Defining Molecular Cornerstones during Fibroblast to iPS Cell Reprogramming in Mouse

Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, 185 Cambridge Street, Boston, MA 02114, USA.
Cell stem cell (Impact Factor: 22.27). 04/2008; 2(3):230-40. DOI: 10.1016/j.stem.2008.02.001
Source: PubMed


Ectopic expression of the transcription factors Oct4, Sox2, c-Myc, and Klf4 in fibroblasts generates induced pluripotent stem (iPS) cells. Little is known about the nature and sequence of molecular events accompanying nuclear reprogramming. Using doxycycline-inducible vectors, we have shown that exogenous factors are required for about 10 days, after which cells enter a self-sustaining pluripotent state. We have identified markers that define cell populations prior to and during this transition period. While downregulation of Thy1 and subsequent upregulation of SSEA-1 occur at early time points, reactivation of endogenous Oct4, Sox2, telomerase, and the silent X chromosome mark late events in the reprogramming process. Cell sorting with these markers allows for a significant enrichment of cells with the potential to become iPS cells. Our results suggest that factor-induced reprogramming is a gradual process with defined intermediate cell populations that contain the majority of cells poised to become iPS cells.

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    • "Studies of the mechanisms that underlie somatic cell reprogramming have revealed multi-step processes involving proliferation and cell-cell adhesion, along with molecular changes such as downregulation of lineage-specific genes and eventual upregulation of pluripotency markers (Plath and Lowry, 2011). Cell-surface markers were associated with reprogramming stages such as emergence of the embryonic stem cell (ESC) marker SSEA-1 (stage-specific embryonic antigen 1) (Polo et al., 2012; Stadtfeld et al., 2008a). Secondary reprogramming systems (Woltjen et al., 2009) helped define initiation, maturation, and stabilization as key stages in reprogramming toward pluripotency (David and Polo, 2014). "
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    ABSTRACT: As the quintessential reprogramming model, OCT3/4, SOX2, KLF4, and c-MYC re-wire somatic cells to achieve induced pluripotency. Yet, subtle differences in methodology confound comparative studies of reprogramming mechanisms. Employing transposons, we systematically assessed cellular and molecular hallmarks of mouse somatic cell reprogramming by various polycistronic cassettes. Reprogramming responses varied in the extent of initiation and stabilization of transgene-independent pluripotency. Notably, the cassettes employed one of two KLF4 variants, differing only by nine N-terminal amino acids, which generated dissimilar protein stoichiometry. Extending the shorter variant by nine N-terminal amino acids or augmenting stoichiometry by KLF4 supplementation rescued both protein levels and phenotypic disparities, implicating a threshold in determining reprogramming outcomes. Strikingly, global gene expression patterns elicited by published polycistronic cassettes diverged according to each KLF4 variant. Our data expose a Klf4 reference cDNA variation that alters polycistronic factor stoichiometry, predicts reprogramming hallmarks, and guides comparison of compatible public data sets. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Stem Cell Reports 03/2015; 26(4). DOI:10.1016/j.stemcr.2015.02.004 · 5.37 Impact Factor
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    • "It is known that XCR is a late event during reprogramming to iPSCs (Payer et al., 2013; Stadtfeld et al., 2008), but the exact dynamics of XCR and how the epigenetic hallmarks of the Xi change in this process have remained unclear. Most insight into the molecular events of reprogramming to iPSCs have been gained from gene expression studies of populations of cells undergoing reprogramming and of subpopulations isolated using cell surface markers (O'Malley et al., 2013; Golipour et al., 2012; Polo et al., 2012; Samavarchi-Tehrani et al., 2010; Stadtfeld et al., 2008; Mikkelsen et al., 2008; reviewed in Buganim et al., 2013). These studies indicated that reprogramming is a multistep process with two predominant ''waves'' of gene expression changes: an early wave marked by enhanced proliferation and a mesenchymal-to-epithelial transition (MET), characterized by Cdh1 (E-cadherin) expression (Polo et al., 2012; Samavarchi-Tehrani et al., 2010; Li et al., 2010), and a late wave, characterized by reactivation of pluripotency genes such as Nanog (O'Malley et al., 2013; Buganim et al., 2012; Golipour et al., 2012; Polo et al., 2012). "
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    ABSTRACT: Reprogramming to iPSCs resets the epigenome of somatic cells, including the reversal of X chromosome inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X inactivation, whereas others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming. Copyright © 2014 Elsevier Inc. All rights reserved.
    Cell 12/2014; 159(7):1681-1697. DOI:10.1016/j.cell.2014.11.040 · 32.24 Impact Factor
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    • "However, the colonies induced by SeVdp(GKOSM) were negative for SSEA-1 as opposed to the ones induced by SeVdp(KOSM), which were positive for SSEA-1 (Figure 1B). Expression of Thy1, Snail1, Snail2, and Tgfb3 was repressed 6 days after infection in the cells induced by SeVdp(GKOSM) and SeVdp(KOSM) (Figure 1C), consistent with the downregulation of somatic cell-specific genes at the early stage of reprogramming (Stadtfeld et al., 2008). On the other hand, at day 49, expression of mesenchymal-specific genes such as Cdh2, Zeb1, Zeb2, and Twist1 remained higher in SeVdp(GKOSM)-induced cells than in SeVdp(KOSM)-induced ones (Figure 1C), indicating that the SeVdp(GKOSM)-induced cells have not completed the mesenchymal-to-epithelial transition (MET), which normally occurs during iPSC generation (Li et al., 2010; Samavarchi-Tehrani et al., 2010). "
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    ABSTRACT: The detailed mechanism of reprogramming somatic cells into induced pluripotent stem cells (iPSCs) remains largely unknown. Partially reprogrammed iPSCs are informative and useful for understanding the mechanism of reprogramming but remain technically difficult to generate in a predictable and reproducible manner. Using replication-defective and persistent Sendai virus (SeVdp) vectors, we analyzed the effect of decreasing the expression levels of OCT4, SOX2, KLF4, and c-MYC and found that low KLF4 expression reproducibly gives rise to a homogeneous population of partially reprogrammed iPSCs. Upregulation of KLF4 allows these cells to resume reprogramming, indicating that they are paused iPSCs that remain on the path toward pluripotency. Paused iPSCs with different KLF4 expression levels remain at distinct intermediate stages of reprogramming. This SeVdp-based stage-specific reprogramming system (3S reprogramming system) is applicable for both mouse and human somatic cells and will facilitate the mechanistic analysis of reprogramming.
    Stem Cell Reports 11/2014; 3(5). DOI:10.1016/j.stemcr.2014.08.014 · 5.37 Impact Factor
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