Generation of Human-Induced Pluripotent Stem Cells from Gut Mesentery-Derived Cells by Ectopic Expression of OCT4/SOX2/NANOG

Abstract

Induced pluripotent stem (iPS) cells have been generated from human somatic cells by ectopic expression of defined transcription factors. Application of this approach in human cells may have enormous potential to generate patient-specific pluripotent stem cells. However, traditional methods of reprogramming in human somatic cells involve the use of oncogenes c-MYC and KLF4, which are not applicable to clinical translation. In the present study, we investigated whether human fetal gut mesentery-derived cells (hGMDCs) could be successfully reprogrammed into induced pluripotent stem (iPS) cells by OCT4, SOX2, and NANOG alone. We used lentiviruses to express OCT4, SOX2, NANOG, in hGMDCs, then generated iPS cells that were identified by morphology, presence of pluripotency markers, global gene expression profile, DNA methylation status, capacity to form embryoid bodies (EBs), and terotoma formation. iPS cells resulting from hGMDCs were similar to human embryonic stem (ES) cells in morphology, proliferation, surface markers, gene expression, and epigenetic status of pluripotent cell-specific genes. Furthermore, these cells were able to differentiate into cell types of all three germ layers both in vitro and in vivo, as shown by EB and teratoma formation assays. DNA fingerprinting showed that the human iPS cells were derived from the donor cells, and are not a result of contamination. Our results provide proof that hGMDCs can be reprogrammed into pluripotent cells by ectopic expression of three factors (OCT4, SOX2, and NANOG) without the use of oncogenes c-MYC and KLF4.

Introduction

Mouse and human somatic cells have been reprogrammed in vitro into induced pluripotent stem (iPS) cells through viral transduction of defined transcription factors (Park et al., 2008; Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Yu et al., 2007). The break-through has greatly improved the prospects of generating patient specific pluripotent stem cells for therapeutic purposes without therapeutic cloning, an approach with technical complications as well as ethical controversies (Jaenisch, 2004). With the fast progress in this field, different methods of delivering reprogramming factors have been developed, including using nonintegrating adenoviruses (Stadtfeld et al., 2008c), transient transfection of plasmids (Okita et al., 2008), episomal expression vectors (Yu et al., 2009), a polycistronic cassette containing all four factors (Carey et al., 2009), and excisable transposons (Kaji et al., 2009; Woltjen et al., 2009). More recently, generation of mouse and human iPS cells by direct delivery of reprogramming proteins was also reported (Kim et al., 2009a; Zhou et al., 2009). However, iPS cell generation without integrating virus-based protocols is very slow and inefficient, and requires further optimization. In addition, generation of iPS cells with a minimal number of factors may further hasten the application of iPS cells in treating human diseases in the future.

Human iPS cells are similar to embryonic stem (ES) cells in terms of gene expression, pluripotency, and epigenetic status, and hold great potential for use in regenerative medicine and in vitro disease modeling (Takahashi et al., 2007; Yu et al., 2007). Until now, a variety of mouse cell types, including fibroblasts (Takahashi and Yamanaka, 2006), neural progenitor cells (Kim et al., 2008, 2009c), liver and stomach cells (Aoi et al., 2008), pancreatic β-cells (Stadtfeld et al., 2008a), and lymphocytes (Hanna et al., 2008) have been reprogrammed to pluripotency. Similarly, for human somatic cells, fibroblasts (Takahashi et al., 2007; Yu et al., 2007), keratinocytes (Aasen et al., 2008), and blood cells (Giorgetti et al., 2009; Haase et al., 2009; Loh et al., 2009), neural progenitor cells (Kim et al., 2009b) and adipose cells (Sun et al., 2009) have been reported to yield successful iPS cell populations thus far. It is unclear whether other cell type, fetal gut mesentery-derived cells (hGMDCs), for example, can be reprogrammed into iPS cells. The hGMDCs derived from aborted fetus, which is easily available, are new cell resource for reprogramming.

The transcription factors OCT4, SOX2, and NANOG have essential roles in early development and are required for the propagation of undifferentiated ES cells in culture (Avilion et al., 2003; Chambers et al., 2003; Mitsui et al., 2003; Nichols et al., 1998). Genome-wide chromatin immunoprecipitation analysis has showed that OCT4, SOX2, and NANOG co-occupy many target genes in both mouse and human ES cells (Boyer et al., 2005; Loh et al., 2006). Therefore, a question remains as to whether these three molecules could can work together to reprogram human somatic cells to pluripotency. Recently, it has been shown that OCT4, SOX2, and KLF4 are sufficient to allow reprogramming of both mouse and human somatic cells to iPS cells, although at much lower efficiencies (<0.001% of input cells) than when c-MYC is included (Nakagawa et al., 2008). However, because the use of oncogene KLF4 is still included, this combination of factors is still not ideal. Here we have established human iPS cell lines from hGMDCs using three factors OCT4, SOX2, and NANOG without using of oncogenes c-MYC or KLF4. Reprogramming efficiency of hGMDCs with three factors is similar to that of four factors-iPS cells derived from fibroblasts (>0.01 of input cells) (Takahashi et al., 2007; Yu et al., 2007).

Among the three transcription factors, OCT4 and SOX2 are essential for the generation of iPS cells (Takahashi et al., 2007; Takahashi and Yamanaka, 2006). Surprisingly, NANOG, a member of the core transcriptional network in ES cells (Boyer et al., 2005; Loh et al., 2006), is dispensable for reprogramming mouse and human fibroblasts (Lowry et al., 2008; Takahashi and Yamanaka, 2006). In our three-factor system, NANOG is required for reprogramming hGMDCs to pluripotency. Three-factor hGMDC-iPS cells may provide a useful cell model for investigating the role of NANOG in reprogramming as well as regulating pluripotency.

Materials and Methods

Cell culture

Human fetal natural aborted samples were obtained with couples consent from the Beijing Maternity Hospital. Permission to use human embryonic tissue was granted by Peking University Health Science Center Ethical Committee. Human fetal gut mesentery tissue fragments were dissected at 10–12 weeks of gestational age, washed with sterile phosphate-buffered saline (PBS), mechanically separated into small pieces, and dissociated with 0.25% trypsin–EDTA (Gibco, Grand Island, NY, USA). Tissue was cultured on Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA)-coated plates using Dulbecco's modified Eagle's medium (HyClone; Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone), 2 mM L-glutamine (Hyclone) and 1% nonessential amino acids (Hyclone). Cells were passaged upon reaching 95% confluence with 0.25% trypsin–EDTA and targeted for reprogramming between passages 3 and 5. 293T cells were maintained in the same medium. H1 human ES cells and iPS cells were maintained in human ES medium containing Dulbecco's modified Eagle's medium (Gibco) supplemented with 20% serum replacement (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1% nonessential amino acids (Hyclone), 2 mM glutamine (Hyclone), and 4 ng/mL FGF2 (Peprotech, London, UK). Human iPS cells were subcultured every 5–7 days by enzymatic dissociation using 1 mg/mL Collagenase IV (Gibco) and maintained on feeder layers of mouse embryo fibroblasts (MEFs) that were mitotically inactivated by mitomycin C (Sigma, St. Louis, MO, USA).

Flow cytometry analysis

FITC-conjugated antihuman antibodies for CD45, CD71, HLA-DR, and CD117, as well as PE-conjugated antihuman antibodies for CD34, CD44, CD38, and CD90 (Pharmingen, San Jose, CA, USA), were applied for characterization of hGMDCs. Adherent cells were dissociated by 0.25% Trypsin–EDTA treatment into single cell suspension and stained with PE or FITC labeled antibodies at 4°C for 30 min. Control samples were stained with isotype-matched control antibodies. Stained cells were analyzed on a FACScaliburTM Flow Cytometer (Becton-Dickinson, Mountain View, CA, USA) using CellQuest software.

Lentivirus production and infection

293T cells were plated at 6 × 10⁶ cells per 100-mm dish and incubated overnight. The generation and structure of lentiviruses expressing OCT4, SOX2, and NANOG has been described in detail elsewhere (Yu et al., 2007). To produce infectious viral particles, 293T cells were transfected with the lentiviral vectors (12 μg) and packaging plasmids MD.G (4 μg) and CMVΔR8.91 (16 μg) with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Viral supernatants were harvested on 2 consecutive days beginning 24 h after transfection. A total of 30 mL of supernatant was typically harvested per virus. The supernatants were filtered through a 0.45-μm syringe filter (Millipore, Billerica, MA, USA), and loaded into a Amcion®Ultra-15 Centrifugal Filter (Millipore) for concentration. Viral supernatants were concentrated approximately 100-fold by centrifugation at 6000 rpm for 20 min at 4°C. Viral concentrates were stored at −80°C. Infections were carried out on 35-mm tissue cultured plates in 1 mL of ES medium containing 5μg/mL polybrene (Sigma) with 5–10 μL of each viral concentrate. hGMDCs were infected at a density of 5 × 10⁴ cells/plate. The medium was replaced 24 h after infection. The infection was repeated three times. Cells were dissociated by trypsin and transferred to MEF feeder layers.

RNA isolation and reverse transcription

Total RNA was purified with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) as per the manufacturer's instructions. Approximately 1 μg of total RNA from each sample was used for Oligo(dT)20—primed reverse transcription (SuperScript TM III First-Strand Synthesis System for RT-PCR, Invitrogen). Quantitative PCR reactions were carried out with Power SYBR®Green Realtime PCR Master Mix (TOYOBO, Osaka, Japan) and run on a Mx3000P QPCR System (Stratagene). PCR products were resolved on (1.5%) agarose gels and visualized by ethidium bromide staining. Images were taken using a Bio-Rad Gel document system. Primer sequences are shown in Supplementary Table S1.

Provirus integration

To examine the presence of transgenes in iPS clones, primers specific for each transgene were used to amplify the three proviruses. Specifically, primers Tg OCT4 and SP3 (vector-specific) were used to amplify the OCT4 transgene, Tg NANOG, and SP3 targeted the NANOG transgene, and Tg SOX2 and SP3 targeted the SOX2 transgene. GAPDH were used as a positive control. PCR on genomic DNA were carried out for 5 min at 95°C, 35 cycles of 58°C for 1min, 72°C for 1 min, 95°C, for 1 min, and 72°C for 10 min.

Alkaline phosphatase staining and immunocytochemistry

Alkaline phosphatase (AP) staining was performed using the Alkaline phosphatase kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. For immunocytochemistry, cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, the cells were treated with PBS containing 10% normal bovine serum albumin (Sigma) and 0.1% Triton X-100 for 30 min at room temperature, then incubated with primary antibodies at 4°C overnight. Primary antibodies included SSEA-1 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), SSEA-3 (1:100, Santa Cruz), SSEA-4 (1:100, Chemicon, Temecula, CA, USA), TRA-1-60 (1:100, Chemicon), TRA-1-81 (1:100, Chemicon), NANOG (1:100, Abcam, Cambridge, UK), OCT4 (1:100, Santa Cruz), SOX2 (1:100, Chemicon), VIMENTIN (1:100, Santa Cruz), GFAP (1:100, Chemicon), PDX-1 (1:100, Chemicon), DESMIN (1:100, Santa Cruz), and α-SMA (1:1000, Sigma). Normal mouse or rabbit serum was used as a negative control. Localization of antigens was visualized with antirabbit or antimouse IgG secondary antibodies conjugated with fluorescein (Santa Cruz). Nuclei were counterstained with DAPI.

In vitro differentiation

For spontaneous differentiation through EB formation, hGMDC-iPS cells cultured in human ES media without FGF2 and transferred to nontissue culture-treated plates. After 8 days in suspension culture, EBs were transferred to gelatin-coated plates and cultured in differentiation medium for another 8 days.

Bisulfite sequencing

Genomic DNA (1 μg) was treated with CpGenome DNAmodification kit (Chemicon), according to the manufacturer's recommendations. Treated DNA was purified with QIAquick column (Qiagen, Hilgen, Germany). The promoter regions of the human OCT4, NANOG genes were amplified by PCR. The PCR products were subcloned into pCR2.1-TOPO. Five clones of each sample were verified by sequencing with the M13 universal primer. Primer sequences used for PCR amplification were provided in Supplementary Table S1.

Teratoma formation

hGMDC-iPS cells were harvested by collagenase IV treatment, suspended in PBS, and injected subcutaneously into severe combined immunodeficient (SCID) mice. Six to 8 weeks after injection, tumors were explanted, fixed in 4% paraformaldehyde, embedded in paraffin, and examined histologically using hematoxylin and eosin staining.

Microarray experiments

Gene expression profiles were obtained from H1 human ES cells, hGMDC-iPS cells, hGMDCs using the human U133 Genome 430 2.0 GeneChip arrays (Affymetrix, Santa Clara, CA, USA) at CapitalBio Corporation. Briefly, total RNA from each sample was subjected to probe preparation and cRNA was hybridized. Arrays were scanned using an AffymetrixGCS3000 device and images were analyzed using the GCOS software. The gene expression raw data were extracted using GeneChip® Operating Software. The signal intensities from the mRNA channel in all the arrays were normalized together using the dChip Analysis software.

Karyotyping and DNA fingerprinting

Standard G-banding chromosome analysis was carried out in the Cytogenetics Laboratory at Peking University. To confirm the hGMDC origins of iPS clones, short tandem repeat (STR) analysis was performed in the center of forensic sciences at Beijing Genomics institute.

Results

Characterization of hGMDCs

hGMDCs showed fibroblast-like morphology (Fig. 1A) and were positive for Vimentin (Fig. 1B). Flow cytometry analysis showed that these cells did not express CD34, CD45, CD38, or HLA-DR but did express high levels of CD117, CD90, CD44, and CD71 (Fig. 1C). Quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) analysis showed that mRNA expression levels of OCT4, SOX2, and NANOG were significantly higher in human ES cells than in hGMDCs, whereas mRNA expression levels of KLF4 and c-MYC were not significantly different (Fig. 1D).

FIG. 1.

Characterization of hGMDCs. hGMDCs showed fibroblast-like morphology (A) and were positive for VIMENTIN (B). Flow cytometry analysis of hGMDCs (C). Q-RT-PCR analysis of expression levels of OCT4, SOX2, NANOG, KLF4, and c-MYC in human ES cells and hGMDCs (D). Scale bars: 50 μm.

Generation of hGMDC-iPS cells

hGMDCs were transduced at a density of 5 × 10⁴ cells per 35-mm plate using lentiviruses containing human OCT4, SOX2, and NANOG genes. The next day after transduction, the cells were trypsinized and plated onto mitomycin C-treated feeder cells and cultured using human ES medium. Approximately 5 days later, 20–30 colony-like cell aggregates containing 40–50 cells each appeared under bright-field microscopy (Fig. 2A). Over the next week, the number and size of colonies increased gradually. However, the morphology of these colonies was slightly different from concurrently cultured ES cell morphology in that colonies derived from hGMDCs were not as compact and did not have a high nucleus to cytoplasm ratio (Fig. 2B and C). About day 20 posttransduction, several of these colonies began to exhibit more typical ES-cell like features. Colonies became more compact and flatter. Cells within the colonies exhibited morphology more similar to that of human ES cells, with a high nucleus-to-cytoplasm ratio and prominent nucleoli (Fig. 2D and E). At day 25, colonies most resembling ES cell colonies were lifted from the plate and mechanically dissociated into small clumps on top of new feeder layers. These colonies were indistinguishable from ES cells of the H1 line (Fig. 2F) (Thomson et al., 1998). Of the original 5 × 10⁴ mesenchymal cells transduced per experiment, we routinely observed a yield of 5–10 human ES-like colonies (efficiency: about 0.01–0.1% of input cells) (Table 1). All ES cell-like iPS colonies were successfully expanded over a time period of 5 months. In this study, two hGMDC-iPS clones (clone1 and clone2) with minimal differentiation were selected for detailed analysis.

FIG. 2.

Induction of iPS Cells from hGMDCs. About day 5 after transduction, some small colonies containing about 40–50 cells emerged (A). Image of non-ES-like colony (B). Image of non-ES-like colony with high magnification (C). Image of ES-like colony (D). Image of ES-like colony with high magnification (E). Morphology of hGMDC-iPS colonies at passage 2 (F). Scale bars: 50 μm.

Table 1.

Summary of hGMDC-iPS Cell Induction Experiments

Exp. ID	No. of initial cell	No. of non-ES-like colony	No. of ES-like colony	Efficiency (%)
1	50,000	97	5	0.010
2	50,000	89	10	0.020
3	50,000	90	8	0.016

Different factor combinations on hGMDC-iPS induction

In addition to three-factor reprogramming, we also tried different two-factor combinations and single factor transgene delivery to reprogram hGMDCs. Our data showed three factors are both sufficient and necessary to generate ES-like colonies, whereas combination of OCT4 and SOX2, OCT4 and NANOG, or OCT4 alone could only generate non ES-like cell aggregates, none of which can be propagated to ES cell morphology or pluripotency. Combination of NANOG and SOX2, SOX2 alone, and NANOG alone proved incapable of even generating any cell aggregates following transduction (Fig. 3 and Table 2).

FIG. 3.

Different factor combinations on hGMDC-iPS induction. Three factors could generated ES-like colonies (A), whereas combination of OCT4 and SOX2 (B), OCT4 and NANOG (C), or OCT4 alone (D) could only generate non ES-like cell aggregates. Combination of NANOG and SOX2 (E), SOX2 alone (F), and NANOG alone (G) proved incapable of even generating any cell aggregates following transduction. Scale bars: 50 μm.

Table 2.

Summary of Factor Combinations on hGMDC-iPS Cell Induction

Factor combination	No. of initial cell	No. of non ES-like colony	No. of ES-like colony
ONS	50,000	93	5
OS	50,000	82	0
ON	50,000	75	0
NS	50,000	0	0
O	50,000	79	0
N	50,000	0	0
S	50,000	0	0

O, S, and N represent OCT4, SOX2, and NANOG, respectively.

hGMDC-iPS cells express human ES cell markers

To confirm that colonies exhibiting ES-like morphology expressed proteins associated with pluripotent cells, iPS colonies were stained for a number of surface and intracellular markers. Immunohistochemistry showed that colonies were positive for OCT4, SOX2, NANOG, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and AP. By contrast, colonies were negative for SSEA-1 (Fig. 4A). Q-RT-PCR analysis showed that expression of the pluripotency markers OCT4, SOX2, NANOG, FGF4, REX1, GDF3, and DPPA4 in the iPS cell line were at levels significantly higher than the parental hGMDC population and equivalent to those in H1 hES cell line (Fig. 4B). Genomic integration of the OCT4, SOX2, and NANOG transgenes was confirmed by PCR analysis (Fig. 4C). We examined the expression of total and endogenous OCT4, SOX2, and NANOG by Q-RT-PCR, and showed that the viral OCT4, SOX2, and NANOG transgenes were efficiently silenced in all iPS cell clones examined (Fig. 4D).

FIG. 4.

Human ES cell marker expressions in hGMDC-iPS cells. Staining of hGMDC-iPS colonies were positive for AP, OCT4, SOX2, NANOG, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 expression. By contrast, colonies were negative for SSEA-1 (A). Expression levels of pluripotency marker genes OCT4, SOX2, NANOG, FGF4, REX1, GDF3, and DPPA4 in hGMDCs, human ES cells and hGMDC-iPS cells relative to GAPDH assessed by Q-RT-PCR (B). Integration of OCT4, SOX2, and NANOG transgenes in hGMDCs-iPS cells was confirmed by PCR using transgene-specific primers, with H1 human ES cells as negative controls. GAPDH was used as a loading control (C). Expression levels of total and endogenous OCT4, SOX2, and NANOG (relative to GAPDH) were assessed by Q-RT-PCR (D). Human ES cells and hGMDCs were used as positive and negative controls. “Endogenous” indicates that primers were included in the 3′ untranslated region measure expression of the endogenous gene only, whereas “total” indicates that primers in coding regions measure expression of both the endogenous gene and the transgene if present. Scale bars: 50 μm.

DNA microarray analysis

To understand compare gene profiles of hGMDC-iPS cells with human ES cells, gene microarry analysis was carried out. Hierarchical clustering analysis clearly indicated that hGMDC-iPS cells were significantly similar to human ES cells, and distinct from the hGMDCs, from which they were derived (Fig. 5A). Scatter plots of the global gene expression profiles obtained from cDNA microarrays further demonstrated that hGMDC-iPS cells exhibit a distribution pattern of gene expression comparable to ES cells, hence completely different from hGMDCs (Fig. 5B). The microarray data are available from GEO (Gene Expression Omnibus) under accession number GSE18180.

FIG. 5.

The gene expression profile and methylation analysis of OCT4 and NANOG promoter regions of hGMDC-iPS cells. Hierarchical cluster analysis of the microarray data from human ES cells, hGMDCs, and hGMDC-iPS cells (A). Scatter plots comparing global gene expression profiles between human ES cells, hGMDCs and hGMDC-iPS cells (B). Bisulfite genomic sequencing of the promoter regions of OCT4 and NANOG. Open and closed circles indicate unmethylated and methylated CpGs (C).

Promoters of ES cell-specific genes are active in hGMDC-iPS cells

To further compare the three factor-induced iPS cells with human ES cells, we analyzed methylation states of CpG dinucleotides in the OCT4 and NANOG promoters. In adult somatic cells of the body, OCT4 and Nanog promoters are typically silenced. Bisulfite genomic sequencing analysis showed that OCT4 and NANOG promoter regions were demethylated in harvested iPS cells relative to parental hGMDCs and were similar to epigenetic profiles of OCT4 and NANOG promoters in human ES cells (Fig. 5C). These findings indicate that promoters of transcription factors associated with pluripotency were unsilenced in derived iPS cells.

In vitro differentiation of hGMDC-iPS cells

To assess pluripotency of hGMDC-iPS cells in vitro, we generated EBs from the iPS cells using a suspension culture. iPS cells were observed to form EBs in suspension in an identical fashion to human ES cells after 8 days (Fig. 6A). EBs were transferred to gelatin-coated plates and cultured for an additional 8 days. Attached cells were observed to have a number of different morphologies. Immunofluorescence staining showed that the detected cells were positive for GFAP (ectoderm), β III-TUBULIN (ectoderm), DESMIN (mesoderm), α-SMA (mesoderm), AFP (endoderm), and PDX-1 (endoderm) (Fig. 6B). RT-PCR confirmed that these differentiated cells expressed AFP (endoderm), PDX1 (endoderm), GATA4 (mesoderm), BRACHYURY (mesoderm), VIMENTIN (mesoderm), MAP2 (ectoderm), and PAX6 (ectoderm). Expression of OCT4 and NANOG was markedly decreased (Fig. 6C).

FIG. 6.

In vitro and in vivo differentiation of hGMDC-iPS cells. hGMDC-iPS cells form embryoid bodies in suspension culture (A). Immunofluorescence staining shows differentiation of hGMDC-iPS cells into cells expressing markers of the three germ layers (B). RT-PCR analysis of various differentiation markers for the three germ layers (C). Hematoxylin and eosin staining shows a teratoma from hGMDC-iPS cells containing multiple tissues (D). Scale bars: 50 μm.

Teratoma formation from hGMDC-iPS cells

To test pluripotency in vivo, we transplanted human iPS cells subcutaneously into SCID mice. Six weeks after injection, palpable bulges under the skin were observed. Histological examination showed that the tumor contained derivatives from three germ layers including neural epithelium, bone, cartilage, muscle, and glandular epithelium (Fig. 6D).

Karyotyping and fingerprinting analysis of hGMDC-iPS cells

To assess genomic integrity, karyotyping was conducted on the iPS cell population. Cytogenetic analysis showed normal karyotypes (Supplementary Fig. S1A), and DNA fingerprinting analysis verified that these cells were indeed derived from the parental hGMDCs and not a result of contamination from existing human ES cell lines (Supplementary Fig. S1B).

Optimization of lentiviral transduction for hGMDCs

Because induction of iPS cells requires use of lentivirus with high transduction efficiencies, a GFP-expressing lentivirus was added in all transduction experiments to monitor infection efficiency. Over 90% of the hGMDCs transduced were found to express GFP, OCT4, SOX2, and NANOG at high levels (Supplementary Fig. S2).

Discussion

In this study, we identified fetal hGMDCs to be negative for the expression of hematopoietic markers such as CD34, CD45, CD38, and HLA- DR, but positive for CD44, CD90, and CD71, which are generally considered for markers of mesenchymal stem cells, and mesenchymal marker VIMENTIN (Chagraoui et al., 2003; Charbord et al., 2002). In addition, these cells also expressed CD117, a marker regarded as one of the key criterion for the diagnosis of gastrointestinal stromal tumors (Yantiss et al., 2000). Compared to human ES cells, hGMDCs did not express OCT4, SOX2, and NANOG; they did express KLF4 and c-MYC at levels comparable to ES cells. We therefore attempted to reprogram hGMDCs only by overexpression of OCT4, SOX2, and NANOG without the need for the oncogenes c-MYC or KLF4. Furthermore, we show here, for the first time, to induce pluripotency in human fetal gut mesentery-derived cells (hGMDCs).

Murine iPS cells were first obtained by applying a drug selection scheme for clones that express endogenous ES cell-specific genes (Okita et al., 2007; Wernig et al., 2007). However, it was later shown that such a genetic selection approach is unnecessary for obtaining iPS cells closely resembling ES cells (Meissner et al., 2007). Several groups have subsequently reported that human iPS cells can be obtained by selection of ES-like colonies based on colony morphology alone (Takahashi et al., 2007; Yu et al., 2007). During the course of reprogramming experiments conducted in this study, we observed many non-ES-like colonies following transduction. Although the proportion of ES-like colonies was relatively low out of the total number of colonies, we found that colonies that most resembled ES cells with compact morphology, high nucleus-to-cytoplasm ratios, and prominent nucleoli could be propagated to pluripotent cell lines successfully without exceptions.

After establishing that three factors were sufficient for the successful induction of hGMDCs to a pluripotent state, we also tested whether a two-factor combination or a single factor could induce pluripotency. Our data showed three factors are both sufficient and necessary to generate ES-like colonies. The combination of OCT4 and SOX2 without NANOG could only generate non-ES-like cell aggregates, which were negative for TRA-1-60, TRA-1-81, SSEA-3, and SSEA-4 (Supplementary Fig. S3). None of these colonies can be propagated to ES cell morphology or pluripotency. NANOG, a member of the core transcriptional network in ES cells (Boyer et al., 2005; Loh et al., 2006), is a homeodomain-containing transcription factor. Its expression, similar to OCT4, decreases rapidly as ES cells differentiate(Chambers et al., 2003; Mitsui et al., 2003). Forced expression of NANOG is sufficient to drive LIF/STAT3-independent self-renewal of undifferentiated ES cells (Chambers et al., 2003). NANOG overexpression also enables feeder-independent growth of human ES cells and improves their cloning efficiency (Darr et al., 2006). Curiously, NANOG also greatly improves the efficiency of nuclear reprogramming by ES cell fusion (Silva et al., 2006). However, NANOG is not one of the transcription factors employed to reprogram mouse fibroblasts (Maherali et al., 2007; Okita et al., 2007; Takahashi and Yamanaka, 2006; Wernig et al., 2007). Moreover, NANOG was reported not to influence the efficiency of reprogramming mouse and human fibroblasts (Lowry et al., 2008; Takahashi and Yamanaka, 2006). In contrast, Thomson et al. reported that NANOG showed a beneficial effect in clone recovery from human somatic cells (Yu et al., 2007). In our three-factor system, NANOG is indispensable for reprogramming hGMDCs to pluripotency. Therefore, three-factor hGMDC-iPS cells may provide a useful system for studying the role of NANOG in reprogramming as well as regulating pluripotency.

The continuity and shutdown of the transgene expression are also essential for faithful reprogramming. Transgene expression must be maintained during the first 10 to 14 days (Brambrink et al., 2008; Stadtfeld et al., 2008b). In this regard, integrating viruses including retroviruses and lentiviruses have advantage over plasmids and adenoviruses. Here we detected genomic integration of lentiviruses-delivered OCT4, SOX2, and NANOG transgenes in hGMDC-iPS cells examined. However, faithful reprogramming requires that exogenous transgene expression should be quenched and then replaced by the endogenous genes after this initial stage (Yamanaka, 2009). The failure to silence exogenous transgene expression could renders so-called partially reprogrammed cells, which possess ES cell morphology and express some ES cell markers, but have a limited ability to differentiate (Mikkelsen et al., 2008; Silva et al., 2008). In this study, the transgene expressions of OCT4, SOX2, and NANOG were efficiently silenced in hGMDC-iPS cells examined, indicating that the maintenance of three-factor hGMDC-iPS cells is independent of continued transgene expression.

The reprogramming efficiency in our study was similar to that in the case of four-factor reprogramming human somatic cells reported by others (>0.01% of input cells) (Takahashi et al., 2007; Yu et al., 2007). However, we have established hGMDC-iPS cells using neither KLF4 nor c-MYC. Because gut mesentery tissues can be available from patient biopsy, this study will improve the feasibility to use patients' iPS cells for treating human diseases in the future.

Footnotes

Acknowledgments

The authors thank Yinan Liu and Xiaoyan Zhang for flow cytometry analysis, Qihua He for confocal analysis, and Junhua Zou for G-banding chromosome analysis. This work was supported by the Chinese National 973 Project (2006CB943603) and (2006CB503905), the Chinese National 863 Project (2006AA02A114), and the Natural Science foundation of the Beijing Ministry of Science and Technology (D0750701350704).

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

This work was performed in Peking University Stem Cell Research Center.

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