A Combined Epigenetic and Non-Genetic Approach for Reprogramming Human Somatic Cells

Reprogramming of somatic cells to different extents has been reported using different methods. However, this is normally accompanied by the use of exogenous materials, and the overall reprogramming efficiency has been low. Chemicals and small molecules have been used to improve the reprogramming process during somatic cell nuclear transfer (SCNT) and induced pluripotent stem (iPS) cell generation. We report here the first application of a combined epigenetic and non-genetic approach for reprogramming somatic cells, i.e., DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and human embryonic stem cell (hESC) extracts. When somatic cells were pretreated with these inhibitors before exposure to hESC (MEL1) extracts, morphological analysis revealed a higher rate of hESC-like colony formation than without pretreatment. Quantitative PCR (qPCR) demonstrated that pluripotency genes were upregulated when compared to those of somatic cells or treated with hESC extracts alone. Overall changes in methylation and acetylation levels of pretreated somatic cells suggests that epigenetic states of the cells have an effect on reprogramming efficiency induced by hESC extracts. KnockOutserum replacement (KOSR™) medium (KO-SR) played a positive role in inducing expression of the pluripotency genes. hESC extracts could be an alternative approach to reprogram somatic cells without introducing exogenous materials. The epigenetic pre-treatment of somatic cells could be used to improve the efficiency of reprogramming process. Under differentiation conditions, the reprogrammed cells exhibited differentiation ability into neurons suggesting that, although fully reprogramming was not achieved, the cells could be transdifferentiated after reprogramming.     

Regulation of Induced Pluripotent Stem (iPS) Cell Induction by Wnt/β-Catenin Signaling

Wnt signaling has been implicated in promoting somatic cell reprogramming. However, its molecular mechanisms remain unknown. Here we report that Wnt/β-catenin enhances iPSCs induction at the early stage of reprogramming. The augmented reprogramming induced by β-catenin is not due to increased total cell population or activation of c-Myc. In addition, β-catenin interacts with reprogramming factors Klf4, Oct4, and Sox2, further enhancing expression of pluripotency circuitry genes. These studies reveal novel mechanisms underlying the regulation of reprogramming somatic cells to pluripotency by Wnt/β-catenin signaling.     

定量分析诱导山羊体细胞重编程过程中端粒酶的表达变化

动物体细胞重编程为诱导多能干细胞(iPS)是目前干细胞生物学研究的热点。文中重点对山羊体细胞重编程过程中端粒酶(TERT)基因的相对表达量进行了检测,探讨了山羊重编程细胞的形成与端粒酶基因表达的关系。从关中奶山羊胎儿皮肤分离得到的胎儿成纤维细胞(GEF),其增殖能力较强,核型正常(60条XY),通过转录因子在体外诱导得到山羊重编程细胞。利用Real-timeRT-PCR方法首先对关中奶山羊胎儿各种组织的TERT表达进行了检测,结果表明睾丸组织中TERT的表达显著高于上皮组织(P0.01),在山羊胎儿的其他组织中TERT也有不同程度的表达。对原代重编程细胞和4株不完全重编程细胞株的TERT表达检测结果发现,碱性磷酸酶(AP)阳性的重编程细胞端粒酶表达量要显著高于AP阴性的重编程细胞(P0.01)。这一结果揭示,激活端粒酶活性并使其保持较高的表达水平对体细胞的重编程至关重要。     

Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts

The expression of 4 pluripotency genes (Oct4, Sox2, c-Myc and Klf4) in mouse embryonic fibroblasts can reprogramme them to a pluripotent state. We have investigated the expression of these pluripotency genes when human somatic 293T cells are permeabilized and incubated in extracts of mouse embryonic stem (ES) cells. Expression of all 4 genes was induced over 1–8 h. Gene expression was associated with loss of repressive histone H3 modifications and increased recruitment of RNA polymerase II at the promoters. Lamin A/C, which is typically found only in differentiated cells, was also removed from the nuclei. When 293T cells were returned to culture after exposure to ES cell extract, the expression of the pluripotency genes continued to rise over the following 48 h of culture, suggesting that long-term reprogramming of gene expression had been induced. This provides a methodology for studying the de-differentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells to a pluripotent state without genetically altering them.     

Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells

We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated "all-iPSC mice" by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating "all-iPSC mice" was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a "generic" epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines.     

The Epigenetic Reprogramming Roadmap in Generation of iPSCs from Somatic Cells

Reprogramming of somatic cells to induced pluripotent stem cells(iPSCs) is a comprehensive epigenetic process involving genome-wide modifications of histones and DNA methylation. This process is often incomplete, which subsequently affects i PSC reprograming,pluripotency, and differentiation capacity. Here, we review the epigenetic changes with a focus on histone modification(methylation and acetylation) and DNA modification(methylation) during i PSC induction. We look at changes in specific epigenetic signatures, aberrations and epigenetic memory during reprogramming and small molecules influencing the epigenetic reprogramming of somatic cells. Finally,we discuss how to improve i PSC generation and pluripotency through epigenetic manipulations.     

Identification of spectral modifications occurring during reprogramming of somatic cells

Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However, research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly, this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose.     

Molecular insights into the heterogeneity of telomere reprogramming in induced pluripotent stem cells

Rejuvenation of telomeres with various lengths has been found in induced pluripotent stem cells (iPSCs). Mechanisms of telomere length regulation during induction and proliferation of iPSCs remain elusive. We show that telomere dynamics are variable in mouse iPSCs during reprogramming and passage, and suggest that these differences likely result from multiple potential factors, including the telomerase machinery, telomerase-independent mechanisms and clonal influences including reexpression of exogenous reprogramming factors. Using a genetic model of telomerase-deficient (Terc(-/-) and Terc(+/-)) cells for derivation and passages of iPSCs, we found that telomerase plays a critical role in reprogramming and self-renewal of iPSCs. Further, telomerase maintenance of telomeres is necessary for induction of true pluripotency while the alternative pathway of elongation and maintenance by recombination is also required, but not sufficient. Together, several aspects of telomere biology may account for the variable telomere dynamics in iPSCs. Notably, the mechanisms employed to maintain telomeres during iPSC reprogramming are very similar to those of embryonic stem cells. These findings may also relate to the cloning field where these mechanisms could be responsible for telomere heterogeneity after nuclear reprogramming by somatic cell nuclear transfer.     

Epigenetic predisposition to reprogramming fates in somatic cells

Reprogramming to pluripotency is a low‐efficiency process at the population level. Despite notable advances to molecularly characterize key steps, several fundamental aspects remain poorly understood, including when the potential to reprogram is first established. Here, we apply live‐cell imaging combined with a novel statistical approach to infer when somatic cells become fated to generate downstream pluripotent progeny. By tracing cell lineages from several divisions before factor induction through to pluripotent colony formation, we find that pre‐induction sister cells acquire similar outcomes. Namely, if one daughter cell contributes to a lineage that generates induced pluripotent stem cells (iPSCs), its paired sibling will as well. This result suggests that the potential to reprogram is predetermined within a select subpopulation of cells and heritable, at least over the short term. We also find that expanding cells over several divisions prior to factor induction does not increase the per‐lineage likelihood of successful reprogramming, nor is reprogramming fate correlated to neighboring cell identity or cell‐specific reprogramming factor levels. By perturbing the epigenetic state of somatic populations with Ezh2 inhibitors prior to factor induction, we successfully modulate the fraction of iPSC‐forming lineages. Our results therefore suggest that reprogramming potential may in part reflect preexisting epigenetic heterogeneity that can be tuned to alter the cellular response to factor induction.     

Nanog overcomes reprogramming barriers and induces pluripotency in minimal conditions

Induced pluripotency requires the expression of defined factors and culture conditions that support the self-renewal of embryonic stem (ES) cells. Small molecule inhibition of MAP kinase (MEK) and glycogen synthase kinase 3 (GSK3) with LIF (2i/LIF) provides an optimal culture environment for mouse ES cells and promotes transition to naive pluripotency in partially reprogrammed (pre-iPS) cells. Here we show that 2i/LIF treatment in clonal lines of pre-iPS cells results in the activation of endogenous Nanog and rapid downregulation of retroviral Oct4 expression. Nanog enables somatic cell reprogramming in serum-free medium supplemented with LIF, a culture condition which does not support induced pluripotency or the self-renewal of ES cells, and is sufficient to reprogram epiblast-derived stem cells to naive pluripotency in serum-free medium alone. Nanog also enhances reprogramming in cooperation with kinase inhibition or 5-aza-cytidine, a small molecule inhibitor of DNA methylation. These results highlight the capacity of Nanog to overcome multiple barriers to reprogramming and reveal a synergy between Nanog and chemical inhibitors that promote reprogramming. We conclude that Nanog induces pluripotency in minimal conditions. This provides a strategy for imposing naive pluripotency in mammalian cells independently of species-specific culture requirements.     

Application of induced pluripotency in cancer studies

As soon as induced pluripotent stem cells (iPSCs) reprogramming of somatic cells were developed, the discovery attracted the attention of scientists, offering new perspectives for personalized medicine and providing a powerful platform for drug testing. The technology was almost immediately applied to cancer studies. As presented in this review, direct reprogramming of cancer cells with enforced expression of pluripotency factors have several basic purposes, all of which aim to explain the complex nature of cancer development and progression, therapy-resistance and relapse, and ultimately lead to the development of novel anti-cancer therapies. Here, we briefly present recent advances in reprogramming methodologies as well as commonalities between cell reprogramming and carcinogenesis and discuss recent outcomes from the implementation of induced pluripotency into cancer research.