核移植与诱导多能干细胞技术在体细胞重编程研究中的应用

体细胞重编程是指分化的体细胞在特定的条件下被逆转后恢复到多能性或全能性状态,或者形成多能干细胞系,或者形成早期胚胎然后发育成一个新的个体的过程。诱导体细胞重编程的方法有许多,如核移植（nuclear transfer,NT）、细胞融合、细胞培养和通过导入特定因子获得诱导多能干（induced pluripotent stem,iPS）细胞的方法等。其中核移植和iPS技术是到目前为止诱导体细胞为多能干细胞最为完全、最具有运用于临床再生医学潜能的方法。然而,它们的效率都很低,机制也不清楚,如何将两个方法结合在一起,提高重编程的效率,揭示重编程的机制,进而促进其在患者特异性治疗中的运用将是下阶段的努力方向。     

Human-Induced Pluripotent Stem Cells: In Quest of Clinical Applications

In the field of regenerative medicine, the development of induced pluripotent stem (iPS) cells may represent a potential strategy to overcome the limitations of human embryonic stem cells (ESCs). iPS cells have the potential to mimic human disease, since they carry the genome of the donor. Hypothetically, with iPS cell technology it is possible to screen patients for a genetic cause of disease (genetic mutation), develop cell lines, reprogram them back to iPS cells, finally differentiate them into one or more cell types that develop the disease. Although the creation of multiple lineages with iPS cells can seem limitless, a number of challenges need to be addressed in order to effectively use these cell lines for disease modeling. These include the low efficiency of iPS cell generation without genetic alterations, the possibility of tumor formation in vivo, the random integration of retroviral-based delivery vectors into the genome, and unregulated growth of the remaining cells that are partially reprogrammed and refractory to differentiation. The establishment of protein or RNA-based reprogramming strategies will help generate human iPS cells without permanent genetic alterations. Finally, direct reprogramming strategies can provide rapid production of models of human ??diseases in a dish??, without first passing the cells through a pluripotent state, so avoiding the challenges of time-consumming and labor-intensive iPS cell line generation. This review will overview methods to develop iPS cells, current strategies for direct reprogramming, and main applications of iPS cells as human disease model, focusing on human cardiovascular diseases, with the aim to be a potential information resource for biomedical scientists and clinicians who exploit or intend to exploit iPS cell technology in a range of applications.     

Mouse meningiocytes express Sox2 and yield high efficiency of chimeras after nuclear reprogramming with exogenous factors

Induced pluripotent stem cell technology, also termed iPS, is an emerging approach to reprogram cells into an embryonic stem cell-like state by viral transduction with defined combinations of factors. iPS cells share most characteristics of embryonic stem cells, counting pluripotency and self-renewal, and have so far been obtained from mouse and humans, including patients with genetic diseases. Remarkably, autologous transplantation of cell lineages derived from iPS cells will eliminate the possibility of immunological rejection, as well as current ethical issues surrounding human embryonic stem cell research. However, before iPS can be used for clinical purposes, technical problems must be overcome. Among other considerations, full and homogeneous iPS reprogramming is an important prerequisite. However, despite the fact that cells from several mouse tissues can be successfully induced to iPS, the overall efficiency of chimera formation of these clones remains low even if selection for Oct4 or Nanog expression is applied. In this report, we demonstrate that cells from the mouse meningeal membranes express elevated levels of the embryonic master regulator Sox2 and are highly amenable to iPS. Meningeal iPS clones, generated without selection, are fully and homogeneously reprogrammed based on DNA methylation analysis and 100% chimera competent. Our results define a population of somatic cells that are ready to undergo iPS, thus highlighting a very attractive cell type for iPS research and application.     

Cell therapy using induced pluripotent stem cells or somatic stem cells: this is the question

A lot of effort has been developed to bypass the use of embryonic stem cells (ES) in human therapies, because of several concerns and ethical issues. Some unsolved problems of using stem cells for human therapies, excluding the human embryonic origin, are: how to regulate cell plasticity and proliferation, immunological compatibility, potential adverse side-effects when stem cells are systemically administrated, and the in vivo signals to rule out a specific cell fate after transplantation. Currently, it is known that almost all tissues of an adult organism have somatic stem cells (SSC). Whereas ES are primary involved in the genesis of new tissues and organs, SSC are involved in regeneration processes, immuno-regulatory and homeostasis mechanisms. Although the differentiating potential of ES is higher than SSC, several studies suggest that some types of SSC, such as mesenchymal stem cells (MSC), can be induced epigenetically to differentiate into tissue-specific cells of different lineages. This unexpected pluripotency and the variety of sources that they come from, can make MSC-like cells suitable for the treatment of diverse pathologies and injuries. New hopes for cell therapy came from somatic/mature cells and the discovery that could be reprogrammed to a pluripotent stage similar to ES, thus generating induced pluripotent stem cells (iPS). For this, it is necessary to overexpress four main reprogramming factors, Sox2, Oct4, Klf4 and c-Myc. The aim of this review is to analyze the potential and requirements of cellular based tools in human therapy strategies, focusing on the advantage of using MSC over iPS.     

The promise of human induced pluripotent stem cells for research and therapy

Induced pluripotent stem (iPS) cells are human somatic cells that have been reprogrammed to a pluripotent state. There are several hurdles to be overcome before iPS cells can be considered as a potential patient-specific cell therapy, and it will be crucial to characterize the developmental potential of human iPS cell lines. As a research tool, iPS-cell technology provides opportunities to study normal development and to understand reprogramming. iPS cells can have an immediate impact as models for human diseases, including cancer     

疾病特异诱导多能干细胞研究进展

通过病毒或非病毒转导体系,在小鼠和人的体细胞中人为表达几个与细胞多能性相关的转录因子,从而使细胞达到类似于胚胎干细胞（embryonic stem cells,ESCs）状态,是近年来新发展起来的体细胞重编程技术。这些被重编程的细胞称为诱导多能干细胞（induced pluripotent stem cells,iPS细胞）。这项技术为获得患者和疾病特异的多能干细胞提供了新的途径。患者和疾病特异的iPS细胞的获得,不仅在避免免疫排斥的宿主特异的细胞移植治疗上有广泛前景,并对了解疾病发生机理、药物筛选和毒性研究有着重要的意义。该文综述从iPS细胞技术的发明入手,着重讨论疾病iPS细胞的研究进展及其在应用于治疗时亟需解决的问题。     

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.     

microRNA在诱导体细胞重编程中的作用

microRNA是调控基因转录后水平的一类长度约为22个核苷酸的非编码小分子RNA。大量研究证实, microRNAs广泛分布于真核生物, 其在细胞的分化发育、生长代谢等各种活动中都起着重要的调节作用。诱导多能性干细胞(Induced pluripotent stem cell, iPS)是将体细胞诱导成为具有胚胎干细胞性质的多潜能干细胞。iPS过程的核心为体细胞表观遗传状态发生重编程, 因此, 探明体细胞重编程机制对建立完善的iPS技术具有重要理论和实际意义。利用病毒载体将Oct4、Sox2、Klf4和c-Myc等因子导入体细胞的方法已不断发展, 但“基因组整合”及原癌基因的参与增加了诱导细胞的致癌率。随着使用腺病毒、质粒或蛋白诱导等“非整合型”方法及L-myc的替换均可获得具有多潜能性的干细胞, 癌变的风险大大降低。但其发生的理论机制仍不十分清楚。最近的研究证实, microRNAs影响体细胞的重编程过程, 特别是miR302/367、miR200、miR-34和miR290/295等家族的microRNAs在体细胞诱导为iPS过程中发挥重要作用。文章就近年microRNA在诱导多能干细胞中的作用进行综述。     

诱导性多潜能干细胞(iPS cells)——现状及前景展望

主要从 iPS细胞发展历程、获得 iPS细胞的几个关键步骤 (如基因导入方式、诱导 iPS细胞所需因子组合与小分子化合物运用和体细胞种类选择等)、病人或疾病特异性 iPS细胞、iPS细胞体内外诱导分化与其衍生物的临床应用和制备无遗传修饰的(genetic modification-free) iPS细胞的可行性与前景等方面对 iPS细胞最新研究进展做评述．日本和美国研究小组先后用4种基因将小鼠(2006年8月)和人(2007年11～12月)的体细胞在体外重编程为诱导性多潜能干细胞(induced pluripotent stem cells,iPS cells),此后在短短两年多时间内,iPS 细胞的研究和关注度呈爆炸式增长．体细胞重编程、去分化和多潜能干细胞来源等一系列热点问题再次成为干细胞和发育生物学等研究的热点和焦点．与胚胎干细胞(embryonic stem cells,ES cells)一样,iPS细胞在体内可分化为3个胚层来源的所有细胞,进而参与形成机体所有组织和器官．迄今,在体外已由 iPS细胞定向诱导分化出功能性的多种成熟细胞．因此,iPS细胞研究不仅具有重要理论意义,而且在再生医学、组织工程和药物发现与评价等方面极具应用价值．     

Efficient generation of iPS cells from skeletal muscle stem cells

Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that "epigenetic memory" significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle.     

Proliferation Rate of Somatic Cells Affects Reprogramming Efficiency

The discovery of induced pluripotent stem (iPS) cells provides not only new approaches for cell replacement therapy, but also new ways for drug screening. However, the undefined mechanism and relatively low efficiency of reprogramming have limited the application of iPS cells. In an attempt to further optimize the reprogramming condition, we unexpectedly observed that removing c-Myc from the Oct-4, Sox-2, Klf-4, and c-Myc (OSKM) combination greatly enhanced the generation of iPS cells. The iPS cells generated without c-Myc attained salient pluripotent characteristics and were capable of producing full-term mice through tetraploid complementation. We observed that forced expression of c-Myc induced the expression of many genes involved in cell cycle control and a hyperproliferation state of the mouse embryonic fibroblasts during the early stage of reprogramming. This enhanced proliferation of mouse embryonic fibroblasts correlated negatively to the overall reprogramming efficiency. By applying small molecule inhibitors of cell proliferation at the early stage of reprogramming, we were able to improve the efficiency of iPS cell generation mediated by OSKM. Our data demonstrated that the proliferation rate of the somatic cell plays critical roles in reprogramming. Slowing down the proliferation of the original cells might be beneficial to the induction of iPS cells.