Current concepts in reprogramming somatic cells to pluripotent state

Recently considerable interests have been roused in nuclear reprogramming by somatic cell nuclear transfer using an egg cytoplasm and/or by other means, such as fusion, cell extracts treatment and genes transfections. However, the very mechanism of reprogramming still remains elusive. Epigenetic modifications, which play a significant role in normal mammalian development in vivo is also involved in the process of reprogramming in vitro. The latter shares some of the other features observed in nuclear reprogramming in vivo. In this review, we discuss the main epigenetic changes involved in nuclear reprogramming and currently available approaches to achieve nuclear reprogramming in vitro and its future prospects.     

细胞重编程与表观遗传学调控

细胞重编程是生命科学研究的热点之一,目前体细胞核移植、细胞融合和特定转录因子诱导等方法都可以实现体外细胞重编程,而在细胞重编程过程中表观遗传学发挥关键的调控作用,因此对重编程过程中表观遗传学调控机制开展深入研究具有重要的意义。本文简要综述细胞重编程的研究现状和表观遗传学调控细胞重编程机制的研究进展,并对小分子化合物和microRNA提高细胞重编程效率的最新进展进行了介绍。     

Epigenetic reprogramming in mouse pre-implantation development and primordial germ cells

Epigenetic modifications are crucial for the identity and stability of cells, and, when aberrant, can lead to disease. During mouse development, the genome-wide epigenetic states of pre-implantation embryos and primordial germ cells (PGCs) undergo extensive reprogramming. An improved understanding of the epigenetic reprogramming mechanisms that occur in these cells should provide important new information about the regulation of the epigenetic state of a cell and the mechanisms of induced pluripotency. Here, we discuss recent findings about the potential mechanisms of epigenetic reprogramming, particularly genome-wide DNA demethylation, in pre-implantation mouse embryos and PGCs.     

体细胞核移植后表观遗传重编程的异常及其修复

体细胞核移植(Somatic cell nuclear transfer, SCNT)是指将高度分化的体细胞移入到去核的卵母细胞中发育并最终产生后代的技术。然而, 体细胞克隆的总体效率仍然处于一个较低的水平, 主要原因之一是由于体细胞供体核不完全的表观遗传重编程, 包括DNA甲基化、组蛋白乙酰化、基因组印记、X染色体失活和端粒长度等修饰出现的异常。使用一些小分子化合物以及Xist基因的敲除或敲低等方法能修复表观遗传修饰错误, 辅助供体核的重编程, 从而提高体细胞克隆效率, 使其更好地应用于基础研究和生产实践。文章对体细胞核移植后胚胎发育过程中出现的异常表观遗传修饰进行了综述, 并着重论述了近年来有关修复表观遗传错误的研究进展。     

细胞重编程中的表观遗传分子机制

成体细胞可以通过核移植、细胞融合或者特定因子导入的方式实现重编程回到多能性状态。在重编程的过程中,表观遗传水平的调控机制起到了非常关键的作用。通过回顾重编程的研究进展来探讨表观遗传学在重编程中的调控机制。     

A novel variant of Oct3/4 gene in mouse embryonic stem cells

OCT4 is a highly conserved gene and plays an important role during early embryonic development and differentiation. Similar to human OCT4, mouse Oct4 gene generates variants. Oct4A is a master regulator of self-renewal in pluripotent stem cells. In this study, we have identified a novel Oct4 spliced variant, designated mouse Oct4B, encoding 3 isoforms, termed Oct4B-247aa, Oct4B-190aa and Oct4B-164aa. Furthermore, we have examined the expression pattern of these isoforms in non-pluripotent cells and their function in somatic cell reprogramming. The results revealed the isoforms 247aa, 164aa localized mainly in nucleus and 190aa expressed dotted in the cytoplasm. In contrast to Oct4A, Oct4B does not function in somatic reprogramming as that of Oct4A. Taken together, our data for first time described the intact coding sequence of mouse Oct4B and its function in somatic cell reprogramming. These findings will be important for further analysis of the epigenetic mechanisms of reprogramming and highlight the necessity of discriminating Oct4 isoforms in future stem cell research.     

Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers

In mammalian development, epigenetic modifications, including DNA methylation patterns, play a crucial role in defining cell fate but also represent epigenetic barriers that restrict developmental potential. At two points in the life cycle, DNA methylation marks are reprogrammed on a global scale, concomitant with restoration of developmental potency. DNA methylation patterns are subsequently re-established with the commitment towards a distinct cell fate. This reprogramming of DNA methylation takes place firstly on fertilization in the zygote, and secondly in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. In each reprogramming window, a unique set of mechanisms regulates DNA methylation erasure and re-establishment. Recent advances have uncovered roles for the TET3 hydroxylase and passive demethylation, together with base excision repair (BER) and the elongator complex, in methylation erasure from the zygote. Deamination by AID, BER and passive demethylation have been implicated in reprogramming in PGCs, but the process in its entirety is still poorly understood. In this review, we discuss the dynamics of DNA methylation reprogramming in PGCs and the zygote, the mechanisms involved and the biological significance of these events. Advances in our understanding of such natural epigenetic reprogramming are beginning to aid enhancement of experimental reprogramming in which the role of potential mechanisms can be investigated in vitro. Conversely, insights into in vitro reprogramming techniques may aid our understanding of epigenetic reprogramming in the germline and supply important clues in reprogramming for therapies in regenerative medicine.     

Natural and artificial routes to pluripotency

Pluripotent cells of the blastocyst inner cell mass (ICM) and their in vitro derivatives, embryonic stem (ES) cells, contain genomes in an epigenetic state that are poised for subsequent differentiation. Their chromatin is hyperdynamic in nature and relatively uncondensed. In addition, a large number of genes are expressed at low levels in both ICM and ES cells. Also, the chromatin of naturally pluripotent cells contains specialized histone modification patterns such as bivalent domains, which mark genes destined for later developmentally-regulated expression states. Female pluripotent cells contain X chromosomes that have yet to undergo the process of X chromosome inactivation. Collectively, these features of very early embyronic chromatin are required for the successful specification and production of differentiated cell lineages. Artificial reprogramming methods such as somatic nuclear transfer (SCNT), ES cell fusion-mediated reprogramming (FMR), and induced pluripotency (iPS) yield pluripotent cells that recapitulate many features of naturally pluripotent cells, including many of their epigenetic features. However, the route to pluripotent epigenomic states in artificial pluripotent cells differs drastically from that of their natural counterparts. Here, we compare and contrast the differing routes to pluripotency under natural and artificial conditions. In addition, we discuss the intrinsically metastable nature of the pluripotent epigenome and consider epigenetic aspects of reprogramming that may lead to incomplete or inaccurate reprogrammed states. Artificial methods of reprogramming hold immense promise for the development of autologous cell graft sources and for the development of cell culture models for human genetic disorders. However, the utility of artificially reprogrammed cells is highly dependent on the fidelity of the reprogramming process and it is therefore critically important to assess the epigenetic similarities between embryonic and induced pluripotent stem cells.     

Parental epigenetic control of embryogenesis: a balance between inheritance and reprogramming?

At fertilization, fusion of two differentiated gametes forms the zygote that is capable of forming all of the varied cell lineages of an organism. It is widely thought that the acquisition of totipotency involves extensive epigenetic reprogramming of the germline state into an embryonic state. However, recent data argue that this reprogramming is incomplete and that substantial epigenetic information passes from one generation to the next. In this review we summarize the changes in chromatin states that take place during mammalian gametogenesis and examine the evidence that early mammalian embryogenesis may be affected by inheritance of epigenetic information from the parental generation.     

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.     

Epigenetic reprogramming in mammalian nuclear transfer

With the exception of lymphocytes, the various cell types in a higher multicellular organism have basically an identical genotype but are functionally and morphologically different. This is due to tissue-specific, temporal, and spatial gene expression patterns which are controlled by genetic and epigenetic mechanisms. Successful cloning of mammals by transfer of nuclei from differentiated tissues into enucleated oocytes demonstrates that these genetic and epigenetic programs can be largely reversed and that cellular totipotency can be restored. Although these experiments indicate an enormous plasticity of nuclei from differentiated tissues, somatic cloning is a rather inefficient and unpredictable process, and a plethora of anomalies have been described in cloned embryos, fetuses, and offspring. Accumulating evidence indicates that incomplete or inappropriate epigenetic reprogramming of donor nuclei is likely to be the primary cause of failures in nuclear transfer. In this review, we discuss the roles of various epigenetic mechanisms, including DNA methylation, chromatin remodeling, imprinting, X chromosome inactivation, telomere maintenance, and epigenetic inheritance in normal embryonic development and in the observed abnormalities in clones from different species. Nuclear transfer represents an invaluable tool to experimentally address fundamental questions related to epigenetic reprogramming. Understanding the dynamics and mechanisms underlying epigenetic control will help us solve problems inherent in nuclear transfer technology and enable many applications, including the modulation of cellular plasticity for human cell therapies.     

综述: 诱导重编程过程中的表观遗传重塑

多能干细胞(pluripotent stem cell,PSC)是一类具有自我更新能力和多向分化潜能的细胞,具有广泛的临床应用前景．诱导性多功能干细胞(induced pluripotent stem cell,iPS cell)的获得,解决了传统方式中的细胞来源和伦理学等问题,从理论研究和应用上实现了体细胞重编程的重大突破,也为疾病发生机制研究、药物筛选、个性化药物选择、细胞治疗和再生医学等研究创造了难得的机会,从而开启了多能干细胞应用的新纪元．iPS过程中有很多问题尚未得到解决,尤其是诱导重编程的分子机制方面,这也是近年来干细胞领域研究的热点．其中如何实现表观遗传的重编程被认为是亟待解决的核心问题之一．本文结合我们的研究,主要介绍诱导重编程领域表观遗传修饰重塑机制的研究进展,并展望未来研究中大规模信息整合分析的重要性．     

Regulation of somatic cell reprogramming through inducible mir-302 expression

Global demethylation is required for early zygote development to establish stem cell pluripotency, yet our findings reiterate this epigenetic reprogramming event in somatic cells through ectopic introduction of mir-302 function. Here, we report that induced mir-302 expression beyond 1.3-fold of the concentration in human embryonic stem (hES) H1 and H9 cells led to reprogramming of human hair follicle cells (hHFCs) to induced pluripotent stem (iPS) cells. This reprogramming mechanism functioned through mir-302-targeted co-suppression of four epigenetic regulators, AOF2 (also known as KDM1 or LSD1), AOF1, MECP1-p66 and MECP2. Silencing AOF2 also caused DNMT1 deficiency and further enhanced global demethylation during somatic cell reprogramming (SCR) of hHFCs. Re-supplementing AOF2 in iPS cells disrupted such global demethylation and induced cell differentiation. Given that both hES and iPS cells highly express mir-302, our findings suggest a novel link between zygotic reprogramming and SCR, providing a regulatory mechanism responsible for global demethylation in both events. As the mechanism of conventional iPS cell induction methods remains largely unknown, understanding this microRNA (miRNA)-mediated SCR mechanism may shed light on the improvements of iPS cell generation.