Abstract: Two independent studies by Li et al., 2010Li R. Liang J. Ni S. Zhou T. Qing X. Li H. He W. Chen J. Li F. Zhuang Q. et al.Cell Stem Cell. 2010; 7 (this issue): 51-63Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar and Samavarchi-Tehrani et al., 2010Samavarchi-Tehrani P. Golipour A. David L. Sung H.-k. Beyer T.A. Datti A. Woltjen K. Nagy A. Wrana J.L. Cell Stem Cell. 2010; 7 (this issue): 64-77Abstract Full Text Full Text PDF PubMed Scopus (744) Google Scholar in this issue of Cell Stem Cell suggest that a mesenchymal-to-epithelial transition is a critical initiating event during the derivation of induced pluripotent stem cells (iPSCs) from fibroblasts, indicating remarkable similarities between cellular reprogramming, development, and cancer. Two independent studies by Li et al., 2010Li R. Liang J. Ni S. Zhou T. Qing X. Li H. He W. Chen J. Li F. Zhuang Q. et al.Cell Stem Cell. 2010; 7 (this issue): 51-63Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar and Samavarchi-Tehrani et al., 2010Samavarchi-Tehrani P. Golipour A. David L. Sung H.-k. Beyer T.A. Datti A. Woltjen K. Nagy A. Wrana J.L. Cell Stem Cell. 2010; 7 (this issue): 64-77Abstract Full Text Full Text PDF PubMed Scopus (744) Google Scholar in this issue of Cell Stem Cell suggest that a mesenchymal-to-epithelial transition is a critical initiating event during the derivation of induced pluripotent stem cells (iPSCs) from fibroblasts, indicating remarkable similarities between cellular reprogramming, development, and cancer. Overexpression of the transcription factors Oct4, Klf4, Sox2, and c-Myc (OKSM) in somatic cells gives rise to induced pluripotent stem cells (iPSCs) that exhibit molecular and functional similarities to embryonic stem cells (ESCs) (Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (17010) Google Scholar). Thus, iPSCs have great potential for the study and possible treatment of numerous degenerative diseases. The reprogramming of fibroblasts is a slow (2–3 weeks) and inefficient (<1%) process in which somatic cells gradually lose their differentiated identity and assume an embryonic gene expression pattern and growth behavior (Hochedlinger and Plath, 2009Hochedlinger K. Plath K. Development. 2009; 136: 509-523Crossref PubMed Scopus (438) Google Scholar). This conversion is accompanied by striking morphological changes as the cells transition from a single layer of adherent cells to become multilayered epithelial cells, a process reminiscent of a mesenchymal-to-epithelial transition (MET). MET, as well as its reverse process, epithelial-to-mesenchymal transition (EMT), play pivotal roles during organ development and in cancer metastasis by endowing cells with migratory and invasive properties, respectively (Thiery et al., 2009Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (6821) Google Scholar). In this issue of Cell Stem Cell, the labs of Jeffrey Wrana (Samavarchi-Tehrani et al., 2010Samavarchi-Tehrani P. Golipour A. David L. Sung H.-k. Beyer T.A. Datti A. Woltjen K. Nagy A. Wrana J.L. Cell Stem Cell. 2010; 7 (this issue): 64-77Abstract Full Text Full Text PDF PubMed Scopus (744) Google Scholar) and Duanqing Pei (Li et al., 2010Li R. Liang J. Ni S. Zhou T. Qing X. Li H. He W. Chen J. Li F. Zhuang Q. et al.Cell Stem Cell. 2010; 7 (this issue): 51-63Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar) provide compelling molecular and functional evidence demonstrating that MET is indeed a crucial early phase during the reprogramming of murine embryonic fibroblasts (MEF) into iPSCs. When studying either gene expression profiles (Samavarchi-Tehrani et al., 2010Samavarchi-Tehrani P. Golipour A. David L. Sung H.-k. Beyer T.A. Datti A. Woltjen K. Nagy A. Wrana J.L. Cell Stem Cell. 2010; 7 (this issue): 64-77Abstract Full Text Full Text PDF PubMed Scopus (744) Google Scholar) or morphological changes (Li et al., 2010Li R. Liang J. Ni S. Zhou T. Qing X. Li H. He W. Chen J. Li F. Zhuang Q. et al.Cell Stem Cell. 2010; 7 (this issue): 51-63Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar) of MEFs during the initial stages of reprogramming, the Wrana and Pei groups uncovered striking cellular and molecular similarities with the hallmarks that define a MET. This pattern included the upregulation of epithelial genes such as E-cadherin and Epcam as well as the concomitant downregulation of key mesenchymal genes such as Snail and N-Cadherin (Figure 1). Based on the observed gene expression dynamics, Samavarchi-Tehrani and colleagues further identified three consecutive phases of reprogramming, which they coined initiation, maturation, and stabilization. The MET-associated alterations were evident during the initiation phase of reprogramming, when cells are still dependent on exogenous factor expression and occurred before the subsequent maturation and stabilization phases, when cells activate an embryonic gene expression program, suggesting that MET may be one of the earliest changes fibroblasts undergo during cellular reprogramming. The functional importance of the MET in iPSC formation was shown by the Wrana lab, who employed an RNAi screen (Samavarchi-Tehrani et al., 2010Samavarchi-Tehrani P. Golipour A. David L. Sung H.-k. Beyer T.A. Datti A. Woltjen K. Nagy A. Wrana J.L. Cell Stem Cell. 2010; 7 (this issue): 64-77Abstract Full Text Full Text PDF PubMed Scopus (744) Google Scholar). This effort identified several hairpins that abrogated iPSC formation, including members of the TGF-β superfamily such as Smads and BMP receptors, which have previously been implicated in MET (Thiery et al., 2009Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (6821) Google Scholar). Consistent with these findings, treatment of reprogramming cultures with recombinant BMP-7 significantly increased the number and accelerated the speed of iPSC colony formation, whereas exposure to the BMP antagonist Noggin impaired colony formation. To identify possible downstream effectors that may be responsible for BMP's ability to enhance reprogramming, Wrana and colleagues focused on the role of miRNA-200 and miRNA-205, which have recently been shown to control MET by downregulating mesenchymal genes. Overexpression of these miRNAs in MEFs was indeed sufficient to activate key MET genes in combination with OKSM and gave rise to SSEA1+ intermediate cells more rapidly than control cells. Intriguingly, overexpression of these miRNAs led to the rescue of the inhibitory effect of knocking down BMPs on iPSC formation, thus establishing an epistatic link between extracellular BMP signaling, miRNA-200 and miRNA-205, and successful reprogramming. Meanwhile, Li et al., 2010Li R. Liang J. Ni S. Zhou T. Qing X. Li H. He W. Chen J. Li F. Zhuang Q. et al.Cell Stem Cell. 2010; 7 (this issue): 51-63Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar showed that E-Cadherin knockdown or Snail overexpression, both of which inhibit MET, substantially reduces the formation of iPSCs. Further, under these conditions, only dissociated epiblast-like colonies with diminished growth and developmental potentials arose, thus confirming the functional importance of other key effectors of the MET pathway during reprogramming. A clue as to how the reprogramming factors might synergize with the MET pathway comes from the observation that the E-cadherin promoter is bound by Klf4, providing evidence for the direct regulation of an MET-associated gene by one of the reprogramming factors. Of note, Klf4 has been implicated in the maintenance of epithelial tissues, and thus may facilitate reprogramming through exerting an epithelializing effect on fibroblasts. It is also tempting to speculate that pluripotency genes may control the maintenance of an epithelial character of pluripotent inner cell mass cells because in vivo downregulation of these genes during development correlates with the onset of gastrulation and EMT. Together, these data provide strong functional evidence that the manipulation of MET-associated growth factors, miRNAs, and cell adhesion molecules enhance both reprogramming speed and efficiency. Given that a MET seems to be important during reprogramming, one would predict that epithelial cells are more amenable to reprogramming than mesenchymal cells. Indeed, previous reports suggested that skin keratinocytes, an epithelial cell type, give rise to iPSCs more efficiently and faster than fibroblasts (Aasen et al., 2008Aasen T. Raya A. Barrero M.J. Garreta E. Consiglio A. Gonzalez F. Vassena R. Bilic J. Pekarik V. Tiscornia G. et al.Nat. Biotechnol. 2008; 26: 1276-1284Crossref PubMed Scopus (1067) Google Scholar, Maherali et al., 2008Maherali N. Ahfeldt T. Rigamonti A. Utikal J. Cowan C. Hochedlinger K. Cell Stem Cell. 2008; 3: 340-345Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). Li et al. extended these studies by showing that mammary gland epithelial cells convert into iPSCs more readily than fibroblasts. This population expresses abundant endogenous Klf4 and thus the cells do not require ectopic expression of either c-Myc or Klf4 to convert into iPSCs, further supporting a role for these two factors in conferring an epithelial phenotype on mesenchymal cells. However, Klf4 seems to play additional roles besides activating E-Cadherin because the overexpression of E-Cadherin alone could not compensate for the function of Klf4 during iPSC formation. It will certainly be interesting to assess whether epithelial cells that do not express Klf4 are equally amenable to reprogramming by Oct4 and Sox2 alone. The findings by Pei and Wrana may also help to explain recent reports showing that the treatment of fibroblasts with TGF-β inhibitors enhances iPSC formation (Ichida et al., 2009Ichida J.K. Blanchard J. Lam K. Son E.Y. Chung J.E. Egli D. Loh K.M. Carter A.C. Di Giorgio F.P. Koszka K. et al.Cell Stem Cell. 2009; 5: 491-503Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar, Lin et al., 2009Lin T. Ambasudhan R. Yuan X. Li W. Hilcove S. Abujarour R. Lin X. Hahm H.S. Hao E. Hayek A. et al.Nat. Methods. 2009; 6: 805-808Crossref PubMed Scopus (461) Google Scholar, Maherali and Hochedlinger, 2009Maherali N. Hochedlinger K. Curr. Biol. 2009; 19: 1718-1723Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). TGF-β is a well-known inducer of EMT, which, in light of the current results, is expected to inhibit reprogramming into iPSCs. Interestingly, OKSM activation resulted in reduced expression of TGF-β receptors 2 and 3 in MEFs and decreased secretion of TGF-β1 into the media, suggesting that the reprogramming factors counteract this major EMT pathway. c-Myc expression alone appeared to be sufficient to downregulate TGF-βR1 and TGF-βR2 expression, which the authors hypothesize might be how c-Myc activation enhances reprogramming. In further support of this notion, the TGF-β receptor inhibitor Alk5i substantially increased reprogramming in the absence of c-Myc but had less of an effect in the presence of c-Myc (Ichida et al., 2009Ichida J.K. Blanchard J. Lam K. Son E.Y. Chung J.E. Egli D. Loh K.M. Carter A.C. Di Giorgio F.P. Koszka K. et al.Cell Stem Cell. 2009; 5: 491-503Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar, Maherali and Hochedlinger, 2009Maherali N. Hochedlinger K. Curr. Biol. 2009; 19: 1718-1723Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). By using two complementary approaches, the Wrana and Pei groups conclusively demonstrate that MET is an important rate-limiting step during the conversion of fibroblasts into iPSCs. The acquisition of an epithelial fate during cellular reprogramming appears to be tightly linked with the pluripotent state and may reflect requirements for cell-cell interactions that initiate and sustain pluripotency. The observation that epithelial cells are more amenable to reprogramming suggests that somatic cells with pre-exisiting features of pluripotent cells require fewer changes in order to reach pluripotency. This notion is consistent with previous findings showing that immortalized fibroblasts as well as adult progenitors reprogram at high efficiency into iPSCs, presumably because these cells already exhibit indefinite growth potential and express shared transcriptional or epigenetic regulators, respectively. Lastly, the presented studies underscore the notion that both intracellular as well as extracellular factors impinge on somatic cells during reprogramming. An interesting question that remains to be addressed is whether nonmesenchymal cell types such as blood cells undergo MET-associated changes as well or whether alternative cellular processes need to be initiated. K.H. is an advisor for iPierian. A Mesenchymal-to-Epithelial Transition Initiates and Is Required for the Nuclear Reprogramming of Mouse FibroblastsLi et al.Cell Stem CellJune 17, 2010In BriefEpithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate determination. Fibroblasts, a product of EMT, can be reset into induced pluripotent stem cells (iPSCs) via exogenous transcription factors but the underlying mechanism is unclear. Here we show that the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition (MET) orchestrated by suppressing pro-EMT signals from the culture medium and activating an epithelial program inside the cells. Full-Text PDF Open ArchiveFunctional Genomics Reveals a BMP-Driven Mesenchymal-to-Epithelial Transition in the Initiation of Somatic Cell ReprogrammingSamavarchi-Tehrani et al.Cell Stem CellJune 17, 2010In BriefSomatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expression of defined embryonic factors. However, little is known of the molecular mechanisms underlying the reprogramming process. Here we explore somatic cell reprogramming by exploiting a secondary mouse embryonic fibroblast model that forms iPSCs with high efficiency upon inducible expression of Oct4, Klf4, c-Myc, and Sox2. Temporal analysis of gene expression revealed that reprogramming is a multistep process that is characterized by initiation, maturation, and stabilization phases. Full-Text PDF Open Archive