Cellular reprogramming of somatic cells

The process of 'cell reprogramming' can be achieved by somatic cell nuclear transfer, cell fusion with embryonic stem cells, exposure to stem cell extracts, or by inducing pluripotentcy mediated by defined factors giving rise to what are termed induced pluripotent stem cells. More recently, the fate of a somatic cell can be directly induced to uptake other cell fates, termed lineage-specific reprogramming, without the need to de-differentiate the cells to a pluripotent state. In this review we will describe the different methods of reprogramming somatic cells.     

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。     

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。     

细胞抽提物诱导的体细胞重编程

体细胞重编程是指在特定条件下将已分化的体细胞去分化逆转回原始多能状态或转分化为其他类型细胞。目前诱导体细胞重编程的方法主要有：体细胞核移植、细胞融合、特定转录因子转染、细胞抽提物诱导等。近年来抽提物诱导体细胞重编程越来越受到研究者的关注。利用此法, 通过重编程不仅可以获得需要类型的细胞, 而且方便识别与重编程有关的细胞因子, 探求重编程的机制。文章简略概述诱导体细胞重编程的方法, 并重点阐述细胞抽提物诱导体细胞重编程的研究进展。     

Mechanisms of nuclear reprogramming by eggs and oocytes: a deterministic process?

Differentiated cells can be experimentally reprogrammed back to pluripotency by nuclear transfer, cell fusion or induced pluripotent stem cell technology. Nuclear transfer and cell fusion can lead to efficient reprogramming of gene expression. The egg and oocyte reprogramming process includes the exchange of somatic proteins for oocyte proteins, the post-translational modification of histones and the demethylation of DNA. These events occur in an ordered manner and on a defined timescale, indicating that reprogramming by nuclear transfer and by cell fusion rely on deterministic processes.     

Five classic articles in somatic cell reprogramming

Research on somatic cell reprogramming has progressed significantly over the past few decades, from nuclear transfer into frogs' eggs in 1952 to the derivation of human-induced pluripotent stem (iPS) cells in the present day. In this article, I review five landmark papers that have laid the foundation for current efforts to apply somatic cell reprogramming in the clinic.     

Somatic cell reprogramming for regenerative medicine: SCNT vs. iPS cells

Reprogramming of somatic cells to a pluripotent state holds huge potentials for regenerative medicine. However, a debate over which method is better, somatic cell nuclear transfer (SCNT) or induced pluripotent stem (iPS) cells, still persists. Both approaches have the potential to generate patient-specific pluripotent stem cells for replacement therapy. Yet, although SCNT has been successfully applied in various vertebrates, no human pluripotent stem cells have been generated by SCNT due to technical, legal and ethical difficulties. On the other hand, human iPS cell lines have been reported from both healthy and diseased individuals. A recent study reported the generation of triploid human pluripotent stem cells by transferring somatic nuclei into oocytes, a variant form of SCNT. In this essay, we discuss this progress and the potentials of these two reprogramming approaches for regenerative medicine.     

细胞命运转变——谱系重编程技术研究进展

体细胞核移植和诱导多能干细胞技术表明已分化的体细胞可以转变命运。最近的研究再一次验证了成熟体细胞可以通过外源转录因子的导入,直接重编程为其他类型的体细胞或祖细胞。这种重编程技术称为谱系重编程(Lineage reprogramming)。这项技术不仅在再生医学领域具有广阔的应用前景,而且在动物生物技术中也应用广泛。它不但避免了伦理争议,还提供了便利的重编程方法,同时也为基因表达调控的研究提供了重要的手段。文章从谱系重编程的方式、谱系重编程的特点及应用前景等3个方面进行了综述,旨在对相关领域的研究人员起到借鉴作用。     

microRNAs和体细胞重编程

体细胞重编程与microRNAs(miRNAs)均为近年来研究的热点问题。到目前为止,能成功诱导体细胞形成多能性干细胞的体细胞重编程方法有核移植(nuclear transfer,NT)和外源因子诱导形成多能干细胞(induced pluripotent stem cells,iPSc)两种,这两种方法让人们看到了体细胞重编程在细胞治疗方面具有诱人的应用前景。miRNAs是真核生物中存在的一类长度为22nt左右起调控作用的内源性非编码RNA,它在转录后水平调节靶基因的表达,是细胞内基因表达的基本调控机制之一。近年的研究结果表明,miRNAs在干细胞干性维持和分化过程中具有重要的调节作用,从miRNAs角度研究体细胞重编程机理将对体细胞重编程的应用具有重要意义。     

Epigenetic reprogramming: roads to pluripotency

Epigenetic reprogramming provides valuable resources for customized pluripotent stem cells generation, which are thought to be important bases of future regenerative medicine. Here we review the commonly used methods for epigenetic reprogramming: somatic cell nuclear transfer, cell fusion, cell extract treatment, inducing pluripotency by defined molecules, and briefly discuss their advantages and limitations. Finally we propose that mechanisms underlying epigenetic reprogramming and safety evaluation platform will be future research directions.     

A novel method for somatic cell nuclear transfer to mouse embryonic stem cells

Nuclear reprogramming by somatic cell nuclear transfer (SCNT) provides a practical approach for generating autologous pluripotent cells from adult somatic cells. It has been shown that murine somatic cells can also be reprogrammed to a pluripotent-like state by fusion with embryonic stem (ES) cells. Typically, the first step in SCNT involves enucleation of the recipient cell. However, recent evidence suggests that enucleated diploid ES cells may lack reprogramming capabilities. Here we have developed methods whereby larger tetraploid ES cells are first generated by fusion of two mouse ES cell lines transfected with plasmids carrying different antibiotic-resistance cassettes, followed by double antibiotic selection. Tetraploid ES cells grown on tissue culture disks or wells can be efficiently enucleated (up to 99%) using a combination of cytochalasin B treatment and centrifugation, with cytoplasts generated from these cells larger than those obtained from normal diploid ES cells. Also, we show that the enucleation rate is dependent on centrifugation time and cell ploidy. Further, we demonstrate that normal diploid ES cells can be fused to tetraploid ES cells to form heterokaryons, and that selective differential centrifugation conditions can be applied where the tetraploid nucleus is removed while the diploid donor nucleus is retained. This technology opens new avenues for generating autologous, diploid pluripotent cells, and provides a dynamic model for studying nuclear reprogramming in ES cells.     

Epigenetic regulation in pluripotent stem cells: a key to breaking the epigenetic barrier

The differentiation and reprogramming of cells are accompanied by drastic changes in the epigenetic profiles of cells. Waddington''s classical model clearly describes how differentiating cells acquire their cell identity as the developmental potential of an individual cell population declines towards the terminally differentiated state. The recent discovery of induced pluripotent stem cells as well as of somatic cell nuclear transfer provided evidence that the process of differentiation can be reversed. The identity of somatic cells is strictly protected by an epigenetic barrier, and these cells acquire pluripotency by breaking the epigenetic barrier by reprogramming factors such as Oct3/4, Sox2, Klf4, Myc and LIN28. This review covers the current understanding of the spatio-temporal regulation of epigenetics in pluripotent and differentiated cells, and discusses how cells determine their identity and overcome the epigenetic barrier during the reprogramming process.     

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.     

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.