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Bmps Functionally Replace Klf4 and Support Efficient Reprogramming of Mouse Fibroblasts by Oct4 Alone

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Bmps Functionally Replace Klf4 and Support Efficient Reprogramming of Mouse Fibroblasts by Oct4 Alone Cell Research (2011) 21:205-212. © 2011 IBCB, SIBS, CAS All rights reserved 1001-0602/11 $ 32.00 npg ORIGINAL ARTICLE www.nature.com/cr BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone Jiekai Chen1, *, Jing Liu1, *, Jiaqi Yang1, You Chen1, Jing Chen1, Su Ni1, Hong Song1, Lingwen Zeng1, Ke Ding1, Duanqing Pei1 1Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine at Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China Generation of induced pluripotent stem cells by defined factors has become a useful model to investigate the mechanism of reprogramming and cell fate determination. However, the precise mechanism of factor-based repro- gramming remains unclear. Here, we show that Klf4 mainly acts at the initial phase of reprogramming to initiate mesenchymal-to-epithelial transition and can be functionally replaced by bone morphogenetic proteins (BMPs). BMPs boosted the efficiency of Oct4/Sox2-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to ~1%. BMPs also promoted single-factor Oct4-based reprogramming of MEFs and tail tibial fibroblasts. Our studies clarify the contribution of Klf4 in reprogramming and establish Oct4 as a singular setter of pluripotency in differentiated cells. Keywords: Oct4; iPSCs; reprogramming; stem cell; BMPs; MET Cell Research (2011) 21:205-212. doi:10.1038/cr.2010.172; published online 7 December 2010 Introduction roles during the reprogramming process. Taken together, one would expect that Oct4 alone can reprogram somatic Somatic cells can be reprogrammed into induced cells such as mouse fibroblasts into pluripotency as Oct4 pluripotent stem cells (iPSCs) by ectopic expression governs embryonic cell fate [9, 10]. In this study, we ana- of Oct4, Sox2, Klf4 and c-Myc [1-6]. However, the Ya- lyzed the relative contribution of Sox2 and Klf4 towards manaka factors included Klf4 and Myc both not directly reprogramming under a chemically defined system opti- implicated in pluripotency, but did not have the pluripo- mized for robust reprogramming. We showed that Klf4 tency regulator Nanog, suggesting that there is a mecha- plays an important role in triggering MET, which can nistic difference between induction and maintenance of also be initiated by bone morphogenetic proteins (BMPs) pluripotency [1]. We recently reported that mouse fibro- [8, 11]. Then, we demonstrated that Oct4/Sox2 (OS) and blasts undergo a mesenchymal-to-epithelial transition Oct4 alone can reprogram mouse embryonic fibroblasts (MET) orchestrated by Oct4, Sox2 and Myc to suppress (MEFs) and tail tibial fibroblasts (TTFs) efficiently in the snail expression and blunt TGF-β signaling, and Klf4 to presence of BMPs. activate expression of epithelial markers, such as Cdh1, during the initial phase of reprogramming [7], thus, de- Results fining a concrete molecular and cellular step towards pluripotency [7, 8]. The division of labor among the Klf4 mainly acts at the initial phase of reprogramming Yamanaka factors suggests that each factor plays distinct Multifactorial requirement is a great barrier for mechanistic investigation of factor-based reprogram- ming. However, the function of each factor employed *These two authors contributed equally to this work. in reprogramming is poorly understood and at least two Correspondence: Duanqing Pei factors are required (including ectopic and endogenous Tel: +86-20-3201-5201 E-mail: [email protected] expression) in the literature [7, 12-16]. Among the four Received 20 October 2010; accepted 25 October 2010; published online 7 Yamanaka factors, c-Myc was proven dispensable and December 2010 can be replaced by various methods [17-20]. Oct4, Sox2 npg Efficient reprogramming by Oct4 alone 206 and Klf4 are currently considered as indispensable fac- at day 15, a 400-fold plus 9 days improvement), but had tors, as replacement of them would greatly reduce repro- no effect on OK reprogramming and even had inhibi- gramming efficiency. OK-mediated reprogramming of tory effect on OKS reprogramming (Figure 2C and 2D mouse fibroblasts was reported with moderate efficiency, and Supplementary information, Figure S2C). We also by using compounds targeting various pathways such as showed that BMP2, BMP6, BMP7 and BMP9, similar to Bayk8644, CHIR99021, RepSox and A83-01 [7, 12, 13, BMP4, significantly enhanced the OS-mediated repro- 16, 21]. By contrast, OS-mediated reprogramming was gramming (Figure 2E, and Supplementary information, reported only with p53-null fibroblasts [14]. We previ- Figure S2D). We also examined the treatment windows ously reported that OKS-mediated reprogramming can of BMP4 in OS-mediated reprogramming and showed be enhanced by the optimized serum-free medium iSF1 that exposure of BMP4 at days 3-9 is most effective [19]. Further optimization led to improved efficiency of (Figure 2F). OKS-mediated reprogramming and we found that iPSCs We then investigated the role of Cdh1 in OS-mediated can be generated by either OK or OS with very low ef- reprogramming and showed that knockdown of Cdh1 ficiency (Supplementary information, Figure S1A) in a decreased reprogramming efficiency significantly (Sup- newly formulated medium iCD1. The resulting OS or plementary information, Figure S2E), suggesting that OK iPSCs were similar to ESCs and could be differenti- the observed enhancement by BMP4 is Cdh1 dependent. ated into three germ layers in teratoma assay as expected We also tested other compounds including Kenpaullone (Supplementary information, Figure S1B). Remarkably, [22] and showed that none of them can enhance OS- OK-mediated reprogramming appeared to be more ef- mediated reprogramming (Supplementary information, ficient and more rapid than OS (Supplementary informa- Figure S2F). As expected, OS-iPSC clones derived with tion, Figure S1A). BMP4 expressed pluripotent markers such as Oct4, We then examined the requirement of Klf4 or Sox2 Nanog, Rex1 and SSEA-1 (Figure 2G). We then showed in a doxycycline-inducible system. We found that Klf4 that these OS iPSCs contributed to the generation of is required mostly during the initial phase of reprogram- chimeric mice, which underwent germline transmission ming (Figure 1A). We performed transcriptomic analysis (Figure 2H), indicating that the OS iPSCs were fully re- and showed that Klf4 upregulated genes important for programmed. epithelial development (Figure 1B). Consistent with this analysis, OK stimulates robust expression of epithelial BMPs induce efficient Oct4-mediated reprogramming by markers (such as Cdh1 and Occludin), while OS has little promoting MET effect on them (Figure 1C). As MET is an early requisite We then hypothesized that BMPs should enhance step during the reprogramming of MEFs and as Klf4 Oct4-mediated reprogramming by inducing MET. As participates in MET by activating the epithelial program in OS-mediated reprogramming, BMP4 and BMP7 im- [7], we conclude that MET is a rate-limiting step in OS- proved the expression of Cdh1 and EpCAM in Oct4- mediated reprogramming (Figure 1D). transduced MEFs (Figure 3A and Supplementary information, Figure S3A). We then showed that Oct4 BMPs functionally replace Klf4 and support efficient re- alone was able to convert MEFs into iPSCs efficiently programming with OS in iCD1+BMP4 with reasonable kinetics (∼0.05% at BMPs have been implicated in MET by reversing D24 post-infection; Figure 3B). Under the same culture TGF-β-induced EMT in NP1 cells and reported to en- condition, we showed that adult mouse TTFs can also be hance MET during reprogramming [8, 11], and thus may reprogrammed by Oct4 alone reproducibly, demonstrat- functionally substitute Klf4. To test this possibility, we ing that Oct4 is sufficient to induce adult somatic cells to showed that BMP4 and BMP7 can enhance the expres- pluripotency (Figure 3C-3E). We also showed that BMPs sion of epithelial genes (Cdh1, EpCAM, etc.) and inhibit has no effect on the expression level of p53 and p21 the expression of mesenchymal genes (Zeb1, Twist1, (Supplementary information, Figure S3B) and further etc.) in OS-infected MEFs, but cannot promote epithe- showed that no other tested chemical compounds can lial markers further in OK-infected cultures (Figure 2A support Oct4-mediated reprogramming (Supplementary and 2B and Supplementary information, Figure S2A information, Figure S3C). and S2B). These results indicate that BMPs can trigger We then characterized the Oct4-iPSC clones isolated MET in OS-induced reprogramming, but cannot further with iCD1+BMP4 and showed that they expressed pluri- enhance the strong MET activated by Klf4 in OK repro- potent markers Oct4, Nanog, Rex1, SSEA-1, Dppa3 and gramming. Consistently, BMP4 greatly improved the ki- Dnmt3l (Figure 4A and 4B, and Supplementary informa- netics and efficiency of OS reprogramming (up to ∼0.8% tion, Figure S3D). All Oct4-iPSC clones that we obtained Cell Research | Vol 21 No 1 | January 2011 Jiekai Chen et al. npg 207 Figure 1 Klf4 mainly acts at early MET phase of reprogramming. (A) Klf4 is critical in the initial stage of reprogramming. By using inducible system, ectopic expression of Klf4 or Sox2 in OKS-mediated reprogramming was terminated in different days. Representative photos were taken and reprogramming efficiency was scored at day 12. n = 3. Scale bar, 2 mm. (B) Klf4 mainly regulated cell-cell adhesion and induced epithelial markers. MEFs transduced by retroviral OS and lentiviral TetOn- Klf4 were cultured
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