人诱导性多潜能干细胞(iPS细胞)系的建立

掌握建立人iPS细胞系(induced pluripotent stem cells,iPSCs)的技术,以便为人肿瘤细胞重编程为iPS细胞建立技术平台.在人胚胎干细胞的培养条件下,通过携带Oct4、Sox2、c-Myc、Klf44个混合因子的慢病毒感染人皮肤成纤维细胞(CCD-1079SK细胞),从而诱导成干细胞样的克隆.根据人胚胎干细胞的特性进行如下鉴定:克隆形态、碱性磷酸酶活性、核型和CCD-1079SK细胞来源的克隆拟胚体(embryoid bodies,EBs)形成及分化等.结果显示,在人胚胎干细胞的培养环境中,导入Oct4、Sox2、c-Myc、Klf44个因子的CCD-1079SK细胞产生了一株iPSC克隆,这株iPSC克隆在细胞形态、增殖能力、胚胎细胞特异性表面抗原以及基因表达与人胚胎干细胞相似,此外,iPSC克隆在体外悬浮培养中形成拟胚体并分化成3个胚层.人iPS细胞系的成功建立为利用iPS细胞技术开展肿瘤细胞重编程研究奠定了坚实基础.     

Derivation of Patient Specific Pluripotent Stem Cells Using Clinically Discarded Cumulus Cells

Induced pluripotent stem cells (iPSCs) are powerful tools for basic and translational research, as well as regenerative medicine. In routine human in vitro fertilization (IVF) practices, cumulus cells (CCs) are discarded, representing a potential source of biological materials for regenerative medicine. In this study, we derived patient-specific iPSCs using CCs from human infertility clinics for the first time. The human cumulus cell derived iPSCs (hc-iPSCs) were characterized for growth, karyotype, expression of pluripotency genes, and were subjected to embryoid bodies (EBs) and teratoma assays to evaluate their differentiation capacity. Hc-iPSCs display typical iPSC characteristics, and are capable of differentiating into all germ layers in vitro and in vivo. We further show that putative primordial germ cell like cells (PGCLCs) can be derived using hc-iPSCs. Our data demonstrate the feasibility of deriving patient-specific pluripotent stem cells using CCs.     

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．     

体细胞重编程为诱导多能干细胞的研究

通过逆转录病毒将4个基因(Oct4 、 Sox2、c-Myc和Klf4)导入小鼠胚胎成纤维细胞 (mouse embryonic fibroblast, MEF)中,能诱导形成胚胎干细胞样特性的诱导多能干(induced pluripotent stem, iPS)细胞.人类iPS细胞的成功构建开拓了广泛的应用前景.本文简要综述了 iPS细胞的基因筛选,转导基因的选择以及iPS细胞的表观遗传特性等.     

Integration-free Methods for Generating Induced Pluripotent Stem Cells

Induced pluripotent stem （iPS） cells by exogenous expression of four factors, Oct4, can be generated from mouse or human fibroblasts Sox2, Klf4 and c-Myc, and hold great potential for transplantation therapies and regenerative medicine. However, use of retroviral vectors during iPS cell generation has limited the techniques clinical application due to the potential risks resulting from genome integration of transgenes, including insertional mutations and altered differentiation potentials of the target cells, which may lead to pathologies such as tumorigenesis. Here we review recent progress in generating safer transgene-free or integration-free iPS cells, including the use of non-integrating vectors, excision of vectors after integration, DNA-free delivery of factors and chemical induction of pluripotency.     

外周血细胞重编程为诱导性多潜能干细胞的研究进展

诱导性多潜能干细胞(iPSCs)是指分化细胞中导入特定转录因子后逆转恢复到类似胚胎干细胞的具有自我更新、多向分化等潜能的一类细胞。诱导疾病特异性iPSCs是疾病机理、再生医学等领域的研究热点。目前,人iPSCs供体细胞主要来源于皮肤成纤维细胞,需要组织活检、体外增殖等繁琐过程。利用外周血细胞(peripheral blood cells)成功诱导iPSCs,具有取材方便、诱导快速等优点,将极大地促进iPSCs研究。该文在介绍iPSCs诱导方法的基础上,重点阐述了从小鼠B细胞、T细胞,人脐带血细胞,到人外周血细胞重编程为iPSCs的研究进展,分析了该技术的特点和可能存在的问题,并进行了前景展望。     

Overlapping Genes May Control Reprogramming of Mouse Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) and Breast Cancer Stem Cells

Recent findings suggest the possibility that tumors originate from cancer cells with stem cell properties. The cancer stem cell (CSC) hypothesis provides an explanation for why existing cancer therapies often fail in eradicating highly malignant tumors and end with tumor recurrence. Although normal stem cells and CSCs both share the capacity for self-renewal and multi-lineage differentiation, suggesting that CSC may be derived from normal SCs, the cellular origin of transformation of CSCs is debatable. Research suggests that the tightly controlled balance of self-renewal and differentiation that characterizes normal stem cell function is dis-regulated in cancer. Additionally, recent evidence has linked an embryonic stem cell (ESC)-like gene signature with poorly differentiated high-grade tumors, suggesting that regulatory pathways controlling pluripotency may in part contribute to the somatic CSC phenotype. Here, we introduce expression profile bioinformatic analyses of mouse breast cells with CSC properties, mouse embryonic stem (mES) and induced pluripotent stem (iPS) cells with an emphasis on how study of pluripotent stem cells may contribute to the identification of genes and pathways that facilitate events associated with oncogenesis. Global gene expression analysis from CSCs and induced pluripotent stem cell lines represent an ideal model to study cancer initiation and progression and provide insight into the origin cancer stem cells. Additionally, insight into the genetic and epigenomic mechanisms regulating the balance between self-renewal and differentiation of somatic stem cells and cancer may help to determine whether different strategies used to generate iPSCs are potentially safe for therapeutic use.     

诱导性多能干细胞构建策略及其提高重编程效率的方法

以Oct4、Sox2、Klf4和c-Myc等转录因子于体细胞中异位表达,可获得具有胚胎干细胞特性的诱导性多能干细胞(iPSCs),但是iPSCs技术的重编程效率和应用于临床上的安全性却都很低。目前,iPSCs的研究集中于3个方面:一是增加iPSCs技术的重编程效率;二是增加iPSCs应用于临床时的安全性;三是开创新的构建iPSCs的方法。第一个方面通过调整体细胞的表观遗传特性和细胞信号网络来达到;第二个方面可通过减少致癌性因子的使用及选择合适的载体系统来达到。而且,一些小分子化合物和调节细胞信号网络的方法也被用于诱导体细胞重编程为iPSCs。相对于仅仅使用转录因子重编程体细胞为iPSCs,使用小分子化合物或调节细胞信号网络的方法重编程体细胞为iPSCs的效率更高,而且通过这种方法获得的iPSCs的有更高的临床安全性。新构建iPSCs的方式与依赖含转录因子表达载体构建iPSCs的传统模式区别较大,它们的临床安全性或(和)重编程效率也得到了极大提高。使用4个转录因子的重组蛋白或体外合成并修饰的转录因子的mRNA已经能成功构建iPSCs;而使用miRNAs高效率重编程小鼠和人的体细胞为iPSCs的方法则开创了脱离转录因子重编程体细胞的全新策略。     

多顺反子质粒重编程人脂肪干细胞为诱导多能干细胞

为建立多顺反子质粒载体转染技术获得人脂肪干细胞(adipose stem cells,ASCs)来源的诱导多能干细胞(induced pluripotency stem cells,iPSCs),应用2A元件连接Oct4/Sox2/KLF4/c-Myc四因子基因,构建为单一开放阅读框的多顺反子质粒载体．使用该质粒对ASCs进行转染及重编程为iPSC．采用形态学观察、特异性抗体免疫荧光鉴定、体外拟胚体诱导分化和体内畸胎瘤形成等方法进行鉴定．结果显示,ASCs成功重编程为iPSCs,具有与人胚胎干细胞相似的形态学及多向分化潜能;通过拟胚体和畸胎瘤实验证实iPSCs能在体内外分化成三胚层细胞;DNA印迹实验显示质粒载体序列未整合至iPSCs基因组中．因此,通过多顺反子质粒载体重编程技术成功建立的人iPSCs具有多向分化潜能,可减免发生插入突变和免疫排斥问题,为iPSCs在遗传性或退行性疾病的治疗奠定了实验基础．     

Ultra-High-Frequency Reprogramming of Individual Long-Term Hematopoietic Stem Cells Yields Low Somatic Variant Induced Pluripotent Stem Cells

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诱导性多潜能干细胞(iPS细胞)的研究进展

通过转染特定的基因组合可以将已分化的体细胞重编程为多潜能干细胞,这种干细胞称为诱导性多潜能干细胞(induced pluripotent stem cells,iPS cells)。这是近年来干细胞研究领域最令人瞩目的一项新的干细胞制备技术。iPS细胞的出现不仅为体细胞重编程去分化机制的研究提供了新的模型,而且为疾病发生发展相关机制研究与特异的细胞治疗带来了新的希望。就当前获取iPS细胞的方法、影响iPS细胞转化率和多能性维持的一些因素及其研究进展进行综述。     

诱导多潜能干细胞(iPSCs)的研究与应用进展

诱导多潜能干细胞(induced pluripotent stem cells,iPSCs)是体细胞在外源因子作用下,经直接细胞核程序重整而重新获得多潜能的干细胞.iPSCs在疾病的模型建立与机理研究、细胞治疗、药物的发现与评价等方面有着巨大的潜在应用价值.在过去几年中,科学家们致力于改进体细胞重编程技术并取得许多突破.然而,为实现其在临床上的应用,必须克服体细胞重编程效率低和iPSCs成瘤风险两大挑战,而且重编程机制有待进一步阐明.结合iPSCs最新研究成果,评述了有关领域国内外研究进展,重点讨论当前存在问题,并展望未来研究方向.