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WIREs Dev Biol
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Mechanisms underlying the formation of induced pluripotent stem cells

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Human pluripotent stem cells (hPSCs) offer unique opportunities for studying human biology, modeling diseases, and therapeutic applications. The simplest approach so far to generate human PSC lines is through reprogramming of somatic cells from an individual by defined factors, referred to simply as reprogramming. Reprogramming circumvents the ethical controversies associated with human embryonic stem cells (hESCs) and nuclear transfer hESCs (nt‐hESCs), and the resulting induced pluripotent stem cells (hiPSCs) retain the same basic genetic makeup as the somatic cell used for reprogramming. Since the first report of iPSCs by Takahashi and Yamanaka (Cell 2006, 126:663–676), the molecular mechanisms of reprogramming have been extensively investigated. A better mechanistic understanding of reprogramming is fundamental not only to iPSC biology and improving the quality of iPSCs for therapeutic use, but also to our understanding of the molecular basis of cell identity, pluripotency, and plasticity. Here, we summarize the genetic, epigenetic, and cellular events during reprogramming, and the roles of various factors identified thus far in the reprogramming process. WIREs Dev Biol 2016, 5:39–65. doi: 10.1002/wdev.206 This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Methods and Principles Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cell Differentiation and Reversion Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cells and Disease
Sources of pluripotent stem cells. Culture‐derived pluripotent stem cells (PSCs) are generated from different in vivo cell types. (a) Embryonal carcinoma cells (ECCs), derived from germline tumors (teratocarcinomas); (b) embryonic stem cells (ESCs), derived from the inner cell mass (ICM) of preimplantation mouse and human embryos at mouse embryonic day 3.5 (mE3.5) or human embryonic day 5.5 (hE5.5); (c) epiblast stem cells (EpiSCs) and region‐selective pluripotent stem cells (rsPSCs), obtained from early postimplantation mouse embryos at mE5.5–7.5; (d) embryonic germ cells (EGCs) retrieved from mouse and human primordial germ cells (PGCs), respectively, at mE8.5–12.5 or between weeks 3 and 5 of human development (hW3‐5); and (e) germline‐derived PSCs (GSCs), derived from spermatogonial stem cells of mouse neonatal and adult testes. In each of the above columns, the cell of origin of the different pluripotent stem cell lines is labeled in blue. Alternatively, exposing the nuclei of somatic cells to exogenous reprogramming factors can induce PSCs. (f) Nuclear transfer embryonic stem cells (ntESC) are obtained by reprogramming somatic nuclei (pink) with factors contained in an enucleated oocyte (blue), and cultured to the blastocyst stage to derive ntESCs from the ICM; (g) through a similar approach, fusion between a somatic cell (pink) and a PSC (blue) gives rise to cell‐fusion‐derived tetraploid (4N) hybrid ESC (cfESC) lines; (h) alternatively, overexpression of the reprogramming transcription factors, Oct4 (O), Sox2 (S), Klf4 (K), and cMyc (M) in a somatic cell (pink) using viral delivery (blue) allows generating induced pluripotent stem cells (iPSC); and (i) F‐class cells are generated through high and constitutive expression of OSKM in a somatic cell (pink) using an inducible integrated transgene (blue). F‐class cells share molecular and phenotypic features with iPSCs and ESCs.
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Phases of reprogramming. (a) Kinetics of pluripotency‐marker appearance and definition of the reprogramming phases. Alkaline phosphatase and stage‐specific embryonic antigen 1 (SSEA1) positive cells appear around days 2 and 3, respectively, after OSKM (Oct4, Sox2, Klf4, and c‐Myc) transduction, coinciding with the downregulation of the mesenchymal marker Thy1. GFP expressed from the endogenous Oct4 or Nanog loci is detected later, around days 9 and 10, respectively. The virally transduced factors need to be expressed for approximately 12 days to generate stable, transgene‐independent induced pluripotent stem cells (iPSCs). (b) A schematic drawing illustrating the kinetics and the magnitude of the molecular changes observed during reprogramming. Permissive reprogramming cells (positive y‐axis) show a biphasic pattern of protein, mRNA, and microRNA (miRNA) expression changes following the kinetics of individual histone modifications. Bivalent domains are generated gradually after an initial burst and DNA methylation changes occur predominantly at the end of reprogramming. Refractory cells (negative y‐axis) undergo a similar wave of expression changes during the initiation phase but remain relatively stable afterward. Forced expression of OKSM in refractory cells can rescue their ability to form iPSCs. (Adapted with permission from Ref . Copyright 2012 Cell Press [Elsevier])
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Models of reprogramming. Two types of models have been proposed to describe reprogramming: deterministic and stochastic. (a–c) Deterministic models predict that somatic donor cells give rise to induced pluripotent stem cells (iPSCs) with a fixed latency (green arrows of same length). (d–f) Stochastic models predict that somatic donor cells give rise to iPSCs with variable latencies (green arrows of different lengths). In both models, either all (a and d) or only a subset of elite cells (brown) (b, c, e, and f) is permissive to reprogramming and elite cells can be present in the donor population before reprogramming (predetermined) (b and e) or induced upon viral delivery (red dots) (c and f). The latency (green arrows) reflects the absolute time or the number of cell divisions required to produce an iPSC from the donor population.
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