一个转录因子Oct4可以将滋养外胚层来源的滋养层干细胞重编程Reprogramming of Trophoblast Stem Cells into

EMBRYONIC STEMCELLS/INDUCED PLURIPOTENT STEMCELLSReprogramming of Trophoblast Stem Cells into Pluripotent Stem Cells by Oct4

Tong Wu1,2, Haitao Wang2, Jing He2, Lan Kang2, Yonghua Jiang2, Jinchao Liu2, Yu Zhang2, Zhaohui Kou2, Lijun Liu3, Xuehong Zhang3and Shaorong Gao2*

1

College of Biological Sciences, China Agricultural University, Beijing 100094, P.R. China.; National Institute of

3

2

Biological Sciences, NIBS, Beijing 102206, P.R. China.; Center of Reproductive Medicine Research; the First Hospital of Lanzhou University; Gansu; P.R. China.

Key words. Trophoblast stem cells x Pluripotent stem cells x Reprogramming x Cell fate conversion

ABSTRACT

Embryonic stem (ES) cells and trophoblast stem (TS) cells are both derived from early embryos, yet these cells have distinct differentiation properties. ES cells can differentiate into all three germ layer cell types, whereas TS cells can only differentiate into placental cells. It has not been determined whether TS cells can be converted into ES-like pluripotent stem (PS) cells. Here we report that overexpression of a single transcription factor, Oct4, in TS cells is sufficient to reprogram TS cells into a pluripotent state. These Oct4 induced pluripotent stem (OiPS) cells have the epigenetic characteristics of ES cells, including X chromosome reactivation, elevated H3K27 me3

modifications and hypomethylation of promoter regions in Oct4 and Nanog genes. Meanwhile, methylation of promoter region in the Elf5 gene occurred during reprogramming of TS cells. The gene expression profile of OiPS cells was very similar to ES cells. Moreover, OiPS cells can differentiate into the three germ layer cell types in vitro and in vivo. More importantly, chimeric mice with germline transmission could be efficiently produced from OiPS cells. Our results demonstrate that one single transcription factor, Oct4, could reprogram the non-embryonic TS cells into pluripotent stem cells.

INTRODUCTION

The first cell fate determination occurs at the 8-cell embryo stage in mice. Consequently,

blastomeres give rise to two completely different cell types [1, 2]. Some blastomeres develop into trophoblast cells, triggered by both intrinsic and extrinsic signals. Upregulation of

Author contributions: T.W.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; H.W., J.H., L.K., Y.J., J.L., Y.Z., Z.K., L.L., and X.Z.: provision of study material, data analysis and interpretation; S.G.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript.

*Correspondence: Shaorong Gao, Ph.D. #7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, P.R. China. Tel: (86)-10-807267; Fax: (86)-10-80727535, E-mail address: gaoshaorong@nibs.ac.cn; Received September 28, 2010; accepted for publication January 28, 2011. ©AlphaMed Press 1066-5099/2011/$30.00/0 doi: 10.1002/stem.617

Reprogramming of TS cells by Oct4

Cdx2 expression, mediated through the Hippo-Tead4 pathway, and simultaneous allocation of such blastomeres to the outside of the embryo initiate the formation of the trophectoderm [1-8]. The rest of the blastomeres, which express high levels of Oct4 and Sox2, are allocated inside of the embryo and develop into the pluripotent inner cell mass (ICM [2, 9, 10]).

In vitro, two types of stem cells can be established from embryos at the very early blastocyst stage: trophoblast stem (TS) cells and embryonic stem (ES) cells [11-13]. In distinct culture conditions, both types of stem cells undergo self-renewal in vitro. Although both types of stem cells are derived from early embryos, they stably retain the cell lineage restrictions and in turn TS cells can never make embryonic tissues. It has recently been discovered that downregulation of Oct4 in ES cells can convert ES cells into TS-like cells [10]. However, it is not yet known if TS cells can be converted into ES-like pluripotent stem cells.

Although TS and ES cells originate from early embryos, it remains unclear whether non-embryonic derived TS cells can be converted into pluripotent stem cells with pluripotency, and if so, how many transcription factors are required for this cell fate transition.

MATERIALS AND METHODS

FAnimals

The specific pathogen-free (SPF) grade mice were housed in the animal facility of the National Institute of Biological Sciences. All studies adhered to procedures consistent with the National Institute of Biological Sciences Guide for the care and use of laboratory animals.

Trophoblast Stem (TS) Cell Derivation and Culture

Trophoblast stem (TS) cells were derived and maintained as previously described by Dr. Janet Rossant [13, 29]. Briefly, E3.5 blastocysts were flushed from the uterus of 129/sv and OG2 female mice which were mated with Rosa26-M2rtTA male mice. Blastocysts were

Generation of induced pluripotent stem (iPS) then transferred on to mitomycin C (MMC) cells from differentiated somatic cells, through treated ICR MEs with TSC medium overexpression of transcription factors that are containing RPMI10 (Gibco Invitrogen), 20% highly enriched in embryonic stem (ES) cells fetal bovine serum (FBS, Hyclone), 1mM [14-24], not only provides us with a sodium pyruvate (Sigma), 100uM revolutionary approach to potentially generate ȕ-mercaptoethanol (Chemicon), 2mM histocompatible patient-specific iPS cells for L-glutamine (Chemicon) and F4H (25ng/ml regenerative medicine application, but also GF4, Invitrogen and 1.0 ȝg/ml Heparin, prompts us to apply this approach to understand Sigma). After disaggregation at 5 to 7 days cell fate determination. Recently, it has been later, culture medium was changed to 70% demonstrated that certain cell types (such as FCM (Feeder Condition Medium) + 1.5X F4H neural stem cells) can be converted into and refreshed every other day. At the time of pluripotent stem cells through expression of first passage the medium was changed to 70% only one or two transcription factors [25, 26]. FCM +F4H. Moreover, conversion of certain differentiated somatic cells into another type of differentiated cell has recently been demonstrated both in vivoandin vitro [27, 28].

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Reprogramming of TS cells by Oct4

Immunofluorescent Staining and Confocal Microscopy

Cells growing on slides were fixed by 4% paraformaldehyde overnight at 4°C and then permeabilized for 15 min with 0.5% Triton X-100. Slides were blocked in 2.5% BSA for 1hr at room temperature then incubated with the first antibodies to anti-Oct4 (1:500, Santa iPS Cells Generation

The plasmids used and the procedure of iPS Cruz), Nanog (1:1000, Cosmo BioCo), SSEA1 cells derivation was described previously [18, (1:50, Millipore), Cdx2 (1:50, Santa Cruz), 30, 31]. Briefly, 293T cells was transfected with H3K27me3 (1:250, Millipore) and EZH2 (1:50, TetO-UW-Oct4, Sox2, Klf4 and c-Myc Cell Signaling) for 1.5hr at room temperature Fplasmids separately with lentivirus packaging separately. After three times of washing, slides plasmids ps-PAX-2 and pMD2G. Medium were incubated with Alexa-luor goat containing virus was collected twice at 24hrs anti-rabbit or goat anti-mouse IgG secondary and 48hrs after transfection. About 30ml virus antibody (1:1000, Invitrogen) for 1 hr at room medium was centrifuged 25’000 rpm for 1.5hr temperature. DNA was stained with 1 µg/ml at 4°C and resuspended in 4ml 70%FCM +F4H DAPI and the slides were mounted in antifade with 5µg/ml polybrene. 105 TS cells was plated solution.

F

on gelatin coated dish and infected for 10hrs. TS

Fcells were recovered for 12hrs in 70%FCM Stained cells on slides were observed on a LSM +F4H and then medium replaced with ES 510 META microscope (Zeiss) using a Plan medium containing DMEM (Chemicon), 15% Neofluar 40× DIC or 63× Oil DIC objective and

excitation wavelengths of 3 nm and 405 nm. BS, 1mM L-glutamine, 0.1mM

mercaptoethanol, 1% nonessential amino acid All collected images were assembled using (Chemicon), and 1000 U/ml LIF (Chemicon) Adobe Photoshop software (Adobe Systems) supplemented with 1µg/ml doxcycline (Sigma) without any adjustment of contrast and and refreshed everyday. After the ES cell like brightness to the images. colonies appeared, the medium were replaced

with ES medium for 2 to 3 days. Then the RT-PCR and Quantitative PCR

colonies was mechanically picked up and Total RNA was extracted from TS cells or iPS digested to culture on MMC treated MEF cells cells after removal of feeder celles by TRIzol

reagent (Invitrogen) and reverse transcribed by as ES cells.

MMLV system (Promega). RT-PCR was performed as following with 2× PCR Supermix Karyotyping

Karyotyping of TS cells and iPS cells were Solution (Vigorous): 94°C 4min; 30 cycle of preformed as previously described [18]. 94°C 30s, 58°C 30s and 72°C 40s; 72°C 5 min.

Quantitative PCR was performed under the Chromosomal G-band analyses were performed

at Lanzhou University First Hospital IVF instruction of manufacturer with SYBR

PrimeScript RT-PCR kit (Takara) on a ABI Center.

PRISM 7500 Realtime PCR System (Applied Biosystems). All the primer sets used were previously described [14]. In vitro Differentiation of TS Cells

5×105TS cells were plated onto gelatin-coated tissue culture dishes with TSC medium for up to 6 days and total RNA were extracted for analyzing the expression of differentiation related genes by realtime PCR.

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Reprogramming of TS cells by Oct4

Alkaline Phosphatase Staining

AKP staining was performed under the instruction of manufacturer with Leukocyte Alkaline Phosphatase Kit (Sigma).

Teratoma Formation

2×106iPS cells were suspended in 200µl PBS and subcutaneously injected into the groin of SCID mice. 4 to 6 weeks later the teratoma with a diameter of 2cm was dissected for hematoxylin-eosin staining performed at Center for Clinical Laboratory Development of Peking Union Medical College.

Two-round nested PCR was performed to amplify the promoter region of each gene and F

PCR products were purified from agarose gel by Gel Purification kit (Qiagen).

Western Blot Analysis

After removal of feeder cells, iPS cells were washed once by PBS and boiled to 100rC for 5

min in protein loading buffer (BioRad) with 2% ȕ-mercaptoethanol (Amersham). Anti Į-tubulin(1:2000, Sigma) and Histone H3 (1:5000, generated by Antibody Center at National Institute of Biological Sciences, Beijing) antibody were used as endogenous In vitro Differentiation of iPS Cells

105iPS cells were cultured in IMDM (Gibco) control and anti H3K27me3 (1:2000, with 15%BS and 1mM L-glutamine on Millipore), H3K4me3 (1:1000, Millipore),

Ultra-low attachment 6-well plate (Costar). H3K9me3 (1:1000, Millipore), Ezh2 (1:1000, After 4 to 6 days, EBs were trypsinized and Cell Signaling), Suz12 (1:1000, Cell Signaling)

were used. ECL peroxidase labelled anti mouse plated on gelatin-coated tissue culture dishes for

an extra 7 days and then total RNA was or rabbit antibodies (Amersham) were used as

the secondary antibodies. extracted.

Chimeras Generation and Genotyping

D3.5 Blastocysts of ICR mice were flushed and 10 to 15 cells were injected to the cavity of each blastocyst. Then the blastocysts were transplanted into the uterus of pseudo-pregnant ICR mice. The female chimeric mice with 8-10 weeks old produced from iPS cells were mated with male ICR mice to confirm the germline transmission of iPS cells. To test the multipotency of TS cells, D12.5 and D18.5 fetus were obtained by caesarean section and genomic DNA of placenta and embryo were isolated by Phenol : Chloroform : Isoamylalcohol (25:24:1). PCR for genotyping was performed using primers targeting rtTA sequence got from Mouse Genome Informatics Website.

Bisulphite Sequencing

Genomic DNA of cells was modified with the Methylamp DNA Modification kit (Epigentek).

4

Microarray Analysis

Total RNA was extracted from TS cells or iPS cells including 3 biological repeats by TRIzol after removal of feeder cells. Affymetrix Mouse Gene 1.0 ST Array (Affymetrix, Inc.) was used and all were performed at Beijing Capitalbio corporation. The microarray data had been submitted to GEO database (GSE25255).

RESULTS

Derivation and Characterization of Trophoblast Stem (TS) Cells

Firstly, we derived trophoblast stem (TS) cell lines using D3.5 blastocysts from 129/sv and OG2 female mice that were mated with Rosa26-M2rtTA mice [18]. ollowing disaggregation and the first passage of cells, cells cultured in fibroblast-conditioned medium (supplemented with FGF4 and heparin) formed colonies with distinct borders and tight

Reprogramming of TS cells by Oct4

epithelial morphology resembling typical TS for trophoblast giant cells, Figure S1 [13, 34]). cells (Figure 1A). Expression of a TS-specific We also examined the in vivo differentiation transcription factor, Cdx2, was detected in the potential of 129R3 TS cells using chimera established TS cells (Figure 1B). Following experiments. Ten to fifteen 129R3 TS cells karyotype analysis, we chose one TS cell line, were microinjected into ICR mouse blastocysts.

Subsequently, rtTA specific sequences were 129R3, with the correct karyotype (40, XX) and

PCR amplified from placenta genomic DNA typical morphology of TS cells for further study

and genomic DNA from embryos collected at (igure 1C). Using reverse

transcription-polymerase chain reaction 12.5 dpc and 18.5 dpc. The rtTA sequence was (RT-PCR) we found that the 129R3 TS cells only detected in genomic DNA from the

placenta (Figure S2). These results confirmed expressed the majority of TS cell marker genes,

including Cdx2, Eomes and Esrrb, Fgfr2 and that the 129R3 TS cells we established are

multipotent and can only commit to placenta Sox2. The expression levels of these genes were

comparable to levels in the widely used cell lineages both in vitro and in vivo.Cdx2-GFP TS cell line (the Cdx2-GFP TS cell

line was kindly provided by Dr. Janet Rossant, Reprogramming of TS Cells into Induced Figure 1D). Quantitative RT-PCR was further Pluripotent Stem (iPS) Cells

performed and the results indicate that the Reprogramming of 129R3 TS cells was further expression levels of Sox2, c-Myc and Esrrb in investigated using a Doxcycline inducible iPS

system [30, 31]. Since the 129R3 TS cells 129R3 TS cells were similar to the levels in ES

cells, whereas Oct4 and Klf4 expression was express Sox2 and c-Myc at moderate levels, and silenced in TS cells. In addition, the other high expression of Esrrb is also detected in Klf-family members including Klf1 and Klf5 these cells, we hypothesized that fewer F

transcription factors, or even one factor (Oct4), which can substitute Klf4 in iPS generation [32,

33], were expressed at moderate level in TS would be sufficient to reprogram TS cells into cells but lower than ES cells (Figure 1E). The pluripotent stem cells. Evidence from recent

studies has clearly shown that Oct4 alone could results presented above prompted us to test the

possibility of reprogramming the TS cells with reprogram neural stem cells into pluripotent fewer transcription factors in subsequent stem cells because the other three transcription

factors were also expressed at relatively high experiments.

levels in neural stem cells [25, 26]. Although Klf4 is not expressed in TS cells, it has been To examine if the newly derived 129R3 TS cells

possessed the unique TS cell differentiation recently demonstrated that Esrrb could properties, we cultured the 129R3 TS cells in substitute for Klf4 in reprogramming of the TS cell medium without FGF4 and heparin fibroblast cells into pluripotent stem cells [35]. in vitro. Subsequently, 129R3 TS cells Based on these studies and the gene expression differentiated into giant cell-like cells, which characteristics of TS cells, we next examined are large and polyploid and belong to primary the possibility of reprogramming TS cells with

fewer transcription factors. Different trophoblast cells (Figure S1). The differentiated

129R3 TS cells expressed several genes of transcription factor combinations were used,

including all four factors, two factor trophoblast cells, including Mash2 (specific for

diploid trophoblast cells), Tpbpa (specific for combinations (which included Oct4 and one of spongiotrophoblast cells), Gcm1 (specific for the other three factors), and Oct4 alone. After

lentiviral infection, 129R3 TS cells were labyrinthine trophoblast cells) and Pl1 (specific

5

Reprogramming of TS cells by Oct4

OiPS cells and 4 iPS cells expressed allowed to recover for 12 hrs in feeder condition

medium, and then the medium was replaced pluripotent genes including Oct4, Sox2, Nanog,

Ecat and Gdf3 at levels comparable to that of with Doxcycline supplemented ES medium. All

exogenous transcription factors were highly normal R1 ES cells (Figure 2B). In contrast, the

Fexpression of TS-specific Cdx2 was silenced in overexpressed in the TS cells within 48 hours

after infection (Figure S3). After two to three both types of iPS cells. Moreover, endogenous

Oct4, Sox2, Nanog, Klf4 and c-Myc were weeks, primary ES-like colonies appeared in all

groups (Figure S3). Variable reprogramming expressed at similar levels in OiPS, 4F iPS and kinetics and efficiency were observed among ES cells (Figure S8). Expression of Oct4, the different transcription factor combinations Nanog and SSEA-1 at protein level was

detected in OiPS and 4 iPS cells by (Figure S3). More importantly, we succeeded in

immunocytochemistry staining, but Cdx2 was generating ten iPS cell lines from Oct4-infected F

TS cells within two and half weeks (16 days), no longer expressed in the Oct4-induced iPS and the reprogramming kinetics were cells (Figure 2C). comparable to four factors induced

reprogramming (igure 2A; igure S3). Epigenetic Characteristics of Oct4-induced Compared with the prolonged reprogramming PS Cells

DNA-methylation mediated gene regulation kinetics (4-5 weeks) observed in neural stem FF

cells [25], Oct4 conversion of TS cells into plays an important role in determining the ES-like cells appeared relatively easier. expression of transcription factors which are Although the reprogramming efficiency of specifically expressed in either ES or TS cells.

F

Oct4-infected TS cells was significantly lower Oct4 and Nanog are two master pluripotency than that of cells infected with Oct4 combined transcription factors expressed in pluripotent with either Klf4 or Sox2, it is comparable to cells, we next analyzed DNA methylation of the four-factor ME reprogramming [15]. Oct4 and Nanog promoters in 129R3 TS, R1, Interestingly, the combination of Oct4 and OiPS and 4F iPS cells, and bisulfite results c-Myc had no positive effects on illustrated that demethylation of Oct4 and reprogramming efficiency, and moreover, it Nanog promoters occurred in both OiPS and 4F F

prolonged the reprogramming kinetics (Figure iPS cells, whereas Oct4 and Nanog promoters

were highly methylated in 129R3 TS cells S3). Similarly, we could successfully reprogram

the other TS cell line derived from OG2rtTA (Figure 3). Similarly, Elf5 is one of the master embryo into pluripotent stem cells using one transcription factors specifically expressed in

TS cells and moreover, its expression is single transcription factor, Oct4 (Figure S4).

determined by DNA methylation [36].

Oct4-induced iPS cells (OiPS) and four factors Therefore, we further analyzed the DNA induced iPS cells (4 iPS) were methylation status of the Elf5 promoter region indistinguishable from ES cells in their in all the different cell lines. The bilsufite results morphology and were AP positive (Figure 2A, revealed that the Elf5 promoter was Figure S5). After doxcycline withdraw, the unmethylated in TS cells but highly methylated

in R1 ES cells (Figure 3). Interestingly, expression of exogenous genes was silenced in

OiPS and 4F iPS cells (Figure S6). After methylation of the Elf5 promoter was observed karyotype analysis, OiPS-8 and 4F iPS-4 cell in the TS-reprogrammed OiPS and 4F iPS cells

(Figure 3). These results indicated that DNA lines derived from 129R3 TS cells were used for

molecular characterization (Figure S7). Both methylation can be reprogrammed in the

6

Reprogramming of TS cells by Oct4

F

process of cell fate transition from TS cells to iPS cells.

Since the 129R3 TS cell line karyotype is 40, XX, we examined whether other epigenetic modification events, including X chromosome inactivation in TS cells, could be precisely reprogrammed during reprogramming. Female TS cell has one active and one inactive X chromosome, which is in accordance with it’s invivo counterpart: D3.5 of the trophoblast embryo [37, 38]. However, both X chromosomes are active in female ES cells [39, 40]. Since PRC2 complex components Eed and Ezh2-mediated H3K27me3 have an important role in the initiation of X chromosome inactivation [38], we used immunofluorescence to detect nuclear H3K27me3 foci, a hallmark of X chromosome inactivation. Apparently, one X chromosome was inactivated in 129R3 TS cells, and after reprogramming no H3K27 me3 foci were observed in either OiPS or 4F iPS cells, suggesting that the inactivated X chromosome was reactivated (Figure 4A).

were lower than in OiPS and 4F iPS cells as detected by western blot. Similarly, the amount of Ezh2 protein in 129R3 TS cells was much lower than in either R1 ES or iPS cells (Figure 4C). These results demonstrate that both OiPS and 4F iPS cells have the same epigenetic modifications, including X chromosome reactivation and elevated H3K27 me3 modifications, as normal ES cells and are distinct from the parental 129R3 TS cell line.

Gene Expression Profile of Oct4-induced PS Cells

To investigate the global gene expression profile, we conducted microarray analysis on OiPS, 4F iPS, R1 and 129R3 TS cells using Affymetrix Mouse Gene 1.0 ST Array (Affymetrix, Inc.). Pearson correlation analysis was used to cluster the cells and the results demonstrate that the gene expression profile of both OiPS and 4F iPS cells is highly similar to that of R1 ES cells, but distinct from the parental 129R3 TS cells (Figure 5A). The volcano plot analysis was used to compare gene expression profiles between two different cell

Recently, it has been reported that there are lines and the results illustrate that iPS cells are

similar to each other as well as to R1 ES cells distinct histone modifications in three stem cell

lines derived from early embryos: ES, TS and (Figure 5B). We also found that the expression XEN (derived from primitive endoderm) cells level of pluripotent genes in both OiPS and 4F

iPS cells is comparable to that of ES cells [41]. The H3K27me3 level in TS and XEN cells

was much lower than in ES cells due to the (Figure S9A). However, the expression of many reduced expression of the PRC2 components X-chromosome genes was significantly Ezh2, Eed and Ezh1 [41]. Therefore, we up-regulated in both OiPS and 4F iPS cells examined the histone modifications and the compared with R1 ES cells. Among them, 93 expression level of PRC2 and PRC1 genes in OiPS and 35 genes in 4F iPS cells

expressed a greater than 2-fold difference components in our TS cells and in the iPS cells

using both western blot and quantitative compared with R1 ES cells (selected RT-PCR assays. The mRNA levels of Ezh2, representative genes are shown in Figure Ezh1 and Eed in 129R3 TS cells were much S10A). On the other hand, expression of several

Y chromosome genes was lower in both OiPS lower than in either R1 ES or iPS cells, whereas

the expression level of Suz12 and the PRC1 and 4F iPS cells than in R1 ES cells (Figure component Rnf2 was similar in all cell lines S10B). Apparently, up-regulation of X (igure 4B). In addition, H3K27me3 chromosome genes and low expression of Y

chromosome genes in iPS cells was due to the modifications in 129R3 and Cdx2-GFP TS cells

7

Reprogramming of TS cells by Oct4

fact that iPS cells contain two active chromosomes. Several X chromosome genes were expressed at much higher levels in OiPS

cells than in 4F iPS cells (Figure S9B). Results from a recent study have shown that expression of pluripotent genes (including Oct4, Nanog and Sox2) is coordinated with Xist repression during embryogenesis [42]. It has also been reported that Oct4 functions upstream of Xist, and depletion of Oct4 leads to inactivation of both X chromosomes in female cells [43]. Trophoblast stem cells inherit the imprinted X chromosome inactivation of the trophoblast [44], and reprogramming of TS cells by pluripotent genes cause repression of Xist expression, which in turn induces expression of other X-chromosome genes. However, whether reprogramming using different combinations of transcription factors causes different states of X chromosome ‘activation’ needs to be further investigated. We analyzed the expression of imprinted genes and found that the reciprocal IGF2/H19 genes had higher expression level in iPS cells compared with R1 ES cells, whereas Peg5 and Peg10 had lower expression in iPS cells than R1 ES cells (Figure S9C). Through an analysis of published microarray data [45], we found that expression of the H19 gene varied within different iPS cell lines. Moreover, biallelic expression of H19 in human iPS cells has been reported [46]. Expression of Gtl2 imprinted gene clusters has been suggested to be important for iPS cells pluripotency [45, 47]. Our array data showed that expression of Gtl2 and Rian was down-regulated in OiPS and 4F iPS cells, whereas Dlk1 expressed higher and Dio3 expressed at similar level as compared with R1 ES cells (igure S10C). Taken together, the microarray data suggest that successful reprogramming occurred in both Oct4 and four factors induced iPS cells, whereas disruption in the expression of some imprinted genes might also occur during the reprogramming process.

8

Pluripotency of Oct4-induced PS Cells

To investigate the differentiation potential of both OiPS and 4F iPS cells in vitro, we first determined whether both one factor and four factor-induced iPS cells could differentiate into the three germ layers using the embryoid body (EB) differentiation assay. When iPS cells were cultured in low adherent dishes without feeder cells or LIF, EBs could be efficiently generated from iPS cells (Figure S11). After being plated onto tissue culture dishes for an extra 7 days, differentiated cells expressed marker genes including Gata6, Brachyury, and Map2 (from the three germ layers, endoderm, mesoderm, and ectoderm, respectively) as determined by quantitative RT-PCR (Figure S11).

To further investigate the differentiation potential of both OiPS and 4F iPS cells in vivo,we used teratoma formation and chimera-contribution assays. Teratomas were formed 4-5 weeks after subcutaneous transplantation of both types of iPS cells into SCID mice. The teratomas contained tissues from all three germ layers as revealed by hematoxylin and eosin staining (Figure 6A). Finally, we used the chimera-contribution assay to assess the developmental potential of both OiPS and 4F iPS cells. Both OiPS and 4F iPS cells could efficiently generate chimeric mice as determined by the coat color of the mice (Figure 6B). We further assessed the germline transmission capability of these chimeric mice. After mating female chimeric mice with ICR male mice, germline transmission was observed (Figure 6B). Our data demonstrate that OiPS cells have a developmental potential that is comparable to that of normal ES cells.

Reprogramming of TS cells by Oct4

TS cells might also play certain role in replacing Klf4 during reprogramming. Therefore, the

As the first differentiated cell lineage during unique gene expression characteristics of TS embryogenesis, trophoblast cells only cells make it another suitable cell type to be contribute to the placenta in vivo.In vitro, TS reprogrammed by a single transcription factor cells were established and have been widely Oct4. Different from the slow reprogramming used to investigate the mechanism of embryo kinetics observed in NS cells reprogramming, implantation. Interestingly, it was discovered the successful conversion of TS cells into that ES cells derived from the other early pluripotent stem cells only need 16 days. This blastocyst cell lineage (ICM cells) could be phenomenon might indicate that molecular converted into TS-like cells through either events occurred in the process of TS cell forced repression of Oct4 or overexpression of reprogramming might be distinct from NS cell Cdx2 [10]. Therefore, it became imperative to reprogramming. More in-depth investigations understand whether TS cell fate could be need to be performed in the future to explore the

precise molecular mechanism leading to this converted into an ES-like cell fate. Our present

study demonstrates that a single transcription cell fate conversion.

DISCUSSION

factor, Oct4, is sufficient to allow this cell fate conversion.

Interestingly, in the initial experiments, we presumed that knock-down of Cdx2 in TS cells

In early embryos, Oct4 and Cdx2 form a might be beneficial for the conversion of TS complex and the expression level of either of cells into pluripotent stem cells. However, we these transcription factors determines the found that down-regulation of Cdx2 expression earliest cell fate commitment [10]. has no positive effects in this process (data not

shown), which may be due to the fact that Subsequently, Cdx2 is predominantly expressed

in TS cells, and we speculate that down-regulation of Cdx2 in TS cells causes TS

cell differentiation instead of boosting the overexpression of Oct4 in TS cells may repress

expression of Cdx2, thus re-establishing a reprogramming process. pluripotent stem cell transcription regulatory network where Sox2 is moderately expressed in TS cells. Indeed, our present study proved this hypothesis. We propose that the principle in TS cell reprogramming resembles neural stem cells reprogramming but with slight differences [25, 26]. All the rest three reprogramming factors including Sox2, Klf4 and c-Myc, are expressed in NS cells but the Klf4 was found silenced in TS cells even though expression of Sox2 and c-Myc was detected. However, expression of another transcription factor, Esrrb, in TS cells was detected. It has been previously shown that Esrrb is capable of substituting for Klf4 in reprogramming of fibroblasts to pluripotency [35]. Moreover, moderate expression of Klf-family members including Klf1 and Klf5 in

9

Epigenetic reprogramming plays a central role in nuclear reprogramming mediated by either somatic cell nuclear transfer or defined transcription factors [48, 49]. Demethylation of the promoter regions in the pluripotency transcription factors has been persistently utilized to evaluate if the reprogramming is complete. In the process of cell fate transition from TS cells to pluripotent stem cells, demethylation of the promoter regions in the Oct4 and Nanog genes indeed occurred. Moreover, methylation of the promoter region of Elf5 gene, which was unmethylated in TS cells, occurred in the reprogrammed pluripotent stem cells. To our knowledge, this is the first evidence showing that the promoter region of a

Reprogramming of TS cells by Oct4

lineage specific gene has to be methylated in the process of reprogramming.

Recent investigations have demonstrated that distinct epigenetic configurations are persisted in ES and TS cells [41]. H3K4me3/H3K27me3 bivalent marks are found in a specific set of genes encoding developmental regulators in ES ACKNOWLEDGMENTScells, whereas this bivalent mark is not observed

in TS cells. It remains to be determined in the We are grateful to our colleagues in our future to address how this epigenetic laboratory for their assistance with experiments configuration re-established in the and in the preparation of this manuscript. We reprogrammed iPS cells derived from TS cells. also thank Dr. Janet Rossant for giving us great Our system provides some advantages in suggestions during the preparation of the answering such questions because only one manuscript. This project was supported by the transcription factor Oct4 is capable of Ministry of Science and Technology (grants reprogramming the TS cells into iPS cells, 2008AA022311, 2010CB944900 and which excludes the effects of other transcription 2011CB9800). factors which are normally used in fibroblasts reprogramming. Disclosure of Potential Conflicts of Interest

CONCLUSION

In conclusion, the present study demonstrated that a single transcription factor Oct4 is sufficient to convert non-embryonic derived TS cells into pluripotent stem cells with

The authors indicate no potential conflicts of interest.

pluripotency. Our study not only provides an invaluable tool to further decipher the molecular mechanism underlying cell fate determination in early embryos, but also suggests that TS cells from the placenta might be a better cell type for obtaining iPS cells.

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See www.StemCells.com for supporting information available online.

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Figure 1. Derivation and characterization of trophoblast stem (TS) cells.

A. Morphology of trophoblast stem cell line 129R3 derived from 129/sv ×Rosa26-MrtTA blastocysts. A picture of R1 ES cell was shown. Left: 40×; Right 10×.

B. Immunofluorescent staining showing that the 129R3 TS cell line is positive for Cdx2. C. The karyotype of the 129R3 TS cell was 40,XX.

D. RT-PCR results indicating that 129R3 TS cells express trophoblast stem cell marker genes including Cdx2, Sox2, and Esrrb. The Cdx2-GFP TS cells were used as a positive control.

E. Quantitative PCR demonstrates that 129R3 TS cells express transcription factors Sox2, c-Myc and Esrrb at similar levels compared with R1 ES cells; but Klf4 expression level was much lower in TS cells. Oct4 expression was not detected in TS cells.

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Figure 2. Conversion of 129R3 TS cells into pluripotent stem cells by overexpressing Oct4 or the combination of four factors.

A. Morphology of 129R3 TS cells after 7 days of induction, indicating that the cell morphology has changed (top), forming primary colonies at 14 or 16 days (middle) and established iPS cell lines (bottom). Left: Induced by Oct4. Right: Induced by Oct4, Sox2, Klf4 and c-Myc.

B. RT-PCR results showing that OiPS and 4F iPS cells expressed ES cell markers Oct4, Nanog, Sox2, etc.

C. Immunofluorescent staining results indicate that OiPS and 4F iPS cells are positive for Oct4, Nanog and SSEA1 but negative for Cdx2.

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Figure 3. DNA methylation status in the promoter regions of Oct4, Nanog and Elf5.

Bisulfite sequencing results showed that the highly methylated Oct4 and Nanog promoter regions in 129R3 TS cells were demethylated in OiPS, 4F iPS cells. In the contrast, unmethylated promoter region of Elf5 in the 129R3 TS cells was methylated in OiPS and 4F iPS cells. R1 ES cells were used as a positive control. Open and closed circles indicated the unmethylated and methylated CpGs. For each gene, 10 samples were sequenced and each row indicated a repeat.

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Figure 4. Epigenetic characterization of inducible pluripotent stem cells.

A. Immunofluorescent staining results showing that 129R3 TS cells have H3K27me3 foci (a hallmark of X inactivation), whereas both OiPS and 4F iPS cells possess two active X chromosomes. The Right panel is an enlarged region of the left panel.

B. Quantitative PCR demonstrated that 129R3 TS cells express lower levels of Ezh1, Ezh2 and Eed, and similar level of Suz12 and Rnf2 compared with OiPS, 4F iPS cells and R1 ES cells.

C. Western blot indicating that H3K27me3 modification in TS cells is lower than in OiPS and 4F iPS cells, while H3K4me3 and H3K9me3 levels are similar in all cell lines tested. The PRC2 component Ezh2 has a much lower expression level in TS cells compared with both OiPS and 4F iPS cells.

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Figure 5. Global gene expression profile of iPS cells.

A. Hierarchical clustering of global transcriptional profiles generated from microarray results from 129R3, OiPS-8, 4F iPS-4 and R1 cells, indicating greater similarities among OiPS, 4F iPS and R1. B. Volcano plot analysis was used to compare gene expression profiles from two different cell lines. The results show that OiPS and 4F iPS cells are similar to each other as well as to R1, but distinct from 129R3.

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Figure 6. Pluripotency characterization of Oct4-induced PS cells

A. Hematoxylin and eosin staining showing that teratomas produced from OiPS and 4F iPS cells contain tissues from the three germ layers.

B. Chimeric mice of OiPS-8 cells with high chimerism (left) and germ line transmission (right) from the chimeric mice.

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