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。     

体细胞重编程为多能干细胞的研究进展

体细胞通过重编程转变成其他类型的细胞,在再生医学方面具有重要的应用前景。细胞重编程的方法主要有体细胞核移植、细胞融合、细胞提取物诱导、限定因子诱导等,这些方法可以不同程度地改变细胞命运。最近,限定因子诱导的多能干细胞（induced pluripotent stem cell。iPS）为重编程提供了一种崭新的方法,不仅可以避免伦理争议,还提供了一种更为便利的技术,为再生医学开辟了新的天地;同时,iPS技术为研究基因表达调控、蛋白质互作、机体生长发育等提供了一个非常重要的研究手段。本文主要论述了体细胞重编程的方法及iPS细胞的进展、面临的问题和应用前景。     

诱导多能干细胞及其在神经退行性疾病研究中的应用

用干细胞转录因子OCT4、SOX2、c-MYC和KLF4进行体细胞重编程产生具有胚胎干细胞特性的诱导多能干细胞(iPS细胞)是干细胞研究领域的突破性进展。近年来,iPS细胞的研究从产生方法、重编程机理及实际应用方面不断取得进展。由于iPS细胞的产生可取自体细胞,因而克服了胚胎干细胞应用的伦理学和免疫排斥等缺陷,为iPS细胞的临床应用开辟了广阔的前景。该文将对iPS细胞的产生方法、重编程机理及其在神经性退行性疾病的研究与应用进行文献综述,反映近几年iPS细胞最新研究成果,并阐述了用病人iPS细胞模型探讨帕金森氏病、老年性痴呆症、脊髓侧索硬化症、脊髓肌肉萎缩症及舞蹈症等5种常见神经性退行性疾病发病机理的研究现状。     

核重编程机制的影响因素

哺乳动物体细胞核移植技术在农业、生物技术、医药生产和濒危动物保护等方面具有很大的潜力和应用价值,已成为目前发育生物学研究的重要方法。但是核重编程仍是核移植技术的关键因素,制约了重构胚胎干细胞的研究。只有供核发生完全重编程,重构胚胎才能正常发育。核重编程与供核者的年龄,供核细胞的组织来源、分化状态、细胞周期、传代次数,供核细胞的表观遗传标记以及供卵者的年龄、卵子的成熟度等因素有关。创造各种适于核重编程的条件有利于从更高的起点开展核移植胚胎干细胞的研究,提高重枸胚胎干细胞建系效率。     

体细胞重编程逆转为干细胞的研究进展

目前细胞和发育生物学上的研究成果为生物医学研究提供了广泛的前景.将完全分化的细胞重编程,不经过胚胎逆转为多能干细胞状态,这点燃了再生医学应用的新希望,这一成果从法律、道德、伦理等不同方面被人们所接受.通过体细胞克隆胚胎获得干细胞所面临的破坏胚胎的伦理限制,促使研究者去寻求将分化细胞重编程逆转为干细胞的新方法.主要论述了体细胞重编程的原理、过程及不经过胚胎逆转为多能干细胞的方法.     

细胞直接重编程:从一种终末分化细胞直接重编程为另一种终末分化细胞

细胞的直接重编程是指将一种终末分化细胞直接转变为另一种终末分化细胞,这一转变不经过诱导多能干细胞阶段和去分化、再分化等过程。最近的一系列研究结果已经证明了这一研究方法的可行性,这些研究进展不仅为重编程的分子机制研究提供了新视角,也为加速重编程细胞的临床应用带来了希望。本文综述了将成纤维细胞直接重编程为神经细胞、肝细胞、心肌细胞及造血细胞的研究进展,探讨了这一研究方法存在的问题以及将来在该领域的研究方向。     

细胞融合致体细胞重编程机制研究进展

已分化的体细胞能够通过重编程转化回多能干细胞,在细胞移植、疾病细胞模型的制备以及药物筛选等领域具有重要意义。通过干细胞和体细胞的细胞融合,可使体细胞重编程。细胞融合致体细胞重编程速度快、效率高,是一种研究重编程机制的重要手段。对细胞融合致体细胞重编程的机制作一综述。     

小分子化合物诱导细胞重编程研究进展

细胞重编程指细胞内的基因表达由一种类型转变为另一种类型,通常包含两层含义:一是分化的细胞重新恢复到多能性或全能性状态;二是从一种分化的细胞转变为另一种分化的细胞。细胞重编程可为临床患者特异性细胞治疗提供无限的细胞资源。细胞重编程的途径有细胞核移植、转染特定转录因子、小分子化合物诱导等方法。核移植技术由于通常需要使用到卵子,而被认为存在伦理问题;转录因子的导入存在引起宿主基因突变的问题,限制了这一技术的临床应用。然而小分子化合物容易合成、细胞渗透性好,并且生物效应具有可塑性,使用小分子化合物诱导细胞重编程,避免了核移植的伦理问题和基因操作潜在的危害。目前,使用小分子化合物从体细胞诱导获得更安全的i PSCs(induced pluripotent stem cells),ci CMs(chemically induced functional cardiomyocyte cells)和ci NSLCs(chemical-induced neural stem cell-like cells)。对小分子化合物诱导细胞重编程,包括小分子化合物诱导多能干细胞;小分子化合物诱导潜能扩展的多能干细胞,以及小分子化合物诱导细胞转分化等方面的研究做了总结,并对小分子化合物诱导的未来发展做了展望,旨在为今后这方面的研究提供借鉴。     

全化学诱导体细胞重编程和转分化

终末分化细胞可以通过重编程和转分化转变为其他类型的细胞,对再生医学领域的研究有重要意义。化学小分子由于结构多样性,及作用剂量、时间的可控性,其在重编程和转分化领域的应用前景广阔。我国科学家在该方向的研究中也取得了许多引人瞩目的成就。对体细胞重编程和转分化的方式、小分子化合物在体细胞重编程研究领域的应用以及全化学诱导体细胞重编程和转分化研究的最新进展,尤其是我国科学家在本领域的进展进行综述。     

成纤维细胞直接重编程为心肌细胞的研究进展

最近有研究通过直接重编程的方法,采用心脏特异的转录因子和miRNA的不同组合形式,成功地将人和小鼠成纤维细胞转化为心肌样细胞,这些细胞具有与心肌相似的基因表达模式和肌节结构,甚至有少量可以跳动的细胞。直接心肌细胞重编程可将心脏原位成纤维细胞转换成有功能的心肌细胞,成为心血管再生医学的一个全新的方法。该文综述了小鼠和人类成纤维细胞在体内、外直接重编程为心肌细胞研究的发展和现状,对其研究价值、重编程方法、研究过程中的教训、存在的问题及采用这些方法获得的细胞的启示与不足进行了比较分析。     

诱导性多能干细胞向神经细胞分化的研究进展

诱导性多能干细胞(induced pluripotent stem cell,iPS cell)是通过转染外源特定的基因组合来诱导成体细胞重编程为类似于胚胎干细胞的一种多潜能干细胞,iPS细胞与胚胎干细胞不仅在形态上相似,而且在功能方面几乎相同.另外,iPS细胞的诞生克服了胚胎干细胞在临床应用时涉及的移植免疫排斥与伦理道德问题,因此具有重要的临床应用价值.目前iPS在治疗中枢神经系统性疾病方面的研究已取得很大进展,包括iPS细胞向神经细胞诱导分化方法的改进、分化机理的探索以及iPS细胞分化来源神经细胞在神经系统疾病模型中治疗作用的研究等.从iPS细胞的创建及特点、iPS细胞向神经细胞分化的诱导方法及研究新进展方面予以综述.     

A poor imitation of a natural process: A call to reconsider the iPSC engineering technique

Reprogramming somatic cells into a pluripotent state is expected to initiate a new era in medicine. Because the precise underlying mechanism of reprogramming remains unclear, many efforts have been made to optimize induced pluripotent stem cell (iPSC) engineering. However, satisfactory results have not yet been attained. In this review, we focus on recent roadblocks in iPSC reprogramming engineering, such as the inefficiency of the process, tumorigenicity and heterogeneity of the generation. We conclude that cell reprogramming is a naturally occurring phenomenon rather than a biological technique. We will only be able to mimic the natural process of reprogramming when we fully understand its underlying mechanism. Finally, we highlight the alternative method of direct conversion, which avoids the use of iPSCs to generate cell materials for patient-specific cell therapy.     

The neurosteroid dehydroepiandrosterone could improve somatic cell reprogramming

Expression of four major reprogramming transgenes, including Oct4, Sox2, Klf4 and c-myc, in somatic cells enables them to have pluripotency. These cells are iPSC (induced pluripotent stem cell) that currently show the greatest potential for differentiation into cells of the three germ lineages. One of the issues facing the successful reprogramming and clinical translation of iPSC technology is the high rate of apoptosis after the reprogramming process. Reprogramming is a stressful process, and the p53 apoptotic pathway plays a negative role in cell growth and self-renewal. Apoptosis via the p53 pathway serves as a major barrier in nuclear somatic cell reprogramming during iPSC generation. DHEA (dehydroepiandrosterone) is an abundant steroid that is produced at high levels in the adrenal cells, and withdrawal of DHEA increases the levels of p53 in the epithelial and stromal cells, resulting in increased levels of apoptotic cells; meanwhile, DHEA decreases cellular apoptosis. DHEA could improve the efficacy of reprogramming yield due to a decrease in apoptosis via the p53 pathway and an increase in cell viability.     

A poor imitation of a natural process

Reprogramming somatic cells into a pluripotent state is expected to initiate a new era in medicine. Because the precise underlying mechanism of reprogramming remains unclear, many efforts have been made to optimize induced pluripotent stem cell (iPSC) engineering. However, satisfactory results have not yet been attained. In this review, we focus on recent roadblocks in iPSC reprogramming engineering, such as the inefficiency of the process, tumorigenicity and heterogeneity of the generation. We conclude that cell reprogramming is a naturally occurring phenomenon rather than a biological technique. We will only be able to mimic the natural process of reprogramming when we fully understand its underlying mechanism. Finally, we highlight the alternative method of direct conversion, which avoids the use of iPSCs to generate cell materials for patient-specific cell therapy.     

Transitions between epithelial and mesenchymal states during cell fate conversions

Cell fate conversion is considered as the changing of one type of cells to another type including somatic cell reprogramming (de-differentiation), differentiation, and trans-differentiation. Epithelial and mesenchymal cells are two major types of cells and the transitions between these two cell states as epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) have been observed during multiple cell fate conversions including embryonic development, tumor progression and somatic cell reprogramming. In addition, MET and sequential EMT-MET during the generation of induced pluripotent stem cells (iPSC) from fibroblasts have been reported recently. Such observation is consistent with multiple rounds of sequential EMT-MET during embryonic development which could be considered as a reversed process of reprogramming at least partially. Therefore in current review, we briefly discussed the potential roles played by EMT, MET, or even sequential EMT-MET during different kinds of cell fate conversions. We also provided some preliminary hypotheses on the mechanisms that connect cell state transitions and cell fate conversions based on results collected from cell cycle, epigenetic regulation, and stemness acquisition.     

An Experimental Approach to the Generation of Human Embryonic Stem Cells Equivalents

Recently, particular attention has been paid to the human embryonic stem cells (hESC) in the context of their potential application in regenerative medicine; however, ethical concerns prevent their clinical application. Induction of pluripotency in somatic cells seems to be a good alternative for hESC recruitment regarding its potential use in tissue regeneration, disease modeling, and drug screening. Since Yamanaka’s team in 2006 restored pluripotent state of somatic cells for the first time, a significant progress has been made in the area of induced pluripotent stem cells (iPSC) generation. Here, we review the current state of knowledge in the issue of techniques applied to establish iPSC. Somatic cell nuclear transfer, cell fusion, cell extracts reprogramming, and techniques of direct reprogramming are described. Retroviral and lentiviral transduction are depicted as ways of cell reprogramming with the use of integrating vectors. Contrary to them, adenoviruses, plasmids, single multiprotein expression vectors, and PiggyBac transposition systems are examples of non-integrative vectors used in iPSC generation protocols. Furthermore, reprogramming with the delivery of specific proteins, miRNA or small chemical compounds are presented. Finally, the changes occurring during the reprogramming process are described. It is concluded that subject to some limitations iPSC could become equivalents for hESC in regenerative medicine.     

Generation of iPSCs as a Pooled Culture Using Magnetic Activated Cell Sorting of Newly Reprogrammed Cells

Although significant advancement has been made in the induced pluripotent stem cell (iPSC) field, current methods for iPSC derivation are labor intensive and costly. These methods involve manual selection, expansion, and characterization of multiple clones for each reprogrammed cell sample and therefore significantly hampers the feasibility of studies where a large number of iPSCs need to be derived. To develop higher throughput iPSC reprogramming methods, we generated iPSCs as a pooled culture using rigorous cell surface pluripotent marker selection with TRA-1-60 or SSEA4 antibodies followed by Magnetic Activated Cell Sorting (MACS). We observed that pool-selected cells are similar or identical to clonally derived iPSC lines from the same donor by all criteria examined, including stable expression of endogenous pluripotency genes, normal karyotype, loss of exogenous reprogramming factors, and in vitro spontaneous and lineage directed differentiation potential. This strategy can be generalized for iPSC generation using both integrating and non-integrating reprogramming methods. Our studies provide an attractive alternative to clonal derivation of iPSCs using rigorously selected cell pools and is amenable to automation.     

Post-irradiation promotes susceptibility to reprogramming to pluripotent state in human fibroblasts

Ionizing radiation causes not only targeted effects in cells that have been directly irradiated but also non-targeted effects in several cell generations after initial exposure. Recent studies suggest that radiation can enrich for a population of stem cells, derived from differentiated cells, through cellular reprogramming. Here, we elucidate the effect of irradiation on reprogramming, subjected to two different responses, using an induced pluripotent stem cell (iPSC) model. iPSCs were generated from non-irradiated cells, directly-irradiated cells, or cells subsequently generated after initial radiation exposure. We found that direct irradiation negatively affected iPSC induction in a dose-dependent manner. However, in the post-irradiated group, after five subsequent generations, cells became increasingly sensitive to the induction of reprogramming compared to that in non-irradiated cells as observed by an increased number of Tra1-81-stained colonies as well as enhanced alkaline phosphatase and Oct4 promoter activity. Comparative analysis, based on reducing the number of defined factors utilized for reprogramming, also revealed enhanced efficiency of iPSC generation in post-irradiated cells. Furthermore, the phenotypic acquisition of characteristics of pluripotent stem cells was observed in all resulting iPSC lines, as shown by morphology, the expression of pluripotent markers, DNA methylation patterns of pluripotency genes, a normal diploid karyotype, and teratoma formation. Overall, these results suggested that reprogramming capability might be differentially modulated by altered radiation-induced responses. Our findings provide that susceptibility to reprogramming in somatic cells might be improved by the delayed effects of non-targeted response, and contribute to a better understanding of the biological effects of radiation exposure.