Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1

Nature volume 474, pages225–229(2011)Cite this article

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Abstract

Induced pluripotent stem cells (iPSCs) are generated from somatic cells by the transgenic expression of three transcription factors collectively called OSK: Oct3/4 (also called Pou5f1), Sox2 and Klf41. However, the conversion to iPSCs is inefficient. The proto-oncogene Myc enhances the efficiency of iPSC generation by OSK but it also increases the tumorigenicity of the resulting iPSCs2. Here we show that the Gli-like transcription factor Glis1 (Glis family zinc finger 1) markedly enhances the generation of iPSCs from both mouse and human fibroblasts when it is expressed together with OSK. Mouse iPSCs generated using this combination of transcription factors can form germline-competent chimaeras. Glis1 is enriched in unfertilized oocytes and in embryos at the one-cell stage. DNA microarray analyses show that Glis1 promotes multiple pro-reprogramming pathways, including Myc, Nanog, Lin28, Wnt, Essrb and the mesenchymal–epithelial transition. These results therefore show that Glis1 effectively promotes the direct reprogramming of somatic cells during iPSC generation.

Main

The generation of iPSCs is technically simple and highly reproducible3,4 but only a small proportion of cells become iPSCs after introduction of the four transcription factors5. In addition, the generation of iPSCs is slow and requires multiple cell divisions6. Reprogramming towards pluripotency can also be achieved by nuclear transfer to meiotic oocytes7 or mitotic zygotes8: this strategy is technically more demanding but it is efficient, rapid and independent of cell division. These differences may indicate that oocytes and zygotes contain factor(s) that promote reprogramming during the generation of iPSCs.

In this study, we initially evaluated a library of 1,437 human transcription factors for their ability to replace Kruppel-like factor 4 (Klf4) or POU domain, class 5, transcription factor 1 (Pou5f1, also known as Oct3/4) during iPSC generation from mouse skin fibroblasts containing a green fluorescent protein (GFP) reporter driven by the nanog homeobox (Nanog) promoter and enhancers9 (Supplementary Table 1). We found that 18 factors could replace Klf4 reproducibly, although with much lower efficiencies of iPSC generation (Supplementary Table 2); we failed to identify any factors that replaced Oct3/4.

Among these 18 factors, we found that GLIS1, a GLI transcription factor10, markedly increased the number of GFP-positive colonies when it was co-introduced with the ‘OSK’ transcription factors Oct3/4, SRY-box 2 (Sox2) and Klf4 into adult mouse skin fibroblasts (Fig. 1a). The effect of GLIS1 was comparable to that of MYC, as judged by the number of GFP-positive colonies (Fig. 1b). We also observed a synergistic increase in the number of GFP-positive colonies when both GLIS1 and MYC were co-introduced with OSK. Notably, GLIS1 specifically promoted the generation of GFP-positive colonies, but not GFP-negative colonies, which represent either partially reprogrammed cells or transformed cells (Fig. 1c). In contrast, MYC increased the number of GFP-negative colonies more than the number of GFP-positive ones. This undesired effect of MYC was counteracted when GLIS1 was co-expressed.

Figure 1: Promotion of mouse iPSC generation by GLIS1.
figure1

a, Number of Nanog–GFP-positive colonies from mouse skin fibroblasts in a 100-mm dish, 28 d after infection. Three days after infection, fibroblasts were re-seeded on feeder cells. Exp, experiment. b, Number of Nanog–GFP-positive colonies from mouse skin fibroblasts in a 6-well plate, 22 d after infection. c, Proportion of Nanog–GFP-positive colonies to total number of colonies (derived directly from experiments in b). Error bars, s.d.; n = 3; **, P < 0.01. d, Upper panel: chimaeric mouse derived from iPSCs obtained by transfection of MEFs with OSK + GLIS1. Lower panel: coat colour of offspring, showing germline transmission.

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Mouse iPSCs generated with OSK and GLIS1 showed morphologies similar to embryonic stem (ES) cells (Supplementary Fig. 1a). Pluripotency markers such as Nanog were expressed at comparable levels to those in ES cells (Supplementary Fig. 1b) and the iPSCs formed teratomas in nude mice (Supplementary Fig. 1c). Furthermore, they produced germline-competent chimaeras (Fig. 1d and Supplementary Table 3).

In human adult fibroblasts, GLIS1 showed a similar effect: it promoted the generation of ES-cell-like colonies to a comparable degree to MYC when it was co-introduced with OSK (Fig. 2a). Notably, GLIS1 specifically promoted the generation of ES-cell-like colonies with a flat, round shape and a distinct edge, but did not promote the generation of non-ES-cell-like colonies, which were granular with an irregular edge (Fig. 2b and Supplementary Fig. 2). In contrast, MYC increased the number of non-ES-cell-like colonies more than the number of ES-cell-like ones (Fig. 2b). The iPSCs generated with OSK and GLIS1 were similar to ES cells in morphology (Supplementary Fig. 3a) and in their expression of undifferentiated-ES-cell marker genes, such as OCT3/4, SOX2, NANOG and ZFP42 (zinc finger protein 42 homolog (mouse), also known as REX1) (Supplementary Fig. 3b). DNA microarray analyses showed that human iPSCs established with OSK and GLIS1 had similar global gene expression to cells generated with OSK and MYC (OSKM) (Fig. 2c). The promoter region of the OCT3/4 gene showed a hypomethylation pattern (Supplementary Fig. 3c) and the iPSCs differentiated into various cells of the three germ layers in the embryoid body (Supplementary Fig. 3d) and also into teratomas (Fig. 2d). These results demonstrate that GLIS1 strongly and specifically promotes the generation of both mouse and human iPSCs by OSK.

Figure 2: Promotion of human iPSC generation by GLIS1.
figure2

a, Number of ES-cell-like colonies from human dermal fibroblasts 30 d after infection. b, ES-cell-like colonies as a proportion of total colonies. **, P < 0.01 compared to cells expressing OSK alone. Error bars, s.d.; n = 3. c, Scatter plots comparing global gene expression patterns between iPSCs generated by OSK + GLIS1 and adult dermal fibroblasts (AHDF) (left panel), and between iPSCs from OSK + GLIS1 and iPSCs from OSKM (right panel). The green diagonal lines indicate twofold changes between the two samples. The correlation coefficient (R2) is also shown. d, iPSCs generated by OSK + GLIS1 were subcutaneously transplanted into nude mice and teratomas were analysed histologically with haematoxylin and eosin staining.

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We next studied the expression pattern of Glis1 in mouse cells. Analyses of expressed sequence tag (EST) databases predicted that Glis1 expression would be enriched in zygotes, especially in the fertilized ovum (http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Mm.331757 as of 7 December 2010). In addition, the gene expression data from reverse transcription PCR (RT–PCR), provided by the mouse genome database MGI, showed that there was moderate expression of Glis1 in metaphase II oocytes and weak expression in two-cell embryos, but that expression was either absent or at trace levels in embryos at the four-cell to embryonic-day-4.5 stages (http://www.informatics.jax.org/searches/expression.cgi?32989 as of 7 December 2010, also reported in ref. 11 in their Supplementary Table 1). To confirm the specific expression of Glis1 in oocytes and one-cell embryos, we isolated total RNA from oocytes, early embryos and several adult mouse tissues. Real-time PCR detected the highest expression of Glis1 in one-cell embryos and unfertilized eggs. A modest level of expression was detected in two-cell embryos and placentas and weak expression was detected in several adult tissues (Fig. 3a). These data confirmed that Glis1 RNA is enriched in unfertilized eggs and one-cell embryos.

Figure 3: Characterization of Glis1: expression and roles during iPSC generation.
figure3

a, Expression patterns of Glis1 in different mouse tissues. Data are normalized to glyceraldehyde-3-phosphate dehydrogenase expression; Glis1 expression in the kidney is set at a relative level of 1. Error bars, s.d.; n = 4. b, Ninety genes were found to be upregulated more than 20-fold in OSK + Glis1 cells compared to OSK + mock cells (upper panel). These included Foxa2, multiple Wnt-family genes and Esrrb. We also focused on 361 probes for which expression was more than 100-fold higher in ES cells than in fibroblasts. Among these, 32 probes showed an expression level that was more than threefold higher in OSK + Glis1 cells than in OSK + mock cells (lower panel). These included Esrrb, Oct3/4, Mycn, Lin28a and Nanog. c, Expression levels of the Myc-family genes (C, Myc; N, Mycn; L, Mycl1) in OSK + Mock and OSK + Glis1 cells. The green diagonal lines indicate twofold changes between the two cell types.

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We next examined whether endogenous Glis1 has a role during iPSC generation by OSK. We found that Glis1 is expressed at a low level in mouse fibroblasts before and after the introduction of OSK (Supplementary Fig. 4a). We constructed retroviral vectors to express several Glis1 small hairpin RNAs (shRNAs), as well as scrambled controls, and tested the knockdown efficiency of each shRNA retrovirus in skin fibroblasts. We found that shRNA2 and shRNA6 were effective (Supplementary Fig. 4b). We then introduced each of these shRNAs, together with OSK, into mouse embryonic fibroblasts (MEFs) containing the Nanog–GFP reporter. We found that both shRNA2 and shRNA6 significantly decreased the number of GFP-positive colonies (Supplementary Fig. 4c), in contrast to the scrambled control shRNA. These results show that endogenous Glis1 may have a supportive role during the generation of mouse iPSCs by OSK.

Finally, we tried to elucidate how Glis1 enhances iPSC generation by OSK. We previously reported that suppression of the p53 pathway markedly enhanced iPSC generation from both mouse and human cells12. We therefore hypothesized that Glis1 may enhance direct reprogramming by inhibiting p53. If this is the case, Glis1 should not be able to promote iPSC generation in cells with a p53-null background. To test this hypothesis, we introduced OSK plus mock (control) or OSK plus Glis1 into either wild-type or p53-knockout MEFs, both containing the Nanog–GFP reporter. Five days after transduction, we measured the proportion of Nanog–GFP-positive cells by flow cytometry. We found that even in p53-knockout MEFs, in which the generation of Nanog–GFP-positive cells by OSK was increased about 10-fold (to about 2%), the addition of Glis1 further increased the proportion of GFP-positive cells up to about 17% (Supplementary Fig. 5). These data indicate that Glis1 promotes iPSC generation irrespective of p53.

We then used the very high reprogramming efficiency in cells with the p53-null background to elucidate the function of Glis1. We sorted and collected Nanog–GFP-positive cells 5 days after the transduction of OSK plus mock or OSK plus Glis1 into the p53-knockout MEFs. We then conducted microarray analysis to compare the gene expression levels of these cell populations undergoing reprogramming (Fig. 3b, c and Supplementary Table 4). We found that Glis1 markedly increased the expression of several genes whose products have been shown to enhance iPSC generation. These included oestrogen-related receptor, beta (Esrrb)13, several Wnt ligands (Wnt3, Wnt6, Wnt8a and Wnt10a)14, lin-28 homologue A (Lin28a)15, Nanog (ref. 16), Mycn and Mycl1(ref. 17). In contrast, the expression of Myc was suppressed by Glis1 (Fig. 3c). We have previously shown that Mycn and Mycl1 predominantly increase the numbers of ES-cell-like colonies, whereas Myc increases both ES-cell-like and non-ES-cell-like colonies17. Therefore, the altered balance between Mycn/Mycl1 and Myc should contribute, at least in part, to the specific promotion of iPSC generation by Glis1. Glis1 also markedly enhanced the expression of forkhead box A2 (Foxa2), a transcription factor that antagonizes the epithelial-to-mesenchymal transition. Because this transition is a prerequisite for iPSC generation18,19, the activation of Foxa2 should also have a role in the promotion of iPSC generation by Glis1. We confirmed the effect of Glis1 on Nanog, Mycn, Myc, neurogranin and tetraspanin 18 in a p53 wild-type background by quantitative PCR (Supplementary Fig. 6). Taken together, these data demonstrate that Glis1 promotes iPSC generation by activating multiple pro-reprogramming pathways.

We next performed chromatin immunoprecipitation assays to identify the direct transcriptional targets of Glis1. Cell lysates were isolated from p53-knockout MEFs transduced with OSK plus mock or OSK plus Glis1. Candidate target genes identified from the microarray analyses were amplified by PCR (Fig. 4a). We found that significantly higher amounts of Mycn, Mycl1 and Myc were precipitated from the cells transduced with OSK plus Glis1 than from those transduced with OSK plus mock. In contrast, no such specific precipitation was observed with Esrrb, Lin28a, Foxa2 or Nanog. These results indicate that the three Myc genes are direct targets of Glis1, whereas Esrrb, Lin28a, Foxa2 and Nanog may be indirect targets.

Figure 4: Characterization of Glis1: target genes and protein–protein interactions.
figure4

a, Chromatin immunoprecipitation and quantitative PCR analysis were conducted on the basis of microarray data, using a Glis1-specific antibody and PCR primers specific for Mycn, Mycl1, Myc, Nanog, Esrrb, Lin28a and Foxa2. GATA binding protein 4 (Gata4) and NK2 transcription factor related, locus 5 (Nkx2-5) were used as negative controls. IP, immunoprecipitate. Error bars, s.d.; n = 2; *, P < 0.05. b, Constructs encoding Flag-tagged Glis1 or Klf4 and untagged Oct3/4 (left panel), Flag-tagged Glis1 or Klf4 and untagged Sox2 (middle panel) or Flag-tagged Klf4 and Myc-tagged Glis1 (right panel) were transfected into HEK293T cells alone or in combination. Flag-tagged Venus was transfected as a negative control. The cell lysates were immunoprecipitated (IP) with an anti-Flag antibody, followed by an immunoblot analysis (IB). The expression levels in whole-cell lysates were determined by IB (bottom panels).

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We next examined whether Glis1 physically associates with the OSK proteins. Using Flag-tagged Glis1, we saw that Oct3/4 and Sox2 co-purified with Glis1 (Fig. 4b), whereas co-purification was not observed with a Flag-tagged Venus protein. In addition, we observed the co-purification of Flag–Klf4 with Myc-tagged Glis1 (Fig. 4b). The zinc-finger domain of Glis1 and its N-terminal region were required for the interaction with Klf4 (Supplementary Fig. 7). The interaction between Klf4 and Glis1 was further confirmed with an in vitro protein fragment complementation assay (Supplementary Fig. 8). These data indicate that Glis1 can associate with OSK by a protein–protein interaction and thereby might promote the activation of OSK target genes.

In contrast to oocytes and one-cell-stage embryos, we found that the expression of Glis1 was very low in ES cells. We therefore examined the effects of forced expression of Glis1 in mouse ES cells20 and found that this suppressed their proliferation (Supplementary Fig. 9). This effect may have contributed to the smaller number of partially reprogrammed cells observed with OSK plus Glis1, because such cells would fail to silence retroviruses and would still express Glis1 transgenes, which would suppress proliferation.

This study shows that the transcription factor Glis1, which is highly enriched in unfertilized eggs and one-cell-stage embryos, promotes iPSC generation effectively and specifically by activating multiple pro-reprogramming pathways. Glis1 might thus be a link between reprogramming during iPSC generation and reprogramming after nuclear transfer. Furthermore, iPSCs generated by OSK and Glis1 did not cause a marked increase in mortality of chimaeric mice, although this did occur with iPSCs generated by Oct3/4, Sox2, Glis1 and Myc (Supplementary Fig. 10) and with iPSCs generated by OSK and Myc, as reported previously17. The identification of Glis1 might therefore be beneficial for future applications of iPSC technology.

Methods Summary

To screen transcription factors for their effects on iPSC generation, cDNAs were used from the human proteome expression resource (HuPEX) library21. Gateway entry clones of 1,437 human transcription factors were transferred to pMXs-GW retroviral expression vectors using the Gateway LR reaction. MEFs were isolated from 13.5 days post coitum (d.p.c.) embryos and adult skin fibroblasts were isolated from 20-week-old mice. The generation of mouse iPSCs with retroviruses was performed as described previously2,9. Human iPSCs were also generated as described previously22. The shRNA-mediated knockdown was performed as described in ref. 12. Retroviruses (pMXs) were generated with Plat-E packaging cells23. ES cells and iPSCs were cultured on SNL feeder cells24. The analyses of iPSCs, such as RT–PCR, alkaline phosphatase staining, DNA microarrays, in vitro differentiation, teratoma formation, bisulphite genomic sequencing and chimaera experiments, were performed as previously described1,9,22. Animal experiments were approved by committees of Kyoto University and the Japan Science and Technology Agency. To examine whether Glis1 is physically associated with the OSK proteins, immunoprecipitation and immunoblotting analyses were performed, as well as an in vitro protein fragment complementation assay25. In addition, a ChIP analysis was performed on Glis1 to identify its target genes. Sequences of primers and shRNAs are listed in Supplementary Tables 5 and 6, respectively. Microarray data are available through GEO with accession number GSE26431.

Online Methods

cDNA library

cDNAs used to screen for novel factors that alter the efficacy of iPSC generation were obtained from the human proteome expression resource (HuPEX) library21. Among the 33,275 cDNAs, we selected those known to be transcription factors or identified by keyword searches of the Human Gene and Protein Database (HGPD, http://www.HGPD.jp/) and Entrez gene (http://www.ncbi.nlm.nih.gov/gene). We used cDNAs that covered more than 80% of the open reading frame reported in RefSeq and had identity with the reported protein sequence of more than 95% at the amino acid level. cDNAs encoding OCT3/4, SOX2, KLF4 or MYC were excluded. This resulted in 1,437 cDNAs (Supplementary Table 1), which were transferred to the pMXs-GW retroviral expression vector using the Gateway LR reaction.

Cell culture

Mouse iPSCs were maintained in ES cell medium (DMEM containing 15% fetal calf serum (FCS), 1× non-essential amino acids (NEAA), 1 mM sodium pyruvate, 5.5 mM 2-mercaptoethanol (2-ME), 50 units ml−1 penicillin and 50 μg ml−1 streptomycin) on feeder layers of mitomycin-C-treated SNL cells stably expressing the puromycin-resistance gene24. As a source of leukaemia-inhibitory factor (LIF), we used the conditioned medium from Plat-E cell cultures that had been transduced with a LIF-expressing vector. Human iPSCs were generated and maintained in primate ES cell medium (ReproCELL), supplemented with 4 ng ml−1 recombinant human basic fibroblast growth factor, 50 units ml−1 penicillin and 50 μg ml−1 streptomycin. MEFs, mouse skin fibroblasts and human fibroblasts were maintained in DMEM containing 10% FCS, 50 units ml−1 penicillin and 50 μg ml−1 streptomycin. Plat-E cells23 were maintained in DMEM containing 10% FCS, 50 units ml−1 penicillin, 50 μg ml−1 streptomycin, 1 μg ml−1 puromycin and 10 μg ml−1 blasticidin S. We used 13.5 d.p.c. embryos for MEF isolation and 20-week-old mice for the isolation of skin fibroblasts.

Mouse iPSC generation

The generation of mouse iPSCs with retroviruses was performed as previously described2,9 with some modifications. Briefly, Plat-E cells were seeded at 2.5 × 106 cells per 100-mm dish. On the next day, pMXs-based retroviral vectors for each gene were independently introduced into Plat-E cells using the FuGENE 6 transfection reagent. After 24 h, the medium was replaced with 10 ml of DMEM containing 10% FCS. Fibroblasts were seeded at 8 × 105 cells per dish, in 100-mm dishes covered with a layer of gelatin or feeder cells. The next day, virus-containing supernatants from the Plat-E cultures were recovered and mixed together (for example, viruses for Oct3/4, Sox2, Klf4 and Glis1). Fibroblasts were incubated in the virus/polybrene-containing supernatants at a final concentration of 4 μg ml−1 for 24 h. Three days after infection, the medium was changed to ES cell medium supplemented with LIF. Fibroblasts on gelatin-coated dishes were then re-seeded onto dishes with feeder cells. The shRNA-mediated knockdown was performed as previously described12.

Generation of human iPSCs

Human iPSCs were generated as previously described22 with some modifications. Briefly, Plat-E cells were plated at 3.6 × 106 cells per 100-mm dish. The next day, pMXs-based retroviral vectors for each gene were independently introduced into the Plat-E cells using the FuGENE 6 transfection reagent. After 24 h, the medium was replaced with new medium. Human fibroblasts expressing the mouse Slc7a1 (solute carrier family 7 (cationic amino acid transporter, y+ system), member 1) gene were seeded at 8 × 105 cells per 100-mm dish. The next day, virus-containing supernatants were recovered and mixed together. Fibroblasts were incubated in the virus/polybrene-containing supernatants at a final concentration of 4 μg ml−1 for 24 h. Six days after transduction, fibroblasts were harvested by trypsinization and replated at 5 × 104 or 5 × 105 cells per 100-mm dish on SNL feeder cells. The next day, the medium was replaced with primate ES cell medium supplemented with 4 ng ml−1 basic fibroblast growth factor.

Characterization of iPSCs

The RT–PCR analyses, alkaline phosphatase staining, in vitro differentiation, teratoma formation, bisulphite genomic sequencing and chimaera experiments were performed as previously described1,9,22. The primers used for RT–PCR are listed in Supplementary Table 5. In the in vitro differentiation assay, differentiated cells were stained positive for α-fetoprotein (endoderm), α-smooth muscle actin (mesoderm) and nestin (ectoderm). Nuclei were stained with Hoechst. For bisulphite genomic sequencing, the white circles indicate unmethylated CpG dinucleotides, whereas the black circles indicate methylated CpG dinucleotides.

DNA microarray

Total RNAs were labelled with Cy3 and hybridized to either a Whole Mouse Genome Microarray or a Whole Human Genome Microarray (Agilent) according to the manufacturer’s protocol. Arrays were scanned using the G2505C Microarray Scanner System (Agilent). The data were analysed using the GeneSpring GX11.0.1 software program (Agilent). The microarray data are available from the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE26431.

Chromatin immunoprecipitation assay

We used the Active Motif ChIP-IT Express kit for the chromatin immunoprecipitation assay. Genomic DNA and nuclear proteins were fixed with formaldehyde. Immunoprecipitation was performed with either anti-Glis1 (Santa Cruz) or purified goat IgG antibody and the elutes were used as templates for quantitative PCR. We selected DNA fragments containing putative Glis1-binding sites for PCR amplification. The primers used for quantitative PCR in the ChIP assay are listed in Supplementary Table 5.

Immunoprecipitation and immunoblotting analyses

Because the expression levels of Glis1 in ES cells and fibroblasts are low, we were not able to elucidate whether there was an association among the endogenous proteins. HEK293T cells were therefore transfected with each cDNA clone in an expression vector and were lysed in CytoBuster (Novagen). Cell lysates were incubated with an anti-Flag M2 Affinity Gel (Sigma) for 2 h and then removed. The gel suspensions were boiled in sample buffer and analysed by SDS–polyacrylamide gel electrophoresis and immunoblotting. The immunoblot analyses were performed using the following antibodies: anti-Flag M2 (Sigma), anti-Myc (Roche), anti-Oct3/4 (Santa Cruz) and anti-Sox2 (MBL).

In vitro protein fragment complementation assay

We prepared split monomeric Kusabira-Green protein (mKG) fragment proteins (Amalgaam) fused to Glis1 and Klf4 using a wheat-germ cell-free protein synthesis system (CellFree Sciences)25. Each protein solution was dispensed into a 384-well plate. After incubation at 25 °C for 8 h or 23 h, the fluorescence was measured using the Typhoon 9200 (GE Healthcare).

Overexpression of genes in ES cells

The mouse ES cell line MG1.19 was maintained in DMEM containing 10% FCS, 1× NEAA, 1 mM sodium pyruvate, 5.5 mM 2-ME, 50 units ml−1 penicillin, 50 μg ml−1 streptomycin and LIF. The vectors pCAG-IP (Mock) or pCAG-Glis1-IP were introduced into MG1.19 cells using Lipofectamine 2000 on day −1. On day zero, 1 × 105 cells were re-seeded on a gelatin-coated 6-well plate. On day 4, the cell number was counted.

Statistical analyses

A one-way repeated-measures ANOVA and a post-hoc Bonferroni test were used for the analyses of the data in Figs 1c and 2b. The unpaired t-test was used for statistical analysis of the data shown in Fig. 4a (between OSK and OSKGlis1). Differences were considered to be statistically significant for P-values <0.05 (*), <0.01 (**) or <0.001 (***).

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The microarray data are available from the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE26431.

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Acknowledgements

We thank T. Yamamoto, Y. Yamada and the members of our laboratory for valuable scientific discussions and administrative support. We thank M. Nakagawa, H. Seki, M. Murakami, A. Okada, M. Narita, M. Inoue, H. Shiga and T. Matsumoto for technical assistance and H. Suemori (Kyoto University) for human ES cells. This work was supported in part by grants from the New Energy and Industrial Technology Development Organization (NEDO), the Leading Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) of the Japanese Society for the Promotion of Science (JSPS), Grants-in-Aid for Scientific Research from JSPS and MEXT, and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO). S.Y. is a member of scientific advisory boards of iPearian Inc. and iPS Academia Japan.

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Affiliations

  1. Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan

    • Momoko Maekawa
    • , Tomonori Nakamura
    • , Ran Shibukawa
    • , Ikumi Kodanaka
    • , Tomoko Ichisaka
    •  & Shinya Yamanaka

  2. Yamanaka iPS Cell Special Project, JST, Kawaguchi 332-0012, Japan

    • Momoko Maekawa
    • , Ran Shibukawa
    • , Ikumi Kodanaka
    •  & Shinya Yamanaka

  3. Japan Biological Informatics Consortium, Tokyo 135-0064, Japan

    • Kei Yamaguchi
    • , Yoshifumi Kawamura
    •  & Hiromi Mochizuki

  4. Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japan

    • Tomonori Nakamura
    • , Tomoko Ichisaka
    •  & Shinya Yamanaka

  5. Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo 135-0064, Japan

    • Naoki Goshima

  6. Gladstone Institute of Cardiovascular Disease, San Francisco, 94158, California, USA

    • Shinya Yamanaka

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Contributions

M.M. conducted most of the experiments in this study. K.Y. analysed the interactions of proteins. T.N. performed the computer analyses of the DNA microarray data, teratoma experiments, overexpression in ES cells and statistical analysis. R.S. generated mouse iPSCs and characterized mouse and human iPSCs. I.K. generated human iPSCs. T.I. performed the chimaera and teratoma experiments and maintained the mouse lines. Y.K. selected cDNA clones from HuPEX with bioinformatics. H.M. produced the retroviral expression clones. N.G. and S.Y. supervised the project. M.M. and S.Y. wrote the manuscript.

Corresponding authors

Correspondence to Naoki Goshima or Shinya Yamanaka.

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The authors declare no competing financial interests.

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Maekawa, M., Yamaguchi, K., Nakamura, T. et al. Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. Nature 474, 225–229 (2011) doi:10.1038/nature10106

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