Pluripotent Stem Cells Induced from Mouse Somatic Cells by Small-Molecule Compounds

ReportsPluripotent stem cells, such as embryonic stem cells (ESCs), can self-renew and differentiate into all somatic cell types. Somatic cells can be reprogrammed to become pluripotent via nuclear transfer into oocytes or through the ectopic expression of defined factors (1–4). However, exogenous pluripotency-associated factors, especially Oct4, are indis-pensable for establishing pluripotency (5–7), and previous reprogram-ming strategies have raised concerns regarding the clinical applications (8, 9). Small molecules have advantages because they can be cell per-meable, non-immunogenic, more cost-effective, and can be more easily synthesized, preserved, and standardized. Moreover, their effects on inhibiting and activating the function of specific proteins are often re-versible and can be finely tuned by varying the concentrations. Here, we identified small-molecule combinations that were able to drive the re-programming of mouse somatic cells toward pluripotent cells.To identify small molecules that facilitate cell reprogramming, we searched for small molecules that enable reprogramming in the absence of Oct4 using Oct4 promoter-driven GFP expression (OG) mouse embryonic fibroblasts (MEFs), with viral expression of Sox2, Klf4, and c-Myc. After screening up to 10,000 small molecules (table S1A), we identified For-skolin (FSK), 2-Methyl-5-hydroxytryptamine (2-Me-5HT), and D4476 (table S1B) as chemical “substitutes” for Oct4 (Fig. 1, A and B, and figs. S1 and S2). Previously, we had developed a small-molecule combination “VC6T” [VPA, CHIR99021 (CHIR), 616452, Tranylcypromine], that enables reprogramming with a single gene, Oct4(6). We next treated OG-MEFs with VC6T plus the chemical substitutes of Oct4in the ab-sence of transgenes. We found VC6T plus FSK (VC6TF) induced some GFP-positive clusters expressing E-cadherin, a mesen-chyme-to-epithelium transition marker, reminiscent of early reprogram-ming by transcription factors (10, 11)(Fig. 1C and fig. S3). However, theexpression of Oct4 and Nanog was notdetectable and their promoters remainedhypermethylated, suggesting a repressedepigenetic state (fig. S3).To identify small molecules that fa-cilitate late reprogramming, we used adoxycycline (DOX)–inducible Oct4expression screening system, addingDOX only in the first 4-8 days (6).Small-molecule hits, including severalcAMP agonists (FSK, Prostaglandin E2,and Rolipram) and epigenetic modula-tors (3-deazaneplanocin A (DZNep),5-Azacytidine, Sodium butyrate, andRG108), were identified in this screen(fig. S4 and table S1B).To achieve complete chemical re-programming without theOct4-inducible system, these smallmolecules were further tested in thechemical reprogramming of OG-MEFswithout transgenes. When DZNep wasadded 16 days after treatment withVC6TF (VC6TFZ), GFP-positive cellswere obtained up to 65-fold more fre-quently than those treated with VC6TF,forming compact, epithelioid,GFP-positive colonies without clear-cutedges (Fig. 1, D and E, and fig. S5). Inthese cells, the expression levels of mostpluripotency marker genes were ele-vated but still lower than in ESCs, sug-gesting an incomplete reprogramming state (fig. S6). After switching to 2i-medium with dual inhibition (2i) of glycogen synthase kinase-3 and mitogen-activated protein kinase sig-naling after day 28 post-treatment, certain GFP-positive colonies devel-oped an ESC-like morphology (domed, phase-bright, homogeneous with clear-cut edges) (Fig. 1F) (12, 13). These colonies could be further cul-tured for more than 30 passages, maintaining an ESC-like morphology (Fig. 1, G and H). We refer to these 2i-competent, ESC-like, and GFP-positive cells as chemically induced pluripotent stem cells (CiPSCs).Next, we optimized the dosages and treatment duration of the small molecules, and were able to generate 1-20 CiPSC colonies from 50,000 initially plated MEFs (fig. S7). After an additional screen, we identified some small-molecule boosters of chemical reprogramming, among which, a synthetic retinoic acid receptor ligand, TTNPB, enhanced chemical reprogramming efficiency up to 40-fold, to a frequency com-parable to transcription factor-induced reprogramming (up to 0.2%) (fig.S8 and table S1B). Furthermore, using the small-molecule combinationVC6TFZ, we obtained CiPSC lines from mouse neonatal fibroblasts (MNFs), mouse adult fibroblasts (MAFs), and adipose-derived stem cells (ADSCs) with OG cassettes by an efficiency approximately 10 times lower than that obtained from MEFs (fig. S9 and table S3). Moreover, we induced CiPSCs from wild-type MEFs without OG cassettes or any other genetic modifications by a comparable efficiency to that achieved from MEFs with OG cassettes (fig. S9). The CiPSCs were also confirmed to beviral-vector free by genomic PCR and Southern blot analysis (fig. S10).The established CiPSC lines were then further characterized. They grew with a doubling time (14.1-15.1 hours) similar to that of ESCs (14.7 hours), maintained alkaline phosphatase activity, and expressed pluripo-tency markers, as detected by immunofluorescence and RT-PCR (Fig. 2,Pluripotent Stem Cells Induced from Mouse Somatic Cells bySmall-Molecule CompoundsPingping Hou,1* Yanqin Li,1* Xu Zhang,1,2* Chun Liu,1,2* Jingyang Guan,1* Honggang Li,1* Ting Zhao,1† Junqing Ye,1,2† Weifeng Yang,3†Kang Liu,1† Jian Ge,1,2† Jun Xu,1† Qiang Zhang,1,2† Yang Zhao,1‡ Hongkui Deng1,2‡1College of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China. 2School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China. 3Beijing Vitalstar Biotechnology Co., Ltd., Beijing 100012, China.*These authors contributed equally to this work.†These authors contributed equally to this work.‡Corresponding author. E-mail: hongkui_deng@ (H.D.); yangzhao@ (Y.Z.).Pluripotent stem cells can be induced from somatic cells, providing an unlimited cell resource, with potential for studying disease and use in regenerative medicine. However, genetic manipulation and technically challenging strategies such as nuclear transfer used in reprogramming limit their clinical applications. Here, weshow that pluripotent stem cells can be generated from mouse somatic cells at a frequency up to 0.2% using a combination of seven small-molecule compounds. The chemically induced pluripotent stem cells (CiPSCs) resemble embryonic stem cells (ESCs) in terms of their gene expression profiles, epigenetic status, and potential for differentiation and germline transmission. By using small molecules, exogenous “master genes” are dispensable for cell fate reprogramming. This chemical reprogramming strategy has potential use in generating functional desirable cell types for clinical applications. o n J u l y 1 8 , 2 0 1 3 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mA and B, and fig. S11). The gene expression profiles were similar in CiPSCs, ESCs, and OSKM-iPSCs (iPSCs induced by Oct4, Sox2, Klf4 and c-Myc) (Fig. 2C and fig. S12). DNA methylation state and histone modifications at Oct4 and Nanog promoters in CiPSCs were similar to that in ESCs (Fig. 2D and fig. S13). In addition, CiPSCs maintained a normal karyotype and genetic integrity for up to 13 passages (fig. S14 and table S2).To characterize their differentiation potential, we injected CiPSCs into immunodeficient (SCID) mice. The cells were able to differentiate into tissues of all three germ layers (Fig. 3A and fig. S15). When injected into eight-cell embryos or blastocysts, CiPSCs were capable of integra-tion into organs of all three germ layers including gonads and transmis-sion to subsequent generations (Fig. 3, B to E, and fig. S16). Unlike chimeric mice generated from iPSCs induced by transcription factors including c-Myc(14), the chimeric mice generated from CiPSCs were 100% viable and apparently healthy for up to 6 months (Fig. 3F). These observations suggest that the CiPSCs were fully reprogrammed into pluripotency (table S3).We next determined which of these small molecules were critical in inducing CiPSCs. We found four essential small molecules whose indi-vidual withdrawal from the cocktails generated significantly reduced GFP-positive colonies and no CiPSCs (Fig. 4, A to C). These small molecules (C6FZ) include: CHIR (C), a glycogen synthase kinase 3 inhibitor (15); 616452 (6), a transforming growth factor-beta inhibitor (16); FSK (F), a cAMP agonist (fig. S17) (17); and DZNep (Z), an S-adenosylhomocysteine (SAH) hydrolase inhibitor (figs. S18 and S19) (18, 19). Moreover, C6FZ was able to induce CiPSCs from both MEFs and MAFs, albeit by a 10 times lower efficiency than that induced by VC6TFZ (fig. S20 and table S3).To better understand the pluripotency-inducing properties of these small molecules, we profiled the global gene expression during chemical reprogramming and observed the sequential activation of certain key pluripotency genes, which was validated by real-time PCR and immuno-fluorescence (fig. S21). The expression levels of two pluripotency-related genes, Sall4 and Sox2, were most significantly induced in the early phase in response to VC6TF, as was the expression of several extra-embryonic endoderm (XEN) markers Gata4, Gata6, and Sox17 (Fig. 4, D to F, and figs. S22 to S24). The expression of Sall4was enhanced most signifi-cantly as early as 12 hours after small-molecule treatment, suggesting that Sall4 may be involved in the first step toward pluripotency in chemical reprogramming (fig. S22B). We further examined the roles of the en-dogenous expression of these genes in chemical reprogramming, using gene overexpression and knockdown strategies. We found the concomi-tant overexpression of Sall4 and Sox2 was able to activate an Oct4 pro-moter-driven luciferase reporter (fig. S25) and was sufficient to replace C6F in inducing Oct4 expression and generating iPSCs (Fig. 4, G and H, and fig. S26). The endogenous expression of Sall4, but not Sox2, requires the activation of the XEN genes, and vice versa (fig. S27). This suggests a positive feedback network formed by Sall4, Gata4, Gata6, and Sox17, similar to which was previously described in mouse XEN formation (20). Moreover, knockdown of Sall4 or these XEN genes impaired Oct4 acti-vation and the subsequent establishment of pluripotency (fig. S28), in consistent with our previous finding that Gata4 and Gata6 can contribute to inducing pluripotency (21). Taken together, these findings revealed a Sall4-mediated molecular pathway that acts in the early phase of chemical reprogramming (Fig. 4L). This step resembles a Sall4-mediated dedif-ferentiation process in vivo during amphibian limb regeneration (22).We next investigated the role of DZNep, which was added in the late phase of chemical reprogramming. We found that Oct4 expression was enhanced significantly after the addition of DZNep in chemical repro-gramming (Fig. 4D), and DZNep was critical for stimulating the expres-sion of Oct4 but not the other pluripotency genes (Fig. 4I). As an SAH hydrolase inhibitor, DZNep elevates the concentration ratio of SAH to S-adenosylmethionine (SAM) and may thereby repress the SAM-dependent cellular methylation process (fig. S18) (18, 19). Con-sistently, DZNep significantly decreased DNA and H3K9 methylation at the Oct4 promoter, which may account for its role in Oct4 activation (Fig. 4, J and K) (23, 24). As master pluripotency genes, Oct4 and Sox2 may thereby activate other pluripotency-related genes, and fulfill the chemical reprogramming process, along with the activation of Nanog and silencing of Gata6, in the presence of 2i (12, 13, 25, 26) (Fig. 4F and fig. S29). In summary, as a master switch governing pluripotency, Oct4 expression, which is kept repressed in somatic cells by multiple epigenetic modifica-tions, is unlocked in chemical reprogramming by the epigenetic modu-lator DZNep, and stimulated by C6F-induced expression of Sox2and Sall4 (Fig. 4L).Our proof-of-principle study demonstrates that somatic reprogram-ming toward pluripotency can be manipulated using only small-molecule compounds (fig. S30). It reveals that the endogenous pluripotency pro-gram can be established by the modulation of molecular pathways non-specific to pluripotency via small molecules rather than by exogenously provided “master genes.” These findings increase our understanding about the establishment of cell identities and open up the possibility of generating functionally desirable cell types in regenerative medicine by cell fate reprogramming using specific chemicals or drugs, instead of genetic manipulation and difficult-to-manufacture biologics. To date, the complete chemical reprogramming approach remains to be further im-proved to reprogram human somatic cells and ultimately meet the needs of regenerative medicine.References and Notes1. I. Wilmut, A. E. Schnieke, J. McWhir, A. J. Kind, K. H. Campbell,Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–813 (1997). doi:10.1038/385810a0 Medline2. K. Takahashi, S. Yamanaka, Induction of pluripotent stem cells frommouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006). doi:10.1016/j.cell.2006.07.024 Medline3. S. Yamanaka, H. M. Blau, Nuclear reprogramming to a pluripotentstate by three approaches. Nature465, 704–712 (2010).doi:10.1038/nature09229 Medline4. M. Stadtfeld, K. Hochedlinger, Induced pluripotency: History,mechanisms, and applications. Genes Dev.24, 2239–2263 (2010).doi:10.1101/gad.1963910 Medline5. S. Zhu, W. Wei, S. Ding, Chemical strategies for stem cell biology andregenerative medicine. Annu. Rev. Biomed. Eng.13, 73–90 (2011).doi:10.1146/annurev-bioeng-071910-124715 Medline6. Y. Li, Q. Zhang, X. Yin, W. Yang, Y. Du, P. Hou, J. Ge, C. Liu, W.Zhang, X. Zhang, Y. Wu, H. Li, K. Liu, C. Wu, Z. Song, Y. Zhao, Y.Shi, H. Deng, Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules. Cell Res.21, 196–204 (2011).doi:10.1038/cr.2010.142 Medline7. W. Li, E. Tian, Z. X. Chen, G. Sun, P. Ye, S. Yang, D. Lu, J. Xie, T. V.Ho, W. M. Tsark, C. Wang, D. A. Horne, A. D. Riggs, M. L. Yip, Y.Shi, Identification of Oct4-activating compounds that enhance reprogramming efficiency. Proc. Natl. Acad. Sci. U.S.A.109, 20853–20858 (2012). doi:10.1073/pnas.1219181110 Medline8. K. Saha, R. Jaenisch, Technical challenges in using human inducedpluripotent stem cells to model disease. Cell Stem Cell5, 584–595 (2009). doi:10.1016/j.stem.2009.11.009 Medline9. S. M. Wu, K. Hochedlinger, Harnessing the potential of inducedpluripotent stem cells for regenerative medicine. Nat. Cell Biol.13, 497–505 (2011). doi:10.1038/ncb0511-497 Medline10. R. Li, J. Liang, S. Ni, T. Zhou, X. Qing, H. Li, W. He, J. Chen, F. Li,Q. Zhuang, B. Qin, J. Xu, W. Li, J. Yang, Y. Gan, D. Qin, S. Feng, H.Song, D. Yang, B. Zhang, L. Zeng, L. Lai, M. A. Esteban, D. Pei, A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell7, 51–63 (2010). doi:10.1016/j.stem.2010.04.014 Medline11. P. Samavarchi-Tehrani, A. Golipour, L. David, H. K. Sung, T. A.onJuly18,213www.sciencemag.orgDownloadedfromBeyer, A. Datti, K. Woltjen, A. Nagy, J. L. Wrana, Functionalgenomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7, 64–77 (2010). doi:10.1016/j.stem.2010.04.015 Medline 12. J. Silva, O. Barrandon, J. Nichols, J. Kawaguchi, T. W. Theunissen, A. Smith, Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol. 6, e253 (2008). doi:10.1371/journal.pbio.0060253 Medline 13. T. W. Theunissen, A. L. van Oosten, G. Castelo-Branco, J. Hall, A.Smith, J. C. Silva, Nanog overcomes reprogramming barriers andinduces pluripotency in minimal conditions. Curr. Biol. 21, 65–71 (2011). doi:10.1016/j.cub.2010.11.074 Medline14. M. Nakagawa, N. Takizawa, M. Narita, T. Ichisaka, S. Yamanaka,Promotion of direct reprogramming by transformation-deficient Myc. Proc. Natl. Acad. Sci. U.S.A. 107, 14152–14157 (2010). doi:10.1073/pnas.1009374107 Medline 15. Q. L. Ying, J. Wray, J. Nichols, L. Batlle-Morera, B. Doble, J. Woodgett, P. Cohen, A. Smith, The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008). doi:10.1038/nature06968 Medline 16. N. Maherali, K. Hochedlinger, Tgf β signal inhibition cooperates in theinduction of iPSCs and replaces Sox2 and cMyc. Curr. Biol. 19,1718–1723 (2009). doi:10.1016/j.cub.2009.08.025 Medline17. P. A. Insel, R. S. Ostrom, Forskolin as a tool for examining adenylylcyclase expression, regulation, and G protein signaling. Cell. Mol. Neurobiol. 23, 305–314 (2003). doi:10.1023/A:1023684503883 Medline18. P. K. Chiang, Biological effects of inhibitors of S -adenosylhomocysteine hydrolase. Pharmacol. Ther. 77, 115–134 (1998). doi:10.1016/S0163-7258(97)00089-2 Medline 19. R. K. Gordon, K. Ginalski, W. R. Rudnicki, L. Rychlewski, M. C.Pankaskie, J. M. Bujnicki, P. K. Chiang, Anti-HIV-1 activity of3-deaza-adenosine analogs: Inhibition of S -adenosylhomocysteine hydrolase and nucleotide congeners. Eur. J. Biochem. 270, 3507–3517 (2003). doi:10.1046/j.1432-1033.2003.03726.x Medline20. C. Y. Lim, W. L. Tam, J. Zhang, H. S. Ang, H. Jia, L. Lipovich, H. H.Ng, C. L. Wei, W. K. Sung, P. Robson, H. Yang, B. Lim, Sall4 regulates distinct transcription circuitries in different blastocyst-derived stem cell lineages. Cell Stem Cell 3, 543–554 (2008). doi:10.1016/j.stem.2008.08.004 Medline 21. J. Shu, C. Wu, Y. Wu, Z. Li, S. Shao, W. Zhao, X. Tang, H. Yang, L.Shen, X. Zuo, W. Yang, Y. Shi, X. Chi, H. Zhang, G. Gao, Y. Shu, K. Yuan, W. He, C. Tang, Y. Zhao, H. Deng, Induction of pluripotency in mouse somatic cells with lineage specifiers. Cell 153, 963–975 (2013). doi:10.1016/j.cell.2013.05.001 Medline22. A. W. Neff, M. W. King, A. L. Mescher, Dedifferentiation and the roleof sall4 in reprogramming and patterning during amphibian limb regeneration. Dev. Dyn. 240, 979–989 (2011). doi:10.1002/dvdy.22554 Medline23. N. Feldman, A. Gerson, J. Fang, E. Li, Y. Zhang, Y. Shinkai, H.Cedar, Y. Bergman, G9a-mediated irreversible epigenetic inactivationof Oct-3/4 during early embryogenesis. Nat. Cell Biol. 8, 188–194(2006). doi:10.1038/ncb1353 Medline24. J. Chen, H. Liu, J. Liu, J. Qi, B. Wei, J. Yang, H. Liang, Y. Chen, J.Chen, Y. Wu, L. Guo, J. Zhu, X. Zhao, T. Peng, Y. Zhang, S. Chen, X.Li, D. Li, T. Wang, D. Pei, H3K9 methylation is a barrier duringsomatic cell reprogramming into iPSCs. Nat. Genet. 45, 34–42 (2013).doi:10.1038/ng.2491 Medline25. L. A. Boyer, T. I. Lee, M. F. Cole, S. E. Johnstone, S. S. Levine, J. P.Zucker, M. G. Guenther, R. M. Kumar, H. L. Murray, R. G. Jenner, D.K. Gifford, D. A. Melton, R. Jaenisch, R. A. Young, Coretranscriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005). doi:10.1016/j.cell.2005.08.020 Medline 26. C. Chazaud, Y. Yamanaka, T. Pawson, J. Rossant, Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway. Dev. Cell 10, 615–624 (2006). doi:10.1016/j.devcel.2006.02.020 Medline 27. K. Hochedlinger, Y. Yamada, C. Beard, R. Jaenisch, Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causesdysplasia in epithelial tissues. Cell 121, 465–477 (2005). doi:10.1016/j.cell.2005.02.018 Medline28. U. Lichti, J. Anders, S. H. Yuspa, Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. Nat. Protoc. 3, 799–810 (2008). doi:10.1038/nprot.2008.50 Medline 29. A. Seluanov, A. Vaidya, V. Gorbunova, Establishing primary adultfibroblast cultures from rodents. J. Vis. Exp. 2010, 2033 (2010). Medline 30. P. A. Tat, H. Sumer, K. L. Jones, K. Upton, P. J. Verma, The efficientgeneration of induced pluripotent stem (iPS) cells from adult mouse adipose tissue-derived and neural stem cells. Cell Transplant. 19, 525–536 (2010). doi:10.3727/096368910X491374 Medline31. J. L. McQualter, N. Brouard, B. Williams, B. N. Baird, S. Sims-Lucas, K. Yuen, S. K. Nilsson, P. J. Simmons, I. Bertoncello, Endogenous fibroblastic progenitor cells in the adult mouse lung are highly enriched in the sca-1 positive cell fraction. Stem Cells 27, 623–633 (2009). doi:10.1634/stemcells.2008-0866 Medline 32. Y. Zhao, X. Yin, H. Qin, F. Zhu, H. Liu, W. Yang, Q. Zhang, C.Xiang, P. Hou, Z. Song, Y. Liu, J. Yong, P. Zhang, J. Cai, M. Liu, H. Li, Y. Li, X. Qu, K. Cui, W. Zhang, T. Xiang, Y. Wu, Y. Zhao, C. Liu, C. Yu, K. Yuan, J. Lou, M. Ding, H. Deng, Two supporting factors greatly improve the efficiency of human iPSC generation. Cell Stem Cell 3, 475–479 (2008). doi:10.1016/j.stem.2008.10.002 Medline 33. N. Maherali, T. Ahfeldt, A. Rigamonti, J. Utikal, C. Cowan, K.Hochedlinger, A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell 3, 340–345 (2008). doi:10.1016/j.stem.2008.08.003 Medline 34. L. Longo, A. Bygrave, F. G. Grosveld, P. P. Pandolfi, Thechromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism. Transgenic Res. 6, 321–328 (1997). doi:10.1023/A:1018418914106 Medline35. G. Pan, J. Li, Y. Zhou, H. Zheng, D. Pei, A negative feedback loop oftranscription factors that controls stem cell pluripotency and self-renewal. FASEB J. 20, 1730–1732 (2006). doi:10.1096/fj.05-5543fje Medline 36. J. Tan, X. Yang, L. Zhuang, X. Jiang, W. Chen, P. L. Lee, R. K. Karuturi, P. B. Tan, E. T. Liu, Q. Yu, Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectivelyinduces apoptosis in cancer cells. Genes Dev. 21, 1050–1063 (2007). doi:10.1101/gad.1524107 Medline37. T. B. Miranda, C. C. Cortez, C. B. Yoo, G. Liang, M. Abe, T. K. Kelly, V. E. Marquez, P. A. Jones, DZNep is a global histonemethylation inhibitor that reactivates developmental genes not silenced by DNA methylation. Mol. Cancer Ther. 8, 1579–1588 (2009). doi:10.1158/1535-7163.MCT-09-0013 Medline Acknowledgments: We thank X. Zhang, J. Wang, C. Han, Z. Hou, J. Liu,and L. Ai for technical assistance. This work was supported by grants from the National 973 Basic Research Program of China (2012CD966401, 2010CB945204), the Key New Drug Creation and Manufacturing Program (2011ZX09102-010-03), the National Natural Science Foundation of China (90919031), the Ministry of Science and Technology (2011DFA30730, 2013DFG30680), the Beijing Science and Technology Plan (Z121100005212001), the Ministry of Education of China (111 project), and a Postdoctoral Fellowship of Peking-Tsinghua Center for Life Sciences. Microarray and RNA-seq data are deposited in the Gene Expression Omnibus (GEO) database (accession number GSE48243). The authors have filed a patent for the small-molecule combinations used in the chemical reprogramming reported in this paper. Supplementary Materials/cgi/content/full/science.1239278/DC1 Materials and Methods Figs. S1 to S30 Tables S1 to S5 References (27–37) o n J u l y 18, 2013w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m17 April 2013; accepted 11 July 2013 Published online 18 July 2013 10.1126/science.1239278o n J u l y 18, 2013w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mFig. 1. Generation of CiPSCs by small-molecule compounds. (A and B) Numbers of iPSC colonies induced from MEFs infected by SKM (A) or SK (B) plus chemicals or Oct4. Error bars indicate standard deviation (s.d.) (n = 3). (C) Morphology of MEFs for chemical reprogramming on day 0 (D0) and a GFP-positive cluster generated using VC6TF on day 20 (D20) after chemical treatment. (D) Numbers of GFP-positive colonies induced after DZNep treatment on day 36. Error bars indicate the s.d. (n = 2). (E to G) Morphology of a compact, epithelioid, GFP-positive colony on day 32 (D32) after treatment (E), a primary CiPSC colony on day 40 (D40) after treatment (F), and passaged CiPSC colonies (G). (H) Schematic diagram illustrating the process of CiPSC generation. For (C) and (E to G), scale bars, 100 μm. For (D), cells fo r reprogramming were replated on day 12.o n J u l y 1 8 , 2 0 1 3 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mFig. 2. Characterization of CiPSCs. (A and B) Pluripotency marker expression as illustrated by immunofluorescence (A, clone CiPS-25) and RT-PCR (B). Scale bars,100 μm. (C) Hierarchical clustering of global transcriptional profiles. 1-PCC, Pearson correlation coefficient. (D) Bisulfite genomic sequencing of the Oct4 and Nanog promoter regions. MNF-CiPS-7, MNF-derived CiPSC line #7; MAF-CiPS-1, MAF-derived CiPSC line #1. o n J u l y 1 8 , 2 0 1 3 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mFig. 3. Pluripotency of CiPSCs.(A) Hematoxylin and eosin staining of CiPSC-derived teratoma (clone CiPS-30). (B to D) Chimeric mice (B, clone CiPS-34), germline contribution of CiPSCs in testis, (C, clone CiPS-45) and F2 offspring (D, clone CiPS-34). Scale bars, 100 μm. (E) Genomic PCR analyzing pOct4-GFP cassettes in the tissues of chimeras. (F) Survival curves of chimeras. n, total numbers of chimeras studied. o n J u l y 1 8 , 2 0 1 3 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mFig. 4. Step-wise establishment of the pluripotency circuitry during chemical reprogramming. (A and B) Numbers of GFP-positive (A) and CiPSC (B) colonies induced by removing individual chemicals from VC6TFZ. The results of three independent experiments are shown with different colors (white, gray and black).(C) Structures of the essential chemicals. (D and E) The expression of pluripotency-related genes (D) and Gata6, Gata4 and Sox17 (E) as measured by real-time PCR. (F) Gene expression heat map at the single colony level. The value indicates the log2-transformed fold change (relative to Gapdh and normalized to the highest value). (G and H) Oct4 activation (G) and numbers of GFP-positive and iPSC colonies (H) induced by the overexpression of Sall4 and Sox2, with removing C6F from VC6TFZ. (I to K) The expression of pluripotency-related genes (I), DNA methylation (J) and H3K9 dimethylation (K) states of the Oct4promoter in the presence and absence of DZNep on day 32. (L) Schematic diagram illustrating the step-wise establishment of the pluripotency circuitry during chemical reprogramming. Error bars indicate the s.d. (n≥ 2).o n J u l y 1 8 , 2 0 1 3 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o m。