neuron, astrocyte dan oligodendrocyte. RA juga boleh mendorong

© COPYRIG

HT UPM

ix

hUCMCs untuk membezakan kepada neuron dan oligodendrocyte, DMSO

mempunyai peranan yang penting dalam pembezaan astrocyte.

Daripada kajian ini, dapat disimpulkan bahawa teknik pengasingan yang berbeza

boleh mempengaruhi ciri-ciri sel mesenchymal daripada Wharton’s Jelly manusia.

Sel-sel ini boleh berkembang di dalam kultur dengan pelbagai jenis media dan faktor

penumbuhan, dan membeza kepada sel neuron.

© COPYRIG

HT UPM

x

ACKNOWLEDGEMENTS

I would like to express my sincere appreciation and gratitude to my supervisor Dr.

Noorjahan Banu Binti Mohammed Alitheen under whose guidance and

supervision had provided me the opportunity and conducive environment to

complete this study. Her invaluable constructive criticism and continuous support

had built in me the confidence to undertake the laboratory investigations with

patience and optimism throughout the course my studies.

Special thanks go to the members of my supervisory committee Dr. Noureddin

Nematollahi Mahani, Prof. Dr. Abdul Manaf B Ali and Prof. Dr. Abdul Rahman

Bin Omar for their suggestions and useful deliberative discussions to make this

study more comprehensive and meaningful.

Thanks are due to Dr. Noureddin Nematollahi Mahani for extending me the use of

facilities of his laboratory. I would like to thank all the staff in the Department of

Anatomy, Faculty of Kerman Medicine for their numerous help and support during

the course my work. I am equally grateful to the staff of the Flow Cytometry Unit,

Institute of Royan, especially to Mr. Ehsan Janzamin for their kind assistance in the

Flow Cytometry analaysis.

I am immensely and forever grateful to those individuals who have, in one way or

another provided me with the courage and resilience to pave the way towards the

successful completion of this study.

© COPYRIG

HT UPM

xi

Last but not least, I wish to thank my family for their continuous moral support and

encouragement. By remaining close to me through constant contact and being there

when I need them, they made me feel as if I am at home with them even though they

are far away from me.

-----------------------------------------------

© COPYRIG

HT UPM

xii

I certify that a Thesis Examination Committee has met on 11 June 2012 to conduct the final examination of Parvin Salehinejad on her thesis entitled “In Vitro Study of Various Factors in Isolation, Expansion and Differentiation of Mesenchymal Stem Cells from Human Umbilical Cord“ in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Doctor of Philosophy. Members of the Examination Committee were as follows: Mohd. Puad bin Abdullah, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Muhajir bin Hamid, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner)

Cheah Yoke Kqueen, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner)

Alp Can, PhD Professor School of Medicine Universiti Ankara Turkey (External Examiner)

----------------------------------------------- SEOW HENG FONG, PhD

Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date: 9 August 2012

© COPYRIG

HT UPM

xiii

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows:

Noorjahan Banu Binti Mohammed Alitheen, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)

Abdul Manaf B Ali, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)

Abdul Rahman B Omar, PhD Professor Institue of Biosciences Universiti Putra Malaysia (Member) Noureddin Nematollahi Mahani , PhD Professor Faculty of Medicine Universiti Kerman Iran (Member)

----------------------------------------------- BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia

Date:

© COPYRIG

HT UPM

xiv

DECLARATION

I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any degree at Universiti Putra Malaysia or at any other institutions.

------------------------------------ PARVIN SALEHINEJAD

Date: 11 June 2012

© COPYRIG

HT UPM

xv

TABLE OF CONTENTS

Page ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL xi DECLARATION xiii LIST OF TABLES xvii LIST OF FIGURES xix LIST OF ABBREVIATIONS xx CHAPTER 1 INTRODUCTION 1 2 LITERATURE REVIEW 7 2.1 Stem Cells 7

2.1.1 Classifying of Stem Cells 8 2.2 Human Umbilical Cord 14

2.2.1 Development of Umbilical Cord 14 2.2.2 Umbilical Cord Structure 15

2.2.3 Wharton`s Jelly 16 2.2.4 Wharton`s Jelly Compounds 17 2.2.5 Wharton`s Jelly Cells (hUCMCs) 22 2.2.6 hUCMCs Characteristics in Vitro 29 2.2.7 hUCMCs and Tissue Engineering 31

2.2.8 hUCMCs as Compared to the Other MSC 33 2.3 Nervous System 37

2.4 Types of Neural Cells 37 2.4.1 Neurons 38 2.4.2 Neuroglial Cells 42 2.4.3 Schwann Cells 47 2.5 Neural Diseases 48 2.5.1 Current Major Treatment Approaches for Neural Diseases 48 2.5.1.1 Cell Therapy in Neural Diseases 49

3 COMPARISON OF DIFFERENT METHODS FOR ISOLATION OF

MESENCHYMAL STEM CELLS FROM HUMAN UMBILICAL WHARTON`S JELLY 51

3.1 Introduction 51 3.2 Materials and Methods 53 3.2.1 Isolation of Human Umbilical Cord Mesenchymal Cells 53 3.2.2 Detection of Cell Viability 60

3.2.3 Cell Proliferation 60 3.2.4 Detection of Cell Activity 60

© COPYRIG

HT UPM

xvi

3.2.5 Detection of MSCs Phenotype 60 3.2.6 Diagnosis of Stem Cell Identity 61 3.2.7 Diagnosis of MC Identity by Differentiation into Adipocyte 62 3.2.8 Diagnosis of MC Identity by Differentiation into Osteocyte 62 3.2.9 Statistical Analysis 63

3.3 Results 63 3.4 Discussion 76 3.5 Conclusion 86

4 THE EFFECT OF ALPHA-MEM/GLUTAMEX AND DMEM/F12 MEDIA ON EXPANSION IN HUMAN UMBILICAL CORD MESENCHYMAL CELLS 87

4.1 Introduction 87 4.2 Materials and Methods 89 4.2.1 Isolation and Culture of Human Umbilical Cord Mesenchymal Cells 89 4.2.2 Cell Activity Assay 90

4.2.3 Cell Proliferation Growth Curve 90 4.2.4 Detection of Phenotype 91 4.2.5 Adipogenic and Osteogenic Differentiation 91

4.2.6 Statistical Analysis 92 4.3 Results 92 4.4 Discussion 100 4.5 Conclusion 104 5 THE EFFECT OF OF EPIDERMAL GROWTH FACTOR AND

FIBROBLAST GROWTH FACTOR ON EXPANSION IN HUMAN UMBILICAL CORD MESENCHYMAL CELLS 105

5.1 Introduction 105 5.2 Materials and Methods 108 5.2.1 Isolation of Human Umbilical Cord Mesenchymal Cells 108

5.2.2 Culture and Treatment of the Cells with Epidermal Growth Factor (EGF) and Fibroblast Growth Factor (FGF) 109

5.2.3 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 109 5.2.4 Measurement of TERT-mRNA Expression 114 5.2.5 Detection of Cell Cycle by Flow Cytometry 114 5.2.6 Statistical Analysis 115

5.3 Results 115 5.4 Discussion 123 5.5 Conclusion 130

6 DIFFERENTIATION OF HUMAN UMBILICAL CORD

MESENCHYMAL CELLS INTO NEURALCELLS IN VITRO 131 6.1 Introduction 131 6.2 Materials and Methods 133

6.2.1 Isolation and Culture of Human Umbilical Cord Mesenchymal Cells 133

6.2.2 Preparation of Mesenchymal Stem Cells 134 6.2.3 Induction of Neural Differentiation 134 6.2.4 Immunocytochemistry 135

© COPYRIG

HT UPM

xvii

6.2.5 Statistical Analysis 136 6.3 Results 136

6.4 Discussion 146 6.5 Conclusion 152 7 CONCLUSIONS AND FURTHER WORK 153

REFERENCES 157 BIODATA OF STUDENT 179 LIST OF PUBLICATION 180