Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.     

Selective Development of Myogenic Mesenchymal Cells from Human Embryonic and Induced Pluripotent Stem Cells

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are promising sources for the cell therapy of muscle diseases and can serve as powerful experimental tools for skeletal muscle research, provided an effective method to induce skeletal muscle cells is established. However, the current methods for myogenic differentiation from human ES cells are still inefficient for clinical use, while myogenic differentiation from human iPS cells remains to be accomplished. Here, we aimed to establish a practical differentiation method to induce skeletal myogenesis from both human ES and iPS cells. To accomplish this goal, we developed a novel stepwise culture method for the selective expansion of mesenchymal cells from cell aggregations called embryoid bodies. These mesenchymal cells, which were obtained by dissociation and re-cultivation of embryoid bodies, uniformly expressed CD56 and the mesenchymal markers CD73, CD105, CD166, and CD29, and finally differentiated into mature myotubes in vitro. Furthermore, these myogenic mesenchymal cells exhibited stable long-term engraftment in injured muscles of immunodeficient mice in vivo and were reactivated upon subsequent muscle damage, increasing in number to reconstruct damaged muscles. Our simple differentiation system facilitates further utilization of ES and iPS cells in both developmental and pathological muscle research and in serving as a practical donor source for cell therapy of muscle diseases.     

人胚胎干细胞和诱导型人多能干细胞分化的心肌细胞在药物心脏毒性筛选中的应用

心脏毒性是药物研发失败的主要原因之一,也是临床前安全评价研究的难题之一。人胚胎干细胞和诱导型人多能干细胞均具有无限增殖、自我更新和多向分化的特性,为体外心脏毒性筛选实验提供了细胞资源。人胚胎干细胞和诱导型人多能干细胞诱导分化的心肌细胞相似,具有相同的形态结构,且随着培养时间的推移,功能性心、Na^＋、Ca^2＋通道密度逐渐增加、心肌特异性基因ANF、α—MHC、MLC-2α的表达量增加,具有相似的动作电位时程和收缩性等特点,相当于幼稚型心肌细胞。将它们应用于已知作用药物的心脏毒性筛选,检测心肌细胞离子通道、动作电位、心脏损伤标志物、收缩功能的变化,获得与临床相似的结果。因此,建立人胚胎干细胞和诱导型人多能干细胞诱导分化心肌细胞的体外评价模型,大大减少了药物研发的时间和成本,克服了种属间的差异,推动了心脏毒性体外评价方法的发展。     

诱导多能干细胞的研究进展

体细胞诱导成为多能性干细胞(induced pluripotent stem cell,iPS cell)的研究成果被国际生命科学界誉为具有里程碑意义的创新之举.在短短3年多的时间里,这项研究已经在细胞重编程的机理研究、探索疾病的发生发展机制以及临床医学的应用等领域引发了很多突破性的进展,而且,这一非克隆干细胞技术的诞生,成功地避开了长期以来争论不休的伦理问题,极大地推动该领域和相关科学领域的发展.从iPS细胞的研究历程、iPS细胞的构建机理、iPS细胞研究的最新应用成果以及iPS细胞的发展前景和研究方向等方面进行了评.     

Pluripotency of Induced Pluripotent Stem Cells

Induced pluripotent stem （iPS） cells can be generated by forced expression of four pluripotency factors in somatic cells. This has received much attention in recent years since it may offer us a promising donor cell source for cell transplantation therapy. There has been great progress in iPS cell research in the past few years. However, several issues need to be further addressed in the near future before the clinical application of iPS cells, like the immunogenieity of iPS cells, the variability of differentiation potential and most importantly tumor formation of the iPS derivative cells. Here, we review recent progress in research into the pluripotency of iPS cells.     

Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

Background

Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored.

Methodology/Principal Findings

We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation.

Conclusions/Significance

Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance.     

Induced Pluripotent Stem Cells Are Sensitive to DNA Damage

Induced pluripotent stem cells(iPSCs)resemble embryonic stem cells(ESCs)in morphology,gene expression and in vitro differentiation,raising new hope for personalized clinical therapy.While many efforts have been made to improve reprogramming effciency,signifcant problems such as genomic instability of iPSCs need to be addressed before clinical therapy.In this study,we try to fgure out the real genomic state of iPSCs and their DNA damage response to ionizing radiation(IR).We found that iPSC line 3FB4-1 had lower DNA damage repair ability than mouse embryonic fbroblast(MEF)cells,from which 3FB4-1line was derived.After the introduction of DNA damage by IR,the number of c-H2AX foci in 3FB4-1 increased modestly compared to a large increase seen in MEF,albeit both signifcantly(P<0.01).In addition,whole-genome sequencing analysis showed that after IR,3FB4-1 possessed more point mutations than MEF and the point mutations spread all over chromosomes.These observations provide evidence that iPSCs are more sensitive to ionizing radiation and their relatively low DNA damage repair capacity may account for their high radiosensitivity.The compromised DNA damage repair capacity of iPSCs should be considered when used in clinical therapy.     

诱导多功能干细胞的影响因素

将体细胞诱导为多功能干细胞为人类的再生医学提供了一个全新的研究手段,从而可以不用损坏胚胎就能获得可用于治疗各种特殊疾病的细胞。本文比较了近年来关于生成诱导性多能干细胞(induced pluripotent stem cells,iPS细胞)的诱导方法及重编程效率,总结了这些方法的共同点;另外通过对每个不同试验过程的影响因素进行比较,归纳了影响iPS细胞重编程过程的几个因素。     

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。     

Enhanced Telomere Rejuvenation in Pluripotent Cells Reprogrammed via Nuclear Transfer Relative to Induced Pluripotent Stem Cells

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人类及小鼠胚胎干细胞的不同多能性状态

胚胎干细胞(embryonic stem cells,ESCs)是来源于早期胚胎的全能性细胞,在合适条件下具有分化为任何一类成体细胞的潜力。在小鼠中,根据细胞来源的胚胎发育时间,ESCs可以被分为原始态多能性(na(?)ve pluripotency)和始发态多能性(primed pluripotency)两种状态。这两种状态的细胞在发育上相互联系,具有不同的形态、信号依赖、发育性质、基因表达及表观遗传学性质,并且在特定的条件下可以相互转化。人类胚胎干细胞(human embryonic stem cells,hESCs)的发育潜能曾一度被认为低于小鼠胚胎干细胞(mouse embryonic stem cells,mESCs),直到人类原始态胚胎干细胞的发现证明了hESCs可以表现出与mESCs相似的性质。这对于人类胚胎发育的研究及ESCs在临床治疗上的实际应用都具有重要的意义。     

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)¹. While improvements in several gene delivery methods have been described^2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor¹¹, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.     

诱导性多能干细胞与肝样细胞分化

肝脏疾病正逐渐成为全球棘手的医疗问题。肝细胞是肝脏生理活动的主要承担者,在肝脏疾病的研究以及药物的研发和测试方面有着举足轻重的作用。然而,体外分离培养的原代肝细胞面临在体外不能无限增殖和稳定表达肝脏特异基因等问题。有强大的自我更新能力和三胚层分化潜能的诱导性多能肝细胞(iPSCs)能被诱导因子、外源基因和小分子化合物等定向诱导分化为功能性肝细胞。同时,还避免了伦理、宗教以及免疫排斥等诸多问题。本文简要综述了从不同策略诱导iPSCs成为功能性肝细胞的研究方法和成果,并对该领域进行小结和展望。