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• W2010672807 abstract "Somatic 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. Functional analysis by systematic RNAi screening further uncovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. We show that this is linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs that are key regulators of MET. These studies thus define a multistep mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming.PaperClip/cms/asset/b7d91c75-1072-4889-a6bc-59bc8698d7ac/mmc5.mp3Loading ...(mp3, 3.51 MB) Download audio Somatic 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. Functional analysis by systematic RNAi screening further uncovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. We show that this is linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs that are key regulators of MET. These studies thus define a multistep mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. Temporal gene expression reveals three phases during somatic cell reprogramming Mesenchymal-to-epithelial transition (MET) is a hallmark of the initiation phase RNAi screening defines MET and BMP signaling as essential for reprogramming BMP induces miR-200 family miRNAs to drive MET and somatic cell reprogramming The capacity of differentiated cells to reacquire a totipotent state was first revealed when the nuclei of differentiated cells were reprogrammed in enucleated oocytes to generate frogs (Gurdon, 1964Gurdon J.B. The transplantation of living cell nuclei.Adv. Morphog. 1964; 4: 1-43Crossref PubMed Google Scholar). Furthermore, ectopic expression of just four transcription factors, Oct4, Klf4, c-Myc, and Sox2 (OKMS), is sufficient to reprogram somatic cells to induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (17010) Google Scholar). Fully reprogrammed iPSCs have a similar developmental potential as embryonic stem cells (ESCs) and can contribute extensively to the three germ layers and the germline (Zhao and Daley, 2008Zhao R. Daley G.Q. From fibroblasts to iPS cells: Induced pluripotency by defined factors.J. Cell. Biochem. 2008; 105: 949-955Crossref PubMed Scopus (90) Google Scholar). At the molecular level, reprogramming results in large changes in gene expression that remodel the somatic cell properties to a state similar to embryonic stem cells (Maherali et al., 2007Maherali N. Sridharan R. Xie W. Utikal J. Eminli S. Arnold K. Stadtfeld M. Yachechko R. Tchieu J. Jaenisch R. et al.Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.Cell Stem Cell. 2007; 1: 55-70Abstract Full Text Full Text PDF PubMed Scopus (1337) Google Scholar, Mikkelsen et al., 2008Mikkelsen T.S. Hanna J. Zhang X. Ku M. Wernig M. Schorderet P. Bernstein B.E. Jaenisch R. Lander E.S. Meissner A. Dissecting direct reprogramming through integrative genomic analysis.Nature. 2008; 454: 49-55Crossref PubMed Scopus (1133) Google Scholar, Sridharan et al., 2009Sridharan R. Tchieu J. Mason M.J. Yachechko R. Kuoy E. Horvath S. Zhou Q. Plath K. Role of the murine reprogramming factors in the induction of pluripotency.Cell. 2009; 136: 364-377Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar) that include early activation of the pluripotency markers alkaline phosphatase (AP) and SSEA1 (Brambrink et al., 2008Brambrink T. Foreman R. Welstead G.G. Lengner C.J. Wernig M. Suh H. Jaenisch R. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells.Cell Stem Cell. 2008; 2: 151-159Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar) followed by embryonic stem cell factors such as Oct4, Sox2, and Klf4 themselves, as well as Nanog and Sall4 (Maherali et al., 2007Maherali N. Sridharan R. Xie W. Utikal J. Eminli S. Arnold K. Stadtfeld M. Yachechko R. Tchieu J. Jaenisch R. et al.Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.Cell Stem Cell. 2007; 1: 55-70Abstract Full Text Full Text PDF PubMed Scopus (1337) Google Scholar, Okita et al., 2007Okita K. Ichisaka T. Yamanaka S. Generation of germline-competent induced pluripotent stem cells.Nature. 2007; 448: 313-317Crossref PubMed Scopus (3360) Google Scholar, Wernig et al., 2007Wernig M. Meissner A. Foreman R. Brambrink T. Ku M. Hochedlinger K. Bernstein B.E. Jaenisch R. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.Nature. 2007; 448: 318-324Crossref PubMed Scopus (2137) Google Scholar, Xu et al., 2009Xu D. Alipio Z. Fink L.M. Adcock D.M. Yang J. Ward D.C. Ma Y. Phenotypic correction of murine hemophilia A using an iPS cell-based therapy.Proc. Natl. Acad. Sci. USA. 2009; 106: 808-813Crossref PubMed Scopus (214) Google Scholar). Nanog is a component of the stem cell regulatory network that is critical for acquiring the pluripotent state during somatic cell reprogramming (Silva et al., 2009Silva J. Nichols J. Theunissen T.W. Guo G. van Oosten A.L. Barrandon O. Wray J. Yamanaka S. Chambers I. Smith A. Nanog is the gateway to the pluripotent ground state.Cell. 2009; 138: 722-737Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar). Furthermore, failure to suppress differentiation-associated genes or block differentiation signals leads to incomplete reprogramming (Mikkelsen et al., 2008Mikkelsen T.S. Hanna J. Zhang X. Ku M. Wernig M. Schorderet P. Bernstein B.E. Jaenisch R. Lander E.S. Meissner A. Dissecting direct reprogramming through integrative genomic analysis.Nature. 2008; 454: 49-55Crossref PubMed Scopus (1133) Google Scholar). Although a considerable amount is known about the transcriptional networks that regulate ESCs, relatively little is known of the signaling pathways that integrate intrinsic and extrinsic cues to maintain the pluripotent state and control reprogramming. TGF-β-related factors that include the bone morphogenetic proteins (BMPs) are an important family of morphogens that regulate cell fate decisions in stem cells (Varga and Wrana, 2005Varga A.C. Wrana J.L. The disparate role of BMP in stem cell biology.Oncogene. 2005; 24: 5713-5721Crossref PubMed Scopus (162) Google Scholar). In the BMP pathway, ligand binding to the heterotetrameric complexes of type II and type I receptors leads to phosphorylation of receptor-regulated R-Smads 1, 5, and 8 that in turn bind to Smad4 and accumulate in the nucleus to regulate transcription (Attisano and Wrana, 2002Attisano L. Wrana J.L. Signal transduction by the TGF-beta superfamily.Science. 2002; 296: 1646-1647Crossref PubMed Scopus (1084) Google Scholar). In mouse ES cells (mESCs), BMP signaling together with leukemia inhibiting factor (LIF) signaling is important for maintaining the pluripotent state (Ying et al., 2003Ying Q.L. Nichols J. Chambers I. Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.Cell. 2003; 115: 281-292Abstract Full Text Full Text PDF PubMed Scopus (1632) Google Scholar), whereas TGF-β/Activin signaling is critical in human ESCs and mouse stem cells that are derived from the epiblast (EpiSC) (Vallier et al., 2009Vallier L. Mendjan S. Brown S. Chng Z. Teo A. Smithers L.E. Trotter M.W. Cho C.H. Martinez A. Rugg-Gunn P. et al.Activin/Nodal signalling maintains pluripotency by controlling Nanog expression.Development. 2009; 136: 1339-1349Crossref PubMed Scopus (295) Google Scholar). Interestingly, TGF-β receptor-specific small molecule antagonists were recently shown to promote reprogramming by promoting Sox2- and Myc-dependent functions (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.A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog.Cell Stem Cell. 2009; 5: 491-503Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar, Maherali and Hochedlinger, 2009Maherali N. Hochedlinger K. Tgfbeta signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc.Curr. Biol. 2009; 19: 1718-1723Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). TGF-β and BMPs may thus play important regulative roles in controlling distinct stem cell states and reprogramming. Understanding the process of reprogramming and in particular the signaling networks that control progression to a stable pluripotent state has been hampered in part by the low frequency of the event. Here we employed mouse iPSCs generated with a piggyBac transposon system that expresses OKMS in a doxycycline (Dox)-inducible manner (Woltjen et al., 2009Woltjen K. Michael I.P. Mohseni P. Desai R. Mileikovsky M. Hämäläinen R. Cowling R. Wang W. Liu P. Gertsenstein M. et al.piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.Nature. 2009; 458: 766-770Crossref PubMed Scopus (1374) Google Scholar). Secondary mouse embryonic fibroblasts (2° MEFs) from piggyBac iPSC-derived chimeras reprogram efficiently, thus allowing us to apply temporal gene expression profiling coupled to a functional siRNA screen to dissect mechanisms underlying the early stages of reprogramming. This integrative approach reveals three phases of reprogramming that we term initiation, maturation, and stabilization and uncovers an early mesenchymal-to-epithelial transition (MET) that marks initiation. Furthermore, we show that BMP signaling synergizes with OKMS to induce a microRNA (miRNA, miR-) expression signature that is associated with MET and promotes progression through the initiation phase. These studies unveil broad temporal alterations in gene expression during reprogramming and define a critical initiation phase that is regulated by a BMP-miRNA-MET signaling axis. Reprogramming of primary murine somatic cells by ectopic expression of OKMS occurs at low frequency, making the molecular characterization of reprogramming difficult. However, reprogramming occurs in secondary systems with much higher efficiencies (Maherali et al., 2008Maherali N. Ahfeldt T. Rigamonti A. Utikal J. Cowan C. Hochedlinger K. A high-efficiency system for the generation and study of human induced pluripotent stem cells.Cell Stem Cell. 2008; 3: 340-345Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, Wernig et al., 2008Wernig M. Lengner C.J. Hanna J. Lodato M.A. Steine E. Foreman R. Staerk J. Markoulaki S. Jaenisch R. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types.Nat. Biotechnol. 2008; 26: 916-924Crossref PubMed Scopus (335) Google Scholar, Woltjen et al., 2009Woltjen K. Michael I.P. Mohseni P. Desai R. Mileikovsky M. Hämäläinen R. Cowling R. Wang W. Liu P. Gertsenstein M. et al.piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.Nature. 2009; 458: 766-770Crossref PubMed Scopus (1374) Google Scholar). Recently, we established a reprogramming system with Dox-regulated OKMS transgenes delivered via piggyBac transposition (Woltjen et al., 2009Woltjen K. Michael I.P. Mohseni P. Desai R. Mileikovsky M. Hämäläinen R. Cowling R. Wang W. Liu P. Gertsenstein M. et al.piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.Nature. 2009; 458: 766-770Crossref PubMed Scopus (1374) Google Scholar). Chimeric mice from two primary iPSC lines (6C and 1B) were used to isolate chimeric 2° MEF lines (2°-6C and 2°-1B MEFs) in which the GFP+, iPSC-derived 2° MEFs reprogrammed with high efficiency upon Dox induction (see schematic, Figure 1A ). We confirmed the pluripotency of iPSCs derived from the 2°-6C MEFs via embryoid body assays, teratomas, and contribution to diploid chimeric embryos (Figure S1 available online). iPSCs derived from 2°-1B MEFs also contributed extensively to adult chimeric mice (Figure S1E). Thus, secondary iPSCs generated from the piggyBac system are pluripotent, like their primary counterparts. Because our secondary iPSC system reprograms with high efficiency (Woltjen et al., 2009Woltjen K. Michael I.P. Mohseni P. Desai R. Mileikovsky M. Hämäläinen R. Cowling R. Wang W. Liu P. Gertsenstein M. et al.piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.Nature. 2009; 458: 766-770Crossref PubMed Scopus (1374) Google Scholar), we sought to characterize changes in the transcriptome during reprogramming of 2°-6C cell line by microarray analysis at 2, 5, 8, 11, 16, and 21 days after OKMS induction (Table S2). Comparison of the starting MEF population and their iPSC progeny revealed 4,252 genes, out of the 13,389 genes detected, changed expression more than 2-fold with 3,520 genes upregulated and only 732 downregulated. Of these, 61% were the same as those identified in a previous analysis of MEF reprogramming (Sridharan et al., 2009Sridharan R. Tchieu J. Mason M.J. Yachechko R. Kuoy E. Horvath S. Zhou Q. Plath K. Role of the murine reprogramming factors in the induction of pluripotency.Cell. 2009; 136: 364-377Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar), indicating good concordance between the two studies. We next analyzed the Pearson correlation coefficient (PCC) of the transcriptional profile, which showed increasing similarity to the primary iPSC profile through the course of reprogramming (Figure 1B), and unsupervised clustering revealed segregation into temporally distinct early, middle, and late phases (Figure 1B). To further resolve these temporal changes, we clustered genes based on when expression changes occurred (Figure 1C). This showed that a large number of genes changed expression early in the time course of reprogramming (clusters I and II), as well as distinct subsets of genes that were altered at later time points. Embryonic stem cell-associated genes clustered into either the middle or late phases (Table S1) and their patterns of expression were validated by qPCR (Figure S2A). Nanog, an exemplar of the middle cluster, initiated expression after day 5 and then rapidly climbed to a high level that was sustained throughout the reprogramming time course, peaking in 2°-6C iPSCs at levels comparable to primary 6C iPSCs (Figure S2A). Sall4 and Esrrb shared Nanog's expression pattern. Similarly, Rex1, Tcl1, Nodal, and Cripto were expressed at high level in the middle phase although their expression initiated slightly later. In contrast, the late-phase cluster was characterized by induction of Dnmt3l, Lin28, Utf1, and slightly later by Pecam, Stella, and Dppa4 (Furusawa et al., 2006Furusawa T. Ikeda M. Inoue F. Ohkoshi K. Hamano T. Tokunaga T. Gene expression profiling of mouse embryonic stem cell subpopulations.Biol. Reprod. 2006; 75: 555-561Crossref PubMed Scopus (36) Google Scholar, Müller et al., 2008Müller F.J. Laurent L.C. Kostka D. Ulitsky I. Williams R. Lu C. Park I.H. Rao M.S. Shamir R. Schwartz P.H. et al.Regulatory networks define phenotypic classes of human stem cell lines.Nature. 2008; 455: 401-405Crossref PubMed Scopus (267) Google Scholar). Altogether, these studies reveal three phases during OKMS-induced MEF reprogramming that we refer to as initiation, maturation, and stabilization (Figure S2B). Interestingly, no embryonic stem cell factors were expressed in the initiation phase, while the maturation phase, as marked by the beginning of Nanog, Sall4, Esrrb, Rex1, Tcl1, Cripto, and Nodal expression, occurred at approximately day 8, and the stabilization phase, marked by Dnmt3l, Lin28, Utf1, Pecam, Stella, and Dppa4, started at around day 21. Nanog drives the broad changes in the transcriptional program that are associated with the acquisition of pluripotency (Mitsui et al., 2003Mitsui K. Tokuzawa Y. Itoh H. Segawa K. Murakami M. Takahashi K. Maruyama M. Maeda M. Yamanaka S. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.Cell. 2003; 113: 631-642Abstract Full Text Full Text PDF PubMed Scopus (2454) Google Scholar, Sridharan et al., 2009Sridharan R. Tchieu J. Mason M.J. Yachechko R. Kuoy E. Horvath S. Zhou Q. Plath K. Role of the murine reprogramming factors in the induction of pluripotency.Cell. 2009; 136: 364-377Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar) and can push pre-iPSCs to the pluripotent state (Silva et al., 2009Silva J. Nichols J. Theunissen T.W. Guo G. van Oosten A.L. Barrandon O. Wray J. Yamanaka S. Chambers I. Smith A. Nanog is the gateway to the pluripotent ground state.Cell. 2009; 138: 722-737Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar). Nanog is not expressed until after day 5 in our reprogramming system, suggesting that the initiation phase might not be self-sustaining. Indeed, SSEA1 induction and morphological changes induced by OKMS are rapidly lost when OKMS expression is suppressed early in reprogramming (Brambrink et al., 2008Brambrink T. Foreman R. Welstead G.G. Lengner C.J. Wernig M. Suh H. Jaenisch R. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells.Cell Stem Cell. 2008; 2: 151-159Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar, Woltjen et al., 2009Woltjen K. Michael I.P. Mohseni P. Desai R. Mileikovsky M. Hämäläinen R. Cowling R. Wang W. Liu P. Gertsenstein M. et al.piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.Nature. 2009; 458: 766-770Crossref PubMed Scopus (1374) Google Scholar). We therefore explored whether the initiation phase was elastic, thus allowing cells to reattain their parental gene expression profile upon OKMS removal. For this, we induced OKMS for 5 days, followed by 5 days of OKMS withdrawal (Figure 1D). Comparative analysis by PCC (Figure 1D) revealed that 2°-1B and 2°-6C parental MEFs were most similar to each other (PCC = 0.97) and to primary MEFs (PCC = 0.95 and 0.94, respectively). OKMS induction led to a divergence of the gene expression profile of 2°-1B cells (PCC = 0.86), with 3421 genes altered greater than 2-fold. Remarkably, after OKMS withdrawal, 88% of these genes reverted back to expression levels observed in the starting MEF population (Figure S2C; Figure 1D; PCC = 0.97). These results indicate that the initiation phase is not self-sustaining and is elastic, thus allowing rapid reversion to a differentiated state once OKMS expression is removed. The molecular events in the early phases of reprogramming are poorly understood. We therefore focused on the initiation phase, which is induced over the first 5 days of reprogramming, and noted induction of a large number of epithelial-associated genes. These genes include the epithelial junctional protein E-cadherin (Cdh1), as well as Cldns -3, -4, -7, -11, Occludin (Ocln), Epithelial cell adhesion molecule (Epcam), and Crumbs homolog 3 (Crb3), all of which are components of epithelial junctions (Figure 1C). These results, which were validated by qPCR (Figure 2A ), led us to investigate the expression of the mesenchymal regulators: Zn finger transcription factors Snail, Slug, Zeb1, and Zeb2. These factors maintain the mesenchymal phenotype by directly repressing epithelial gene expression (Thiery et al., 2009Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (6821) Google Scholar). Consistent with the gain of epithelial-like markers, these transcription factors were repressed in reprogramming 2°-6C cultures and paralleled the loss of the fibroblast markers Cdh2 and Thy1 (Figure 2A). These findings suggest that initiation of MEF reprogramming involves a mesenchymal-to-epithelial transition (MET). To confirm this, we initiated reprogramming for 5 days and stained early reprogramming colonies with phalloidin, which stains F-actin, together with β-catenin and Cdh1, both of which mark adherence junctions (Figure 2B). In parental MEFs, F-actin was organized into stress fibers characteristic of fibroblasts, Cdh1 expression was undetectable, and β-catenin was diffusely cytosolic and localized to puncta. In contrast, after OKMS induction, colonies with cortical F-actin and both β-catenin and Cdh1 in cell junctional regions were readily apparent, similar to fully reprogrammed 2°-6C iPSC colonies (Figure 2B, arrows). At 5 days, all colonies were positive for AP and SSEA1, both markers of early reprogramming (Figures S3A and S3B). To further confirm MET in reprogramming cells, we FACS-sorted SSEA1-positive (SSEA1+) cells 5, 8, and 11 days after OKMS induction and observed that Cdh1, Epcam, Crb3, and Ocln were all strongly induced in SSEA1+ cells (Figure 2C). We confirmed that these cells also express Nanog, Sall4, and Oct4, but not until day 8 (Figure S3C). Conversely, Snail, Slug, ZEB1, and ZEB2 were all strongly repressed. The initiation phase of fibroblast reprogramming is thus characterized by a coordinated MET that precedes the upregulation of ESC markers. To understand how the transcriptional program induced by OKMS drives the initiation phase of reprogramming, we next devised a systematic genetic RNAi screen with 2°-6C cells. To optimize assay parameters, we used Oct4 siRNA as a positive control and dharmacon control siRNA and Nanog siRNA, which is not induced until after initiation, as negative controls. After transfection, cells were cultured in the presence of Dox for 5 days, after which colonies were stained for AP, an early marker of pluripotency (Brambrink et al., 2008Brambrink T. Foreman R. Welstead G.G. Lengner C.J. Wernig M. Suh H. Jaenisch R. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells.Cell Stem Cell. 2008; 2: 151-159Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar, Okita et al., 2007Okita K. Ichisaka T. Yamanaka S. Generation of germline-competent induced pluripotent stem cells.Nature. 2007; 448: 313-317Crossref PubMed Scopus (3360) Google Scholar, Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (17010) Google Scholar), and DAPI to identify nuclei (Figure 3A ). Knockdown of Oct4, which is expressed from the integrated piggyBac transposon, inhibited colony formation (Figure S4A). In contrast, Nanog siRNA had no effect, consistent with its lack of expression at this early phase. We then developed an automated image analysis strategy to identify colonies based on the distribution of nuclei and AP counterstaining (Figure 3A) and optimized parameters via mock, control, Nanog, and Oct4 transfectants (Figure S4B). Because of the limited proliferative potential of primary cells, we were not able to conduct a genome-wide screen, so we generated a custom siRNA library that targets all signaling genes, transcription factors, and chromatin regulators (4010 genes; Table S3). Each siRNA was assessed in replicate, the results were averaged, and those with less than 15% covariance plotted (Figure 3B). Only 413 siRNA showed covariance greater than 15%, indicating excellent concordance between replicates and a robust screen. Analysis of screen data revealed that knockdown of four Yamanaka factors, Oct4, Klf4, c-Myc, and Sox2, that were present in the library suppressed reprogramming to varying extents (Figure 3B). Of note, p53 knockdown enhanced formation of colonies (Figure S4C), consistent with recent studies showing that p53 suppresses reprogramming (Zhao and Xu, 2010Zhao T. Xu Y. p53 and stem cells: New developments and new concerns.Trends Cell Biol. 2010; 20: 170-175Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). To gain mechanistic insight into key events required for progression through the initiation phase, we explored the siRNAs that suppressed or abolished colony formation. Interestingly, analysis of genes associated with MET revealed that siRNAs targeting Cdh1 and the polarity complex components, Par3 and Crb3, all strongly suppressed the appearance of AP-positive reprogramming colonies (Figure 3C). These findings suggest that MET is a functionally important early event during reprogramming. We also wanted to gain insight into signaling pathways that might regulate initiation and found that knockdown of Smad1, which is a key transcriptional mediator of signaling by BMPs, suppressed formation of AP-positive colonies (Figure 3D). Furthermore, knockdown of the Smad1 partner, Smad4, also inhibited reprogramming, as did knockdown of the BMP type II receptor, BMPRII, and the BMP type I receptor, ALK3 (Figure 3D). We confirmed that the siRNA pools to both the epithelial and BMP pathway components efficiently knocked down their targets and tested the individual siRNAs that comprise the pool, most of which efficiently reduced expression of their target and inhibited reprogramming (Figure S5). These results suggest that efficient progression through the initiation phase of reprogramming is dependent on MET and BMP signal transduction. Our functional genomics screen implicated the BMP pathway as a key regulator of reprogramming initiation, though no exogenous BMP is added to the media. However, BMP derived from serum, the cocultured MEFs, or the reprogramming cells themselves might activate the pathway. To examine this, we analyzed the receptor-activated form of Smad1 with a phosphospecific antibody, which revealed strong Smad1 activation in reprogramming MEFs that was blocked by the BMP receptor antagonist, dorsomorphin (Figure 4A ). Dorsomorphin has poor specificity, so we also used the soluble BMP ligand antagonists Noggin (Herrera and Inman, 2009Herrera B. Inman G.J. A rapid and sensitive bioassay for the simultaneous measurement of multiple bone morphogenetic proteins. Identification and quantification of BMP4, BMP6 and BMP9 in bovine and human serum.BMC Cell Biol. 2009; 10: 20Crossref PubMed Scopus (102) Google Scholar) and the extracellular domain of ALK1 (A1ECD) (David et al., 2008David L. Mallet C. Keramidas M. Lamandé N. Gasc J.M. Dupuis-Girod S. Plauchu H. Feige J.J. Bailly S. Bone morphogenetic protein-9 is a circulating vascular quiescence factor.Circ. Res. 2008; 102: 914-922Crossref PubMed Scopus (280) Google Scholar). Each alone decreased Smad1 activation, while together they strongly suppressed the pathway. Thus, intrinsic BMP signaling occurs during the initiation phase of reprogramming. We next examined whether supplementing BMP or suppressing intrinsic BMP signaling with Noggin/A1ECD-modulated reprogramming. In the absence of OKMS expression, BMP2, BMP7, or BMP9 failed to induce AP in 2°-6C MEFs, whereas they enhanced AP-positive colonies in the presence of Dox-induced OKMS (Figure S6A). Similarly, FACS analysis revealed 41% of OKMS-expressing cells were SSEA1+ by day 5, while BMP7 strongly enhanced the proportion to 68% of the population (Figures 4B and 4C). In stark contrast, only 26% of Noggin/A1ECD-treated cells were SSEA1+ and almost no SSEA1+ colonies were observed (Figures 4B and 4C). Of note, SSEA1 was not induced by BMP7 in the absence of OKMS (Figure S6B). Finally, we examined primary MEFs transfected with OKMS-expressing piggyBac transposons and observed that 2 nM BMP7 stimulated formation of reprogramming colonies by 3-fold (Figure 4D). We also confirmed that reprogrammed primary iPSCs, which were treated with BMP7 during the initiation phase, successfully produced adult chimeric mice (Figure 4E). Our results show that BMP signaling promotes the early stage of reprogramming. This was not due to regulation of proliferation as assessed by phospho-histone H3 (Figure S6Ci) and CyQuant staining (Figure S6Cii). However, Sall4, Nanog, and endogenous Oct4 all showed enhanced expression upon BMP stimulation that was reduced by BMP antagonism (Figure 5A ). Importantly, these genes were not induced by BMP in the absence of OKMS induction. BMP thus synergizes with OKMS to stimulate the onset of Nanog and Sall4 expression. Nanog is required for (Brambrink et al., 2008Brambrink T. Foreman R. Welstead G.G. Lengner C.J. Wernig M. Suh H. Jaenisch R. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells.Cell Stem Cell. 2008; 2: 151-159Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar) and Sall4 promotes (Tsubooka et al., 2009Tsubooka N. Ichisaka T. Okita K. Takahashi K. Nakagawa M. Yamanak" @default.
• W2010672807 created "2016-06-24" @default.
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• W2010672807 date "2010-07-01" @default.
• W2010672807 modified "2023-10-16" @default.
• W2010672807 title "Functional Genomics Reveals a BMP-Driven Mesenchymal-to-Epithelial Transition in the Initiation of Somatic Cell Reprogramming" @default.
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