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Research Article Open Access

The Transcription Factor CP2-like 1 Is Expressed in Very SmallEmbryonic-like Stem Cells and Other Adult Stem Cells:Implications for Cancer Stem Cells

Hye-Yeon Lee1,2,x, Hyein Ju1,2,x, Jinbeom Heo1,2, YongHwan Kim1,2, Jisun Lim1,2,

Seungun Lee1,2, Hwan Yeul Yu1,2, Chae-Min Ryu1,2, Ju-Young Han1,2, Sabine J. Waigel3,

Yinlu Chen4, and Dong-Myung Shin1,2,�

1Department of Biomedical Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul,

05505, Korea. 2Department of Physiology, University of Ulsan College of Medicine, Seoul, 05505, Korea.3Genomics Facility and Department of Medicine, University of Louisville, KY 40202, USA. 4Genomics Facility

and Department of Anatomical Sciences and Neurobiology, University of Louisville, KY, 40202, USA.

Abstract: A population of very small embryonic-like stem cells (VSELs) in adult tissues, similar to embryonic stem cells (ESCs), iscapable of differentiation in in vitro conditions and in vivo animal models into cells of all three germ lineages. The open chromatinstructure of pluripotency genes and genomic imprinting-related epigeneticmechanismsmaintain their pluripotent and quiescentstate, respectively. However, which transcription factors (TFs) are commonly expressed to maintain pluripotency in these SCsremains unknown. Here, by comparing the global transcriptome of VSELs with that of adult stem cells (SCs) [e.g., hematopoieticSC (HSCs)] or ESCs, we demonstrated that transcription factor CP2-like 1 (Tfcp2l1l), a well-known naïve factor for ESCs, is highlyexpressed inmurine VSELs. By analyzing a single-cell-level transcriptomedatabase established fromhighly purifiedmurine bonemarrow (BM)-derived VSELs and HSCs as well as ESCs, we found that expression in a subset of TFs was shared by VSELs andESCs. Among them, Tfcp2l1 was commonly expressed in murine VSELs and ESCs but not in HSCs and terminally differentiatedBM mononuclear cells. During the differentiation of ESCs by forming an embryoid body or by treatment with retinoic acid, theexpression of Tfcp2l1 decreased more rapidly than that of the typical pluripotency-associated TFs, including Oct4, Nanog, andSox2. Ectopic expression of Tfcp2l1 in ESCs enforced expression of Oct4. Taken together, these results suggest that Tfcp2l1functions as a common TF to regulate pluripotency in Oct4-expressing embryonic and adult pluripotent SCs and that dys-regulation of Tfcp2l1 in adult SCs could initiate the transformation into cancer stem cells.

Keywords: Tfcp2l1, Transcription factors, Pluripotency, Oct4, VSELs.

INTRODUCTIONEmbryonic development and subsequent rejuvenation of

adult tissues are regulated by a population of stem cells

(SCs) that undergo self-renewal, maintain their own pool,

and give rise to differentiated progenitors that replace cells

used up during life [1]. Thus, SCs are guardians of tissue/

organ integrity and regulate the life span of an adult

organism. The most important SC population from a

regenerative point of view is pluripotent SCs (PSCs) [2,

3]. According to the definition, PSCs have to fulfill certain

in vitro as well as in vivo criteria, such as (i) giving rise to

cells from all three germ layers; (ii) completing blastocyst

development; and (iii) forming teratomas after inoculation

into experimental animals. PSCs from the inner mass of

blastocysts could be expanded ex vivo as immortalized

embryonic SCs (ESCs) [4, 5], which aremost well-studied

PSCs. Recently, PSCs could be established by somatic cell

reprograming, including the transduction of so-called

Yamanaka factors (Oct4, Sox2, Klf4, and cMyc) [6, 7]

and somatic cell nuclear transfer [8].

To maintain this unique pluripotent property, PSCs

commonly express pluripotency core transcription factors

(TFs) such as Oct4, Nanog, and Sox2 [9], which are turned

off in differentiated somatic cells. These TFs form the

pluripotent core circuitry by reinforcing the expression of

genes, which are involved in keeping PSCs in an undif-

ferentiated status but repressing differentiation-inducing

transcription. Moreover, several genes that are frequently

upregulated in tumors, such as Stat3 [10, 11], E-Ras [12],

xThese authors equally contributed to this work.�Corresponding Author: Dong-Myung Shin, Ph.D., Department of

Biomedical Sciences, Asan Medical Center, University of Ulsan

College of Medicine, Pungnap-2 dong, Songpa-gu, Seoul, 05505,

Korea. Tel: 82-2-3010-2086; Fax: 82-2-3010-8493; E-mail:

d0shin03@amc.seoul.kr

Received: March 5, 2017; Revised: March 28, 2017;

Accepted: March 28, 2017

Journal of Cancer Stem Cell Research (2017), 5:e1001�2017 Creative Commons. All rights reserved ISSN 2329-5872DOI: 10.14343/JCSCR.2017.5e1001http://cancerstemcellsresearch.com

1001; 10/4/2017; 9:33:17

cMyc [13], Klf4 [14], and b-catenin [15, 16], have been

shown to contribute to the long-term maintenance of the

ES cell phenotype and the rapid proliferation of ESCs in

culture.

Recently, a population of very small embryonic-like

SCs (VSELs) has been identified in murine adult tissues,

including the bone marrow (BM), fetal liver, testes, ova-

ries, and human umbilical cord blood [17–21]. VSELs are

smaller than erythrocytes and express several markers of

(i) pluripotency (Oct4, Nanog, Sox2, and SSEA-1), (ii)

epiblasts (Gbx2, Fgf5, and Nodal), and (iii) epiblast-

derived migratory primordial germ cells (PGCs) (Stella,

Blimp1, and Prdm14) [22, 23]. The true expression of

Oct4, Nanog, and Stella inmurineBM-derivedVSELs has

been confirmed by demonstrating the demethylated state

of DNA and enrichment of transcriptionally active histone

codes in the promoters of these genes [22, 24]. Further-

more, epigenetic changes in the expression of some

imprinted genes that are paternally (Igf2-H19 and

RasGRF1) and maternally methylated/imprinted (Igf2R

and KCNQ1) maintain the quiescence of VSELs [24].

VSELs can differentiate into cells from all three germ

layers in in vitro culture conditions. Using several in vivo

tissue regeneration animal models, VSELs can be spec-

ified in vivo into mesenchymal SCs (MSCs) [25], cardi-

omyocytes [26], type II alveolar cells [27], and long-term

engrafting hematopoietic SCs (HSCs) [28, 29]. In normal

physiological condition, proliferation and developmental

potency of VSELs should be tightly modulated by a

unique epigenetic reprogramming in genomic imprints to

protect VSELs from uncontrolled proliferation and

teratoma formation. In pathological situations, however,

unleashed VSELs could contribute to the development of

several malignancies [30]. However, the precise molecu-

lar mechanism by which primitive VSELs control their

pluripotency, proliferation and differentiation potential

remains to be determined.

As mentioned, TFs, including Oct4, Sox2, Klf4, Nanog,

and Stat3, and chromatin regulatory proteins play a major

role in the regulation of self-renewal by maintaining an

ESC-specific gene expression pattern [3]. However, the

precise profile of these pluripotency-associated TFs in

Oct4-expressing adult VSELs is not well understood. In

this regard, we examined TFs that would show common

expression patterns in VSELs and ESCs using single-cell-

level transcriptome databases from several types of PSCs

and adult cells [31]. In this present study, we demonstrated

that TFCP2-like 1 (Tfcp2l1), awell-knownna€ıve factor forESCs,washighly expressed inmurineBM-derivedVSELs.

MATERIALS AND METHODSAnalysis of Microarray Data for Single-cell-level SC

Transcriptome

We employed amicroarray database representing a cDNA

library established from 20 cells of FACS-sorted VSELs,

HSCs, or trypsinized ESC-D3 cells. All the procedures for

20-cell cDNA library synthesis, microarrays, and data

processing have been described in our previous report

[31]. The microarray datasets discussed in the present

study have been deposited in NCBI’s Gene Expression

Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo) and

are accessible through GEO Series accession number

GSE29281. Heatmap analyses with hierarchical

clustering of microarray data were performed using

Partek software (Partek Inc, Saint Louis, MO), and

gene network functional analysis was performed using

Ingenuity pathway analysis (IPA) software version 8.7

(Ingenuity Systems, Inc. Redwood, CA) by core and

comparison analysis for gene networks, bio-functions,

and canonical pathways. Hierarchical agglomerative

clustering with Spearman’s rank correlation coefficient

and average linkage were applied to both rows (samples)

and columns (probe sets), and heatmaps were produced by

arranging the rows and columns according to the

clustering outputs. Gene network analysis was

performed using IPA software by default setting. The

minimum resolution for multiple probes was set at the

experimental p value. Red and green represented

upregulated and downregulated values, respectively.

The bio-function and canonical pathway analysis of the

indicated gene lists was performed using the default

settings, with a threshold value of 0.05 and Fisher’s

exact test for scoring method.

Reverse Transcriptase-polymerase Chain Reaction

(RT-PCR)

Total RNA from various cells was isolated using the

RNeasy Mini Kit (QIAGEN, Valencia, CA), including

treatment with DNase I (QIAGEN). mRNA (400 ng) was

reverse-transcribed with Taqman Reverse Transcription

Reagent (Applied Biosystems, Foster City, CA), accord-

ing to the manufacturer’s instructions. The resulting

cDNA fragments were amplified using Amplitaq Gold at

one cycle of 8 min at 95�C; two cycles of 2 min at 95�C,1 min at 62�C, and 1 min at 72�C; 35 subsequent cycles of30 s at 95�C, 1 min at 62�C, and 1 min at 72�C; and one

cycle of 10 min at 72�C. All primers were designed with

Primer Express software (Applied Biosystems) as at least

one primer included an exon and intron boundary. They

are available upon request.

Real-time Quantitative PCR (RQ-PCR)

For quantification of the expression of the indicated

transcripts, cDNA templates, prepared using both regular

total RNA-reverse transcription and single-cell-level pro-

tocols, were amplifiedwith SYBRGreen PCRMasterMix

(Applied Biosystems) using RQ-PCR on the PikoRealTM

Real-Time PCR System (Thermo Fisher Scientific, Pitts-

burgh, PA). All primers were designed with Primer

Express software (Applied Biosystems) as at least one

primer included an exon–intron boundary. The threshold

2 H. Lee et al.

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1001; 10/4/2017; 9:33:17

cycle (Ct), the cycle number at which the fluorescence of

the amplified gene reaches a fixed threshold, was subse-

quently determined, and relative quantification of the

expression level of target geneswas performed through the

2�DDCt method using the mRNA level of Gapdh as an

endogenous control gene and that of the indicated cells as a

calibrator.

Cultivation of Murine ESCs

ESCs (R1 line) were grown in DMEM-high glucose

medium (HyClone, Pittsburgh, PA) supplemented with

2 mM L-glutamine, 20 mM HEPES, MEM nonessential

amino-acid, penicillin/streptomycin solution (Cellgro,

Pittsburgh, PA), 0.1 mM b-mercaptoethanol (Sigma-

Aldrich, St Louis, MO), 15% heat-inactivated FBS

(Hyclone), and 100 IU/ml ESGRO (Millipore, Billerica,

MA) in gelatin-coated tissue culture dishes as previously

described [3]. Embryoid body (EB) formation was per-

formed using the hanging drop method in Petri dishes. In

total, 1 mM retionic acid (Sigma-Aldrich) was used to

induce the differentiation of ESCs. The undifferentiated

status of ESCs was assessed using the Alkaline Phospha-

tase (AP) Detection Kit (Millipore), according to the

manufacturer’s instructions.

Overexpression of Tfcp2l1 Protein and Western Blot

Analysis

The open reading frame of murine Tfcp2l1 was directly

amplified from a murine ES cell cDNA library with

following the primers: mTfcp2l1_ORF_F: GGAT-

CCGCCACCATG-CTGTTCTGGCACACGCAG and

mTfcp2l1_ORF_R: CTCGAGTCAGAGTCCACACTT-

CAGGAT. The amplified Tfcp2l1 ORF was cloned into

pCMV_3Tag-1 vector for overexpression of the Flag-

tagged Tfcp2l1 protein. The plasmid containing the

Tfcp2l1 construct was transfected into murine ESCs using

Lipofectamine 2000 (Life Technologies, La Jolla, CA). To

examine the expression level of the indicated proteins, cell

extracts (30 mg) were prepared in RIPA lysis buffer (Santa

Cruz Biotechnology, Santa Cruz, CA) and separated on

10% SDS-PAGE gels. The protein expression level was

assessed by probing with monoclonal antibodies specific

to Oct4 (Santa Cruz Biotechnology), Tfcp2l1 (Aviva

Systems Biology, San Diego, CA), Sox2 (Epitomics,

Burlingame, CA), Nanog (Abcam, Cambridge, UK), Tet1

(Millipore), and Flag epitope (Sigma-Aldrich). Relative

protein expression was calculated by normalization to

b-actin (Santa Cruz Biotechnology).

Immunostaining

For immunocytochemistry, ESCs were fixed with 4%paraformaldehyde (Sigma-Aldrich) for 24 h and co-

stained using anti-Oct4 mouse IgG monoclonal antibody

(Millipore) and anti-Tfcp2l1 rabbit IgG polyclonal anti-

body. Immunostaining was visualized using Alexa 488- or

564- conjugated anti-mouse or anti-rabbit antibodies

(Molecular Probes, Grand Island, NY). The nuclei were

counterstained with 40,6-diamino-2-phenylindole (DAPI,

Sigma-Aldrich). The stained samples were photographed

using an inverted fluorescence microscope (EVOS� FL

Color Imaging System, Life Technologies).

Statistical Analysis

All data were analyzed by one- or two-way analysis of

variance (ANOVA) with Bonferroni post-hoc tests.

GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA)

was used to perform all analyses. Statistical significance

was defined as p < 0.05 or p < 0.01.

RESULTSIdentification of the Gene Set for TFs Commonly

Characterizing Murine VSELs and ESCs

VSELs share several molecular natures with ESCs to

maintain pluripotency. Because TF networks determine

the expression of genes involved in many biological

processes [3], we attempted to identify the TFs genes

shared by VSELs and ESCs. To address this issue, we re-

analyzed previously published data (GSE29281), consist-

ing of a transcriptome of 20 FACS-sorted cells from

murine BM-derived VSELs, HSCs, and the ESC line

ESC-D3 [32]. By setting the cutoff as two fold upregulated

or downregulated genes simultaneously inVSEL andHSC

as well as ESC and HSC comparison, with a statistical p-

value cutoff of 0.000491342 (FDR ¼ 0.01 in VSEL and

HSC comparison), we found 589 upregulated and 1553

downregulated genes (Figure 1A). By further filtering the

selected genes using the ontology term "Transcription"

we found that 129 upregulated and 262 downregulated

genes were enriched as pluripotency-related genes. Heat-

map clustering analysis using these selected genes dem-

onstrated that transcriptomes of both PSCs (VSELs and

ESCs) were closely clustered with each other but distant

from those of differentiated adult SCs (HSCs) (Figure 1B),

confirming that this selected gene set properly represents

the TF profile characteristic of PSCs.

Identification of Tfcp2l1 As A Novel Pluripotency-

related TF Shared by VSELs and ESCs

Next, we performed gene network analysis, which is an

all-comprehensive approach for identifying the molecules

interacting with target genes with respect to all biological

events, including transcription regulation, upstream or

downstream regulation, and protein–protein interaction

[31]. To focus on genes involved in the regulation of

pluripotency, putative targets were selected if their gene

network included more than two interacting pluripotency-

related genes (e.g., Oct4, Nanog, Sox2, Rex1, Sall4, and

Stat3) (Figure 1C). Accordingly, we found four putative

target genes, including Tfcp2l1, PR domain-containing

protein 5 (Prdm5), WD repeat-containing protein 77

(Wdr77), and CBP/p300-interacting transactivator with

glu/asp-rich c-terminal domain 1 (Cited1).

Expression of Tfcp2l1 in murine VSELs 3

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1001; 10/4/2017; 9:33:17

By employing the RQ-PCR assay, we confirmed that

Tfcp2l1 transcripts, similar to Oct4 and Nanog ones, were

specifically found inVSELs andESCs, but fewwere found

in MEF and other adult tissue cells (HSCs and BMMNCs)

(Figure 2A). The expression of Prdm5 was detected in

embryonic tissue cells and only in VSELs in adult tissue

cells (Figure 2A and Figure 2B). In contrast, the expres-

sion of Wdr77 and Cited1 was the highest in HSCs and

BMMNCs, respectively, but they were hardly detected in

either VSELs or ESCs. Among the validated genes,

Tfcp2l1 showed the highest enrichment in the expression

dataset (16.24-fold increase in VSELs vs. HSCs) and gene

network analysis; it has recently been reported as the

missing pluripotency-associated TF in both murine [33,

34] and human ESCs [35]. Indeed, the immunofluorescent

staining of undifferentiated ESC colonies demonstrated

that Tfcp2l1 located in the nucleus clearly co-localized

with Oct4 (Figure 2C).

Role of Tfcp2l1 in Maintaining Pluripotency

Next, we examined the change in the expression ofTfcp2l1

during the differentiation of ESCs. We employed two

differentiation methods: EB culture using the hanging

drop method and the addition of retinoic acid (RA) to the

growth medium, which inactivates Oct4 transcription,

which is directly mediated by trans-acting repressors such

as ARP-1, COUP-TF1, and GCNF [36, 37]. During dif-

ferentiation following both EB and RA treatment, the

expression of Tfcp2l1 decreased similar to that of other

PSCmarkers, includingOct4,Nanog, and Sox2 (Figure 3A

and 3B). In particular, we noted that the expression of

Tfcp2l1 was more rapidly repressed than that of other

pluripotency-associated TFs, suggesting the significance

of Tfcp2l1 in controlling pluripotency. The downregula-

tion of Tfcp2l1 during ESC differentiation was confirmed

at the protein level bywestern blot analysis (Figure 3C and

3D).

Next, to determine the relationship between Tfcp2l1

and Oct4, a key TF associated with pluripotency, we

overexpressed Tfcp2l1 in murine ESCs. Western blot

analysis confirmed that Oct4 was efficiently induced in

a dose- and time-dependent manner by the ectopic expres-

sion of Tfcp2l1 (Figure 4A), suggesting that Tfcp2l1 could

positively regulate the expression of Oct4. Accordingly,

ESCs ectopically expressing Tfcp2l1 exhibited a stronger

AP-positive staining profile than control cells (Figure 4B).

Taken together, these data indicate that the expression of

Tfcp2l1 positively correlates with pluripotency in embry-

onic and adult tissue SCs and that Tfcp2l1 plays an

essential role in maintaining self-renewal and an undif-

ferentiated state.

DISCUSSIONThe results presented here demonstrate that a subset of TFs

is shared by murine VSELs and ESCs and that Tfcp2l1

plays a key role in maintaining a pluripotent and undif-

ferentiated state.

Accumulating evidence has reported a population of

adult tissue PSCs, which are referred to by various names,

including (i) MSCs; (ii) multipotent adult progenitor cells

(MAPCs); (iii) marrow-isolated adult multilineage induc-

ible (MIAMI) cells; (iv) multipotent adult SCs (MASCs);

and (v) OmniCytes [23, 38]. However, of late, there has

been much debate challenging the existence of primitive

Figure 1. Gene set characterizing TFs shared by murine VSELs and ESCs. (A) Summary of the analysis of microarray data, focusing on

pluripotency-associated transcription factors (TFs). FDR; false discovery rate, GO; gene ontology, VSEL; very small embryonic-like stem cell,

HSC; hematopoietic stemcell, ESC; embryonic stemcell. (B)Heatmap analysiswith hierarchical clustering for genes related toTFs and enriched

in both PSCs (VSELs and ESCs). Blue, red, and green bars represent HSCs, ESCs, and VSELs respectively. (C) Gene network analysis of

Tfcp2l1. Gene networks are illustrated by overlaying all experimental values for the ESC and HSC comparison datasets. Upregulated and

downregulated genes in the heatmap and gene network analyses are represented as red and green colors, respectively.

4 H. Lee et al.

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1001; 10/4/2017; 9:33:43

PSCs in adult tissues [39]. Thus, further investigation is

required to better explain their precise molecular nature

and to use them as a powerful source in regenerative

medicine. In this regard, by employing genomewide gene

expression databases, we identified the expression profile

of TFs that could commonly characterize both ESCs and

VSELs (Figure 1). Accordingly, the identified gene set

included several well-known pluripotency-associated

TFs, including Zfp42, Oct4, Zeb2, Nanog, Sox2, and

Dnmt3l. However, we noted that this gene set was also

characterized by various genes involved in basic biolog-

ical processes, including cyclin E1, cell division cycle-

associated 2, forkhead box O3, HIRA-interacting protein

3, and paired-like homeodomain 2, with highly ranked

enrichment scores. This indicates that the transcriptional

memory of PSCs in embryonic tissues may, to an unde-

fined level, be preserved in primitive SCs deposited in

adult tissues. Thus, the TF gene set shared by VSELs and

ESCs identified in the present study (Figure 1) could be

employed to characterize the state of pluripotency or

stemness in adult tissues. This interesting possibility

should be validated by further in-depth investigations. Of

importance, several tumors exhibited similar morphologic

andmolecular features to developmentally early tissues. In

addition, accumulating evidences have proven that malig-

nancy arises from continuous serious mutation in normal

stem/progenitor cells, leading to transform of them into

cancer stem cells [40]. Thus, the TF gene set shared by

VSELs and ESCs could be also applicable to investigate

the molecular nature of cancer stem cells found in several

tumor tissues.

Among the TFs associated with pluripotency, Tfcp2l1

showed the highest enrichment scores in fold-change and

gene network analysis. However, its function in adult

tissues has been not well studied. Tfcp2l1 is expressed in

murine and human inner cell mass of blastocysts and is

downregulated shortly after implantation [41, 42]. When

human ESCs were converted into a na€ıve-like state by theoverexpression of Klf2, Klf4, and Oct4, the expression of

Tfcp2l1 was found to be characteristically upregulated

based on analysis of the change in the transcriptome profile

[43]. In a differentiation assay of ESCs, the expression of

Tfcp2l1 rapidly decreased faster than that if other plur-

ipotency-associated TFs (Figure 3). Furthermore, the

ectopic expression of Tfcp2l1, in time- and dose-depen-

dent manners, enforced the expression of Oct4 and AP

Figure 2. Expression of Tfcp2l1 inmurine VSELs and ESCs. (A andB)RQ-PCR (A) and RT-PCR (B) of pluripotency-associated TFs in the

indicated stem cells (SCs) or bone-marrowmononuclear cells (BMMNCs). The relative expression level of genes is represented as the ratio of the

indicated stem cells value to that obtained for ESCs and is shown as the mean � SEM; n ¼ 4, ���p < 0.001, compared with HSCs, two-way

ANOVAwith Bonferroni post-hoc tests. RT-PCR showed consistent expression ofOct4 and Tfcp2l1 in ESC and VSELs.Gapdhwas used as an

internal control. D.W.; distilled water. (C) Representative images of immunofluorescent staining for Oct4 (green) and Tfcp2l1 (red) proteins in

murine ESCs (upper panel; �400 magnification, scale bar ¼ 200 mm, lower panel; �400 magnification, scale bar ¼ 100 mm). Nuclei were

counterstained with DAPI (blue).

Expression of Tfcp2l1 in murine VSELs 5

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1001; 10/4/2017; 9:34:24

activity in undifferentiated ESCcolonies (Figure 4). These

results strongly indicated that Tfcp2l1 may play a role in

generating and stabilizing the murine and human plurip-

otent state. Thus, further studies on the mechanisms of

activation and repression by Tfcp2l1 are required to

resolve this. Interestingly, gene targeting of Tfcp2l1 has

identified an important role of this TF in the ductal

epithelium of several developing organs, including the

kidney and salivary glands [44, 45], suggesting a role of

Tfcp2l1 in adult tissues. In this regard, further investiga-

tion is required to determinewhether themodulation of the

expression or activity of Tfcp2l1 could be reliable in

maintaining pluripotency or ex vivo expansion of murine

or human VSELs.

Tfcp2l1 functions in gene transcription as an activator

or repressor by forming DNA-binding complexes with

Figure 3. Expression of Tfcp2l1 dur-

ing differentiation ofmurineESCs. (A

and B) ESC differentiation was induced

by embryoid body (EB) formation (A) or

retinoic acid (RA) treatment (B). RQ-

PCR results are represented by the rel-

ative expression level as the ratio of the

value of the indicated cells to that of

undifferentiated ESCs and are shown as

the mean � SEM; n ¼ 4. RT-PCR

analysis of the indicated genes is shown

at the bottom of each RQ-PCR result. (C

and D). Western blot analysis of

Tfcp2l1 during ESC differentiation

induced by EB formation (C) or treat-

ment of RA (D). The right-sized and

nonspecific bands in the Nanog and

Tfcp2l1 western blot data are indicated

by an arrow and asterisk, respectively.

b-actin was used as an internal control.

W.B.; Western blot.

Figure 4. Ectopic expression of Tfcp2l1 reinforces

Oct4 expression in murine ESCs. (A) Western blot

analysis of ESCs transfected with the Flag-tagged

Tfcp2l1 expression construct, with the indicated

amount of plasmid and post-transfection periods.

b-actinwas used as an internal control.W.B.;Western

blot. (B) Alkaline phosphatase (AP) staining of ESCs

stably established with the Flag-tagged Tfcp2l1

expression construct (�200 magnification, scale bar

¼ 200 mm). Empty; mock vector, Br; bright field.

6 H. Lee et al.

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com

1001; 10/4/2017; 9:35:14

other CP2 family members; the combination in complex

components determines their transcriptional activity [46].

This implies that theseTFs should be studied as a family by

carefully investigating the networks between familymem-

bers expressed in a given cell type or tissue of interest. A

previous study has reported that other CP2 family mem-

bers such as CP2, NF2d9 and altNF2d9 are also expressed

in murine ESCs [47]. However, these CP2 family genes

were not listed in the TF gene set shared by ESCs and

VSELs (Figure 1B), suggesting that Tfcp2l1 plays a

specific role in PSCs. Thus, further examination is

required to establishwhether CP2 family complexes could

determine the function of this family in PSCs from embry-

onic and adult tissues as well as by somatic cell

reprograming.

In summary, in the present study, we have identified

Tfcp2l1 as a novel pluripotency-related TF shared by

PSCs in embryonic (ESCs) and adult (VSELs) tissues by

analyzing the transcriptomes characteristic to both types

of PSCs. Tfcp2l1 plays an essential role in regulating the

pluripotent state in Oct4C embryonic or adult PSCs. Thus,

the present study provides a crucial conceptional and

technical advance in our understanding of not only how

PSCs maintain their pluripotency but also how their

transcriptional memory may be progressively sustained

throughout embryonic and adult tissue development.

ACKNOWLEDGMENTSThis research was supported by Basic Science Research

Program through the National Research Foundation

of Korea (NRF) funded by the Ministry of

Science, ICT and future Planning (grant number)

(2015R1A2A1A15054754) and co-supported by the

Global High-tech Biomedicine Technology Development

Program of the National Research Foundation (NRF) &

Korea Health Industry Development Institute (KHIDI)

(MSIP&MOHW) (No. 2015M3D6A1065364). Part of this

work was performed with the assistance of the University

of Louisville Microarray Facility, which is supported by

NCRR IDeA Awards INBRE-P20 RR016481 and

COBRE-P20RR018733, the James Graham Brown Foun-

dation, and user fees.

CONFLICT OF INTERESTThe authors have no competing financial interests to

declare.

REFERENCES[1] Ratajczak MZ, Liu R, Ratajczak J, Kucia M, Shin D-M. The role

of pluripotent embryonic-like stem cells residing in adult tissues

in regeneration and longevity. Differentiation 2011;81:153–61.

[2] Niwa H. How is pluripotency determined and maintained?

Development 2007;134:635–46.

[3] Heo J, Lim J, Lee S, Jeong J, Kang H, Kim Y, Kang JW, Yu HY,

Jeong EM, Kim K, et al. Sirt1 regulates DNA methylation and

differentiation potential of embryonic stem cells by antagonizing

Dnmt3l. Cell Rep 2017;18:1930–45.

[4] Evans MJ, Kaufman MH. Establishment in culture of pluripo-

tential cells from mouse embryos. Nature 1981;292:154–6.

[5] Thomson JA, Itskovitz-Eldor J, Shapiro SS,WaknitzMA, Swier-

giel JJ,Marshall VS, Jones JM. Embryonic stem cell lines derived

from human blastocysts. Science 1998;282:1145–7.

[6] Takahashi K, Yamanaka S. Induction of pluripotent stem cells

from mouse embryonic and adult fibroblast cultures by defined

factors. Cell 2006;126:663–76.

[7] Wernig M, Meissner A, Foreman R, Brambrink T, Ku M,

Hochedlinger K, Bernstein BE, Jaenisch R. In vitro reprogram-

ming of fibroblasts into a pluripotent ES-cell-like state. Nature

2007;448:318–24.

[8] Tachibana M, Amato P, Sparman M, Gutierrez NM, Tippner-

Hedges R, Ma H, Kang E, Fulati A, Lee HS, Sritanaudomchai H,

et al. Human embryonic stem cells derived by somatic cell

nuclear transfer. Cell 2013;153:1228–38.

[9] Kim J, Chu J, Shen X, Wang J, Orkin SH. An extended tran-

scriptional network for pluripotency of embryonic stem cells.

Cell 2008;132:1049–61.

[10] Maruyama M, Ichisaka T, Nakagawa M, Yamanaka S. Differ-

ential roles for Sox15 and Sox2 in transcriptional control in

mouse embryonic stem cells. J Biol Chem 2005;280:24371–9.

[11] Niwa H, Burdon T, Chambers I, Smith A. Self-renewal of

pluripotent embryonic stem cells is mediated via activation

of STAT3. Genes Dev 1998;12:2048–60.

[12] Takahashi K, Mitsui K, Yamanaka S. Role of ERas in promoting

tumour-like properties in mouse embryonic stem cells. Nature

2003;423:541–5.

[13] Cartwright P, McLean C, Sheppard A, Rivett D, Jones K, Dalton

S. LIF/STAT3 controls ES cell self-renewal and pluripotency by

a Myc-dependent mechanism. Development 2005;132:885–96.

[14] Li Y, McClintick J, Zhong L, Edenberg HJ, Yoder MC, Chan RJ.

Murine embryonic stem cell differentiation is promoted by

SOCS-3 and inhibited by the zinc finger transcription factor

Klf4. Blood 2005;105:635–7.

[15] Kielman MF, Rindapaa M, Gaspar C, van Poppel N, Breukel C,

van Leeuwen S, Taketo MM, Roberts S, Smits R, Fodde R. Apc

modulates embryonic stem-cell differentiation by controlling the

dosage of [beta]-catenin signaling. Nat Genet 2002;32:594–605.

[16] Lin T, ChaoC, Saito Si,Mazur SJ,MurphyME,Appella E, XuY.

p53 induces differentiation of mouse embryonic stem cells by

suppressing Nanog expression. Nat Cell Biol 2005;7:165–71.

[17] Kucia M, Reca R, Campbell FR, Zuba-Surma E, Majka M,

Ratajczak J, Ratajczak MZ. A population of very small embry-

onic-like (VSEL) CXCR4CSSEA-1COct-4C stem cells identi-

fied in adult bone marrow. Leukemia 2006;20:857–69.

[18] Zuba-Surma EK, Kucia M, Wu W, Klich I, JWL Jr, Ratajczak J,

Ratajczak MZ. Very small embryonic-like stem cells are present

in adult murine organs: ImageStream-based morphological anal-

ysis and distribution studies. Cytometry A 2008;73A:1116–27.

[19] Kucia M, Halasa M, Wysoczynski M, Baskiewicz-Masiuk M,

Moldenhawer S, Zuba-Surma E, Czajka R, Wojakowski W,

Machalinski B, Ratajczak MZ. Morphological and molecular

characterization of novel population of CXCR4C SSEA-4COct-

4C very small embryonic-like cells purified from human cord

blood - preliminary report. Leukemia 2006;21:297–303.

[20] Wojakowski W, Tendera M, Kucia M, Zuba-Surma E, Pacz-

kowska E, Ciosek J, HalasaM, KrM, KazmierskiM, Buszman P,

et al. Mobilization of bone marrow-derived Oct-4C SSEA-4Cvery small embryonic-like stem cells in patients with acute

myocardial infarction. J Am College Cardiol 2009;53:1–9.

[21] Paczkowska E, Kucia M, Koziarska D, Halasa M, Safranow K,

Masiuk M, Karbicka A, Nowik M, Nowacki P, Ratajczak MZ,

Machalinski B. Clinical evidence that very small embryonic-like

Expression of Tfcp2l1 in murine VSELs 7

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com

1001; 10/4/2017; 9:35:14

stem cells are mobilized into peripheral blood in patients after

stroke. Stroke 2009;40:1237–44.

[22] Shin DM, Liu R, Klich I,WuW, Ratajczak J, KuciaM, Ratajczak

MZ: Molecular signature of adult bone marrow-purified very

small embryonic-like stem cells supports their developmental

epiblast/germ line origin. Leukemia 2010;24:1450–61.

[23] YongHwan K, Jaeho J, Hyunsook K, Jisun L, Jinbeom H, Janina

R, Mariusz ZR, Dong-Myung S. The molecular nature of very

small embryonic-like stem cells in adult tissues. Int J Stem Cells

2014;7:55–62.

[24] Shin DM, Zuba-Surma EK,WuW,Ratajczak J,WysoczynskiM,

Ratajczak MZ, Kucia M: Novel epigenetic mechanisms that

control pluripotency and quiescence of adult bone marrow-

derived Oct4C very small embryonic-like stem cells. Leukemia

2009;23:2042–51.

[25] TaichmanRS,Wang Z, ShiozawaY, JungY, Song J, BalduinoA,

Wang J, Patel LR, Havens AM, Kucia M, et al. Prospective

identification and skeletal localization of cells capable of multi-

lineage differentiation in vivo. StemCellsDev 2010;19:1557–70.

[26] Dawn B, Tiwari S, Kucia MJ, Zuba-Surma EK, Guo Y, Sanga-

nalMath SK, Abdel-Latif A, Hunt G, Vincent RJ, Taher H, et al.

Transplantation of bone marrow-derived very small embryonic-

like stem cells attenuates left ventricular dysfunction and

remodeling after myocardial infarction. Stem Cells 2008;26:

1646–55.

[27] Kassmer SH, JinH, Zhang P-X, Bruscia EM,Heydari K, Lee J-H,

Kim CF, Krause DS. Very small embryonic-like stem cells from

the murine bone marrow differentiate into epithelial cells of the

lung. Stem Cells 2013:N/A-N/A.

[28] Ratajczak J, Wysoczynski M, Zuba-Surma E, Wan W, Kucia M,

Yoder MC, Ratajczak MZ. Adult murine bone marrow-derived

very small embryonic-like stem cells differentiate into the

hematopoietic lineage after coculture over OP9 stromal cells.

Exp Hematol 2011;39:225–37.

[29] Ratajczak J, Zuba-Surma E, Klich I, Liu R, Wysoczynski M,

Greco N, Kucia M, Laughlin MJ, Ratajczak MZ. Hematopoietic

differentiation of umbilical cord blood-derived very small

embryonic/epiblast-like stem cells. Leukemia 2011;25:1278–85.

[30] Shin D-M, Suszynska M, Mierzejewska K, Ratajczak J, Ratajc-

zak MZ. Very small embryonic-like stem-cell optimization of

isolation protocols: an update of molecular signatures and a

review of current in vivo applications. Exp Mol Med 2013;45:

e56.

[31] ShinD-M,LiuR,WuW,Waigel SJ, ZachariasW,RatajczakMZ,

Kucia M: Global gene expression analysis of very small embry-

onic-like stem cells reveals that the Ezh2-dependent bivalent

domain mechanism contributes to their pluripotent state. Stem

Cells Dev 2011;21:1639–52.

[32] Shin DM, Liu R,WuW,Waigel SJ, ZachariasW, RatajczakMZ,

Kucia M. Global gene expression analysis of very small embry-

onic-like stem cells reveals that the Ezh2-dependent bivalent

domain mechanism contributes to their pluripotent state. Stem

Cells Dev 2012;21:1639–52.

[33] Ye S, Li P, Tong C, Ying QL. Embryonic stem cell self-renewal

pathways converge on the transcription factor Tfcp2l1. EMBO J

2013;32:2548–60.

[34] Martello G, Bertone P, Smith A. Identification of the missing

pluripotency mediator downstream of leukaemia inhibitory fac-

tor. EMBO J 2013;32:2561–74.

[35] Takashima Y, Guo G, Loos R, Nichols J, Ficz G, Krueger F,

Oxley D, Santos F, Clarke J, Mansfield W, et al.: Resetting

transcription factor control circuitry toward ground-state plur-

ipotency in human. Cell 2014;158:1254–69.

[36] Ben-Shushan E, Sharir H, Pikarsky E, Bergman Y. A dynamic

balance between ARP-1/COUP-TFII, EAR-3/COUP-TFI, and

retinoic acid receptor:retinoid X receptor heterodimers regulates

Oct-3/4 expression in embryonal carcinoma cells. Mol Cell Biol

1995;15:1034–48.

[37] Fuhrmann G, Chung ACK, Jackson KJ, Hummelke G, Baniah-

mad A, Sutter J, Sylvester I, Sch€oler HR, Cooney AJ. Mouse

germline restriction of Oct4 expression by germ cell nuclear

factor. Dev Cell 2001;1:377–87.

[38] Ratajczak MZ, Ratajczak J, Suszynska M, Miller DM, Kucia M,

Shin D-M. A novel view of the adult stem cell compartment from

the perspective of a quiescent population of very small embry-

onic-like stem cells. Circ Res 2017;120:166–78.

[39] Ratajczak MZ, Zuba-Surma E, Wojakowski W, Suszynska M,

Mierzejewska K, Liu R, Ratajczak J, Shin DM, Kucia M. Very

small embryonic-like stem cells (VSELs) represent a real chal-

lenge in stem cell biology: recent pros and cons in the midst of a

lively debate. Leukemia 2014;28:473–84.

[40] Ratajczak MZ, Shin D-M, Liu R, Marlicz W, Tarnowski M,

Ratajczak J, Kucia M. Epiblast/germ line hypothesis of cancer

development revisited: lesson from the presence of Oct-4C cells

in adult tissues. Stem Cell Rev Rep 2010;6:307–16.

[41] Pelton TA, Sharma S, Schulz TC, Rathjen J, Rathjen PD.

Transient pluripotent cell populations during primitive ectoderm

formation: correlation of in vivo and in vitro pluripotent cell

development. J Cell Sci 2002;115:329–39.

[42] GuoG,HussM, TongGQ,WangC, LiSunL, ClarkeND,Robson

P. Resolution of cell fate decisions revealed by single-cell gene

expression analysis from Zygote to Blastocyst. Dev Cell

2010;18:675–85.

[43] Hanna J, Cheng AW, Saha K, Kim J, Lengner CJ, Soldner F,

Cassady JP, Muffat J, Carey BW, Jaenisch R. Human embryonic

stemcellswith biological and epigenetic characteristics similar to

those of mouse ESCs. Proc Natl Acad Sci 2010;107:9222–7.

[44] Yamaguchi Y, Yonemura S, Takada S. Grainyhead-related tran-

scription factor is required for duct maturation in the salivary

gland and the kidney of the mouse. Development 2006;

133:4737–48.

[45] Yamaguchi Y, Ogura S, Ishida M, Karasawa M, Takada S. Gene

trap screening as an effective approach for identification of Wnt-

responsive genes in the mouse embryo. Dev Dyn 2005;233:484–

95.

[46] To S, Rodda SJ, Rathjen PD, Keough RA. Modulation of CP2

family transcriptional activity by CRTR-1 and sumoylation.

PLoS ONE 2010;5:e11702.

[47] KangHC, Chae JH, Lee YH, ParkM-A, Shin JH, Kim S-H, Ye S-

K, Cho YS, Fiering S, Kim CG. Erythroid cell-specific a-globin

gene regulation by the CP2 transcription factor family. Mol Cell

Biol 2005;25:6005–20.

8 H. Lee et al.

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com