Direct cell reprogramming is a stochastic process amenable to acceleration (original) (raw)
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A Molecular Roadmap of Reprogramming Somatic Cells into iPS Cells
Cell, 2012
Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming.
Deterministic direct reprogramming of somatic cells to pluripotency
Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.
Transcriptional and epigenetic mechanisms of cellular reprogramming to induced pluripotency
Epigenomics, 2016
Enforced ectopic expression of a cocktail of pluripotency-associated genes such as Oct4, Sox2, Klf4 and c-Myc can reprogram somatic cells into induced pluripotent stem cells (iPSCs). The remarkable proliferation ability of iPSCs and their aptitude to redifferentiate into any cell lineage makes these cells a promising tool for generating a variety of human tissue in vitro. Yet, pluripotency induction is an inefficient process, as cells undergoing reprogramming need to overcome developmentally imposed epigenetic barriers. Recent work has shed new light on the molecular mechanisms that drive the reprogramming of somatic cells to iPSCs. Here, we present current knowledge on the transcriptional and epigenetic regulation of pluripotency induction and discuss how variability in epigenetic states impacts iPSCs' inherent biological properties.
2014
Recent reports have proposed a new paradigm for obtaining mature somatic cell types from fibroblasts without going through a pluripotent state, by briefly expressing canonical iPSC reprogramming factors Oct4, Sox2, Klf4, c-Myc (abbreviated as OSKM) in cells expanded in lineage differentiation promoting conditions. Here we apply genetic lineage tracing for endogenous Nanog locus and X chromosome reactivation during OSKM induced trans-differentiation, as these molecular events mark final stages for acquisition of induced pluripotency. Remarkably, the majority of reprogrammed cardiomyocytes or neural stem cells derived from mouse fibroblasts via OSKM mediated trans-differentiation (~>90%), are attained after transient acquisition of pluripotency, and followed by rapid differentiation. Our findings underscore a molecular and functional coupling between inducing pluripotency and obtaining “trans-differentiated” somatic cells via OSKM induction, and have implications on defining molecu...
The Developmental Potential of iPSCs Is Greatly Influenced by Reprogramming Factor Selection
Cell Stem Cell, 2014
Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications.
Scientific Reports, 2020
The four transcription factors OCT4, SOX2, KLF4, and MYC (OSKM) together can convert human fibroblasts to induced pluripotent stem cells (iPSCs). It is, however, perplexing that they can do so only for a rare population of the starting cells with a long latency. Transcription factors (TFs) define identities of both the starting fibroblasts and the end product, iPSCs, and are also of paramount importance for the reprogramming process. It is critical to upregulate or activate the iPSC-enriched TFs while downregulate or silence the fibroblast-enriched TFs. This report explores the initial TF responses to OSKM as the molecular underpinnings for both the potency aspects and the limitation sides of the OSKM reprogramming. The authors first defined the TF reprogramome, i.e., the full complement of TFs to be reprogrammed. Most TFs were resistant to OSKM reprogramming at the initial stages, an observation consistent with the inefficiency and long latency of iPSC reprogramming. Surprisingly, ...
The potential for clinical application of pluripotent embryonic stem cells is immense but hampered by moral and ethical complications. Recent advances in the reprogramming of somatic cells by defined factors to a state that resemble embryonic stem cells have created tremendous excitement in the field. Four factors, Sox2, Oct4, Klf4 and c-Myc, when exogenously introduced into somatic cells, can lead to the formation of induced pluripotent stem (iPS) cells that have the capacity for self-renewal and differentiation into tissues of all three germ layers. In this review, we focus on the role of Krüppel-like factors (KLFs) in regulating somatic cell reprogramming. KLFs are zinc finger-containing transcription factors with diverse biological functions. We first provide an overview of the KLF family of regulatory proteins, paying special attention to the established biological and biochemical functions of KLF4 and KLF5. We then review the role of KLFs in somatic cell reprogramming and delineate the putative mechanism by which KLFs participates the establishment and self-renewal of iPS cells. Further research is likely to provide additional insight into the mechanisms of somatic cell reprogramming and refinement of the technique with which to generate clinically relevant iPS cells.
Advances in Reprogramming Somatic Cells to Induced Pluripotent Stem Cells
Stem Cell Reviews and Reports, 2010
Traditionally, nuclear reprogramming of cells has been performed by transferring somatic cell nuclei into oocytes, by combining somatic and pluripotent cells together through cell fusion and through genetic integration of factors through somatic cell chromatin. All of these techniques changes gene expression which further leads to a change in cell fate. Here we discuss recent advances in generating induced pluripotent stem cells, different reprogramming methods and clinical applications of iPS cells. Viral vectors have been used to transfer transcription factors (Oct4, Sox2, c-myc, Klf4, and nanog) to induce reprogramming of mouse fibroblasts, neural stem cells, neural progenitor cells, keratinocytes, B lymphocytes and meningeal membrane cells towards pluripotency. Human fibroblasts, neural cells, blood and keratinocytes have also been reprogrammed towards pluripotency. In this review we have discussed the use of viral vectors for reprogramming both animal and human stem cells. Currently, many studies are also involved in finding alternatives to using viral vectors carrying transcription factors for reprogramming cells. These include using plasmid transfection, piggyback transposon system and piggyback transposon system combined with a non viral vector system. Applications of these techniques have been discussed in detail including its advantages and disadvantages. Finally, current clinical applications of induced pluripotent stem cells and its limitations have also been reviewed. Thus, this review is a summary of current research advances in reprogramming cells into induced pluripotent stem cells.
Two-phase analysis of molecular pathways underlying induced pluripotent stem cell induction
Stem cells (Dayton, Ohio), 2011
Induced pluripotent stem cells (iPSCs) can be reprogrammed from adult somatic cells by transduction with Oct4, Sox2, Klf4, and c-Myc, but the molecular cascades initiated by these factors remain poorly understood. Impeding their elucidation is the stochastic nature of the iPS induction process, which results in heterogeneous cell populations. Here we have synchronized the reprogramming process by a two-phase induction: an initial stable intermediate phase following transduction with Oct4, Klf4, and c-Myc, and a final iPS phase following overexpression of Sox2. This approach has enabled us to examine temporal gene expression profiles, permitting the identification of Sox2 downstream genes critical for induction. Furthermore, we have validated the feasibility of our new approach by using it to confirm that downregulation of transforming growth factor β signaling by Sox2 proves essential to the reprogramming process. Thus, we present a novel means for dissecting the details underlying ...
Sequential Expression of Pluripotency Markers during Direct Reprogramming of Mouse Somatic Cells
Cell Stem Cell, 2008
Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4 and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox) inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, were only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step towards understanding some of the molecular events governing epigenetic reprogramming.