Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst

Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.     

Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer

Embryonic stem (ES) cells are fully pluripotent in that they can differentiate into all cell types, including gametes. We have derived 35 ES cell lines via nuclear transfer (ntES cell lines) from adult mouse somatic cells of inbred, hybrid, and mutant strains. ntES cells contributed to an extensive variety of cell types, including dopaminergic and serotonergic neurons in vitro and germ cells in vivo. Cloning by transfer of ntES cell nuclei could result in normal development of fertile adults. These studies demonstrate the full pluripotency of ntES cells.     

诱导多能干细胞的研究进展

通过转入特定基因能诱导体细胞重编程成为多能干细胞(Induced Pluripotent Stem Cells,iPSCs),这项技术为干细胞的研究和应用带来了革命性的变化,对于解决长期困扰干细胞研究领域的伦理困境和免疫排斥等问题具有重大意义。然而,目前iPSCs技术本身还不完善,面临致癌性、效率低、以病毒为载体的安全性问题以及移植后存活率低等诸多问题。主要从iPSCs的产生和筛选、应用前景及其存在的问题等3个方面进行了综述,以期为今后关于这方面的研究提供参考。     

胚胎干细胞与胚胎生殖细胞

胚胎干细胞与胚胎生殖细胞同属于胚胎源的多潜能干细胞。胚胎干细胞是从附置前胚胎内细胞团分离而来的,而胚胎生殖细胞来自胎儿原始生殖细胞。二者在现代生命科学的各个领域都有着广阔的应用前景。对胚胎生殖细胞的特性,胚胎干细胞与胚胎生殖细胞的异同以及二者的应用前景作一综述。     

不同来源山羊体细胞重编程效率比较

[目的]检测克隆奶山羊(♀12003)和转基因克隆奶山羊(♂12001)作为供体细胞,对体细胞重编程效率的影响。[方法]采用慢病毒作为载体,携带多能因子来感染正常山羊(g2)、♀12003和♂12001体细胞,统计克隆形成率和碱性磷酸酶阳性克隆率。[结果]与普通山羊(g2)相比,转基因克隆奶山羊耳成纤维细胞的重编程效率低,出现的细胞克隆在传代之前容易分化;克隆奶山羊体细胞与普通山羊体细胞的重编程效率接近,转基因克隆奶山羊(♂12001)的重编程效率极显著低于克隆奶山羊(♀12003)和普通山羊(P0.01)。[结论]转基因克隆奶山羊体细胞可以被重编程为诱导性多能干细胞(Induced pluripotent stem cells,iPSCs),但效率较低。     

抑制体细胞衰老促进诱导性多潜能干细胞（iPSC）生成的研究进展

诱导性多潜能干细胞（induced pluripotent stem cell, iPSC）是指对体细胞实施特定的诱导方法,将体细胞重编程获得的多潜能干细胞。最常用的诱导方法是利用基因导入技术,将与胚胎干细胞（embryonic stem cell, ESC）多潜能性相关基因的转录因子导入体细胞,激活内源多能性基因的表达,实现体细胞重编程。除转基因外,使用某些小分子物质或蛋白质等也可以实现体细胞重编程。iPSC与ESC在形态学、表观遗传学和分化能力上高度相似。由于iPSC来源于普通成体细胞,避免了胚胎干细胞面临的伦理道德和免疫排斥问题,使得这一技术在再生医学和畜牧生产上都具有广阔的应用前景。然而,iPSC的低生成率和高风险性逐渐成为制约该技术发展的主要障碍。生成效率和速率低,大大增加了获得iPSC的难度,工作量大且成本高;高风险性使iPSC无法安全应用于再生医学和生产转基因动物。这成为iPSC科研工作者亟待解决的两大问题。文章介绍了通过抑制体细胞衰老来促进iPSC生成的相关研究进展。该方法对体细胞重编程有显著效果,但同时也存在较大的安全性争议。控制细胞衰老凋亡的Ink4a/Arf位点,p53、pRB、p21等基因和蛋白因子可以及时清除体内损伤细胞,促进细胞衰老凋亡,抑制细胞癌变,组成了维护机体健康的重要调控通路。近年研究表明,抑制促细胞衰老凋亡基因的表达可显著提高iPSC生成的效率和速率,说明肿瘤发生和iPSC生成存在某些相同的调控通路。抑制体细胞衰老的方法主要有3类：改进培养液成分、使用新的转录因子和调节细胞培养环境。通过抑制体细胞衰老,最高可将小鼠iPSC生成效率提高到100%。这为高效获得iPSC,探索iPSC及肿瘤的生成机制提供了新思路。同时,此方法存在较大安全性问题。iPSC最初是由Oct4,Sox2,Klf4,c-Myc等4种转录因子诱导而来,这些因子本身都存在致癌风险。抑癌基因的失活使获得的iPSC致癌风险增大,成为制约该方法广泛应用的最主要障碍。文章概括了2008年以来通过抑制体细胞衰老促进iPSC生成的研究进展、存在问题和应用前景。     

Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons

The generation of pluripotent stem cells from an individual patient would enable the large-scale production of the cell types affected by that patient's disease. These cells could in turn be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. Although recent studies have demonstrated the reprogramming of human fibroblasts to a pluripotent state, it remains unclear whether these induced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease. We have generated iPS cells from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS). These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiate into motor neurons, the cell type destroyed in ALS.     

The past,present and future of bovine pluripotent stem cells: a brief overview

Although the pursuit of bovine embryonic stem cells started more than 26 years ago for the purpose of gene-targeting, true pluripotent stem cells in this economically important species are still elusive. With the rapid advances in genome-editing and cloning using homologously recombined somatic cells, the need for pluripotent stem cells for precise genetic modification in any species became questionable. With the pig being the better model for human regenerative biology, the identification of the commonalities and uniqueness of the pluripotency circuitry across mammalian species may be the main objective for studying pluripotent stem cells in the bovine.     

Molecular coupling of Xist regulation and pluripotency

During mouse embryogenesis, reversion of imprinted X chromosome inactivation in the pluripotent inner cell mass of the female blastocyst is initiated by the repression of Xist from the paternal X chromosome. Here we report that key factors supporting pluripotency-Nanog, Oct3/4, and Sox2-bind within Xist intron 1 in undifferentiated embryonic stem (ES) cells. Whereas Nanog null ES cells display a reversible and moderate up-regulation of Xist in the absence of any apparent modification of Oct3/4 and Sox2 binding, the drastic release of all three factors from Xist intron 1 triggers rapid ectopic accumulation of Xist RNA. We conclude that the three main genetic factors underlying pluripotency cooperate to repress Xist and thus couple X inactivation reprogramming to the control of pluripotency during embryogenesis.     

人类胚胎干细胞研究述评

对人类胚胎干细胞(hESCs)的来源、培养方法、建系条件、生物学特性和鉴定方法、遗传操作及其需解决的问题进行了讨论。提出目前研究的重点在于揭示维持ES细胞多能性和自我更新的机理,进一步优化人类和其他哺乳动物类ES细胞的分离、培养、建系方法,探讨其定向分化机理,建立胚胎干细胞(ESCs)和胚胎生殖细胞(EGCs)的大规模快速扩增技术;完善hESCs向重要功能细胞(生殖细胞)分化的体系;单细胞比对分析ESCs、畸胎瘤细胞(ECSs)、EGCs、类胚体(EBs)、各级生殖细胞和成体细胞的基因及蛋白表达图谱,以探求生殖细胞、成体细胞、ESCs、ECSs、EGCs的本质区别,积极开展将ES细胞用于治疗人类疾病模型的研究。     

诱导多能干细胞体外定向分化为雄性生殖细胞研究进展

诱导多能干细胞（induced pluripotent stem cells, iPS）是借助基因导入技术将某些转录因子导入人或动物的体细胞,使体细胞重编程而得到的多能性细胞。iPS细胞来源丰富,研究表明利用不同的载体诱导系统或不同的转录因子组合,多种体细胞都可被重编程为iPS细胞,如成纤维细胞、肝细胞、角质细胞及脐带血细胞等。作为干细胞中新的一员,iPS细胞在克隆形态、基因表达模式、表面标志物、拟胚体形成、畸胎瘤及嵌合体形成（小鼠）、分化能力等方面与胚胎干细胞（embryonic stem cell, ESC）非常相似。与胚胎干细胞一样,在特定的诱导条件下,iPS细胞在体外可被诱导分化为多种细胞,如心肌细胞、血细胞、生殖细胞等,进一步拉近了干细胞和临床疾病治疗的距离,iPS细胞也成为细胞治疗及组织器官再生最有前景的种子细胞,在细胞替代性治疗、发病机理研究及新药筛选方面具有潜在价值。雄性不育不仅影响人类正常健康生活,对畜牧业的发展也极为不利。国内外的研究成果表明,给予适当的诱导物及诱导条件,人和小鼠的iPS细胞体外可被诱导分化为原始生殖细胞（primordial germ cells, PGCs）、精子细胞及其前体细胞。这些研究不仅避免了胚胎干细胞研究领域存在的取材困难、免疫排斥和伦理道德等问题,还为揭示雄性生殖细胞的发育机制及研究雄性不育提供了较好的研究平台。由人iPS细胞诱导得到的雄性配子可为患者提供自身的雄性配子产生后代,避免了免疫排斥问题,为未来治疗雄性不育带来曙光。iPS细胞体外诱导分化技术对现代畜牧业发展也具有巨大的潜在应用价值,可进行转基因动物的生产。现从不同的诱导剂、不同的培养条件及iPS细胞体外诱导分化优势等方面,对iPS细胞体外定向诱导分化为雄性生殖细胞的研究进展及应用前景进行综述和展望。     

多能干细胞来源的肝细胞诱导分化及其应用研究进展

胚胎干细胞(embryonic stem cells,ESCs)和诱导多能干细胞(induced pluripotent stem cells,iPSCs)是一类具有多能性和自我更新能力的多能干细胞,这些细胞具有无限增殖能力,并有潜能分化为机体三胚层所有类型的细胞。多能干细胞来源的肝细胞能够作为肝细胞的替代物应用于体外药物筛选和肝脏疾病的治疗。文章对多能干细胞向肝细胞的诱导研究、多能性干细胞来源肝细胞的鉴定方案研究及其在药物筛选和疾病治疗方面的研究进行了综述分析,并对未来的研究方向进行了总结与展望。     

胚胎干细胞体外定向诱导分化研究进展

胚胎干细胞是由早期内细胞团分离的一种多潜能细胞 ,在体外分化抑制培养时 ,可保持未分化状态而无限增殖。一旦撤除分化抑制因素 ,或添加适当的诱导剂 ,ES细胞可分化为内胚层、中胚层和外胚层的多种类型的特化细胞。文章综述了体外定向诱导 ES细胞分化为造血细胞、心肌细胞、神经细胞、脂肪细胞、胰岛细胞、内皮细胞、上皮细胞、成骨细胞、软骨细胞和黑素细胞等的途径和方法。     

Research on Isolation and Clone of Embryonic Stem Cell-Like in Bovine

Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary marine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS ,0.1μmol/L Na2SeO3, 0. 1mmol/L β-mercaptoethanol, 1 000ng/ml LIF,10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine), then, we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage marine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0. 125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification, karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.