胚胎干细胞向神经细胞诱导分化的研究

胚胎干细胞具有全能性和无限增殖的能力，有望成为组织工程和细胞治疗的重要种子细胞来源。如何将胚胎干细胞向特定理论诱导分化已成为重要的课题。目前已有将胚胎干细胞诱导分化为神经细胞，并将其应用于动物模型治疗的报道。这些工作对今后神经系统疾病和损伤的治疗意义重大。     

胚胎干细胞的定向心肌分化及其调控机制

胚胎干细胞是具有分化为各种类型组织细胞潜能的全能干细胞,可在体外大量扩增,细胞因子、激素、诱导剂和细胞内转录因子等可诱导和调控胚胎干细胞进行心肌细胞定向分化,这将使干细胞移植治疗心肌损伤性疾病成为可能。探讨胚胎干细胞向心肌细胞的定向分化及其调控机制,进而可在体内外调控干细胞向心肌细胞的定向分化,这将为临床应用干细胞分化新生心肌细胞以治疗心肌损伤性疾病提供理想的细胞来源和可靠的理论依据。     

胚胎干细胞的定向心肌分化及其调控机制

胚胎干细胞是具有分化为各种类型组织细胞潜能的全能干细胞,可在体外大量扩增,细胞因子、激素、诱导剂和细胞内转录因子等可诱导和调控胚胎干细胞进行心肌细胞定向分化,这将使干细胞移植治疗心肌损伤性疾病成为可能.探讨胚胎干细胞向心肌细胞的定向分化及其调控机制,进而可在体内外调控干细胞向心肌细胞的定向分化,这将为临床应用干细胞分化新生心肌细胞以治疗心肌损伤性疾病提供理想的细胞来源和可靠的理论依据.     

胚胎干细胞定向诱导分化为心肌细胞进展

在个体发育过程中，通常把那些具有自我复制能力并能在一定条件下分化形成一种以上类型细胞的多潜能细胞称为干细胞。根据其发育阶段，干细胞可分为胚胎干细胞和成体干细胞。由于胚胎干细胞具有在体外培养下保持未分化状态的增殖能力及分化为多种细胞类型的潜能，使之成为一种研究哺乳动物细胞分化、组织形成过程的基本体系，以及临床移植治疗的新的细胞来源。人们利用胚胎干细胞建立了各种体外分化模型，本文将概述现今对胚胎干细胞定向诱导分化为心肌细胞方面的主要进展和趋势。     

神经前体细胞不是胚胎干细胞向胰岛素分泌细胞分化的必经途径

探讨胚胎干细胞向胰岛素分泌细胞分化的途径,对胰腺组织工程的临床运用有重要意义。将胚胎干细胞在有小鼠胚胎成纤维细胞饲养层和白血病抑制因子的条件下培养扩增后,再将扩增后的胚胎干细胞不经过神经前体细胞阶段直接诱导为胰岛素分泌细胞,并与传统的多阶段诱导(经过神经前体细胞阶段)进行比较。结果发现,胚胎干细胞脱离饲养层细胞后,经过9~10d的分化诱导,可以分化为具有胰岛β-细胞特征的胰岛素分泌细胞。与传统的多阶段诱导方法相比,诱导过程简化,诱导时间缩短,所得到的胰岛素分泌细胞数量无明显差异。说明胚胎干细胞向胰岛素分泌细胞分化存在多条途径。神经前体细胞阶段不是胚胎干细胞向胰岛素分泌细胞分化的必须途径。用传统的多阶段分化诱导法和直接诱导法都可以将胚胎干细胞诱导成胰岛素分泌细胞。     

诱导人孤雌胚胎干细胞分化为胰岛样细胞团

背景:随着人类孤雌胚胎干细胞系的成功建立,对该细胞的进一步分化功能研究成为新的研究热点。目的:研究体外培养的人类孤雌胚胎干细胞诱导分化为胰岛样细胞团的潜能。方法:自主建系孤雌来源人胚胎干细胞和正常来源人胚胎干细胞各1株,运用基于胰腺体内发育规律的改良5阶段诱导法诱导人孤雌胚胎干细胞为胰岛样细胞团,加入不同生长因子及诱导试剂对人胚胎干细胞分5阶段序贯培养。结果与结论:终末分化细胞光镜下呈团状聚集,RT-PCR、免疫荧光染色检测分化细胞表达胰岛细胞特征性的基因与蛋白。胰岛素释放实验提示获得的细胞具有胰岛样生化功能。由人类孤雌胚胎干细胞来源的胰岛样细胞团具备胰岛的基本特征,是未来治疗1型糖尿病的可用材料。     

不同培养体系可逆改变人类胚胎干细胞的分化倾向

背景:人类胚胎干细胞可以在饲养细胞依赖性培养体系和化学限定性培养体系下维持未分化状态,能够在体内外诱导分化成三胚层来源的细胞类型。目的:比较饲养细胞和化学限定性培养体系对人类胚胎干细胞特性的影响。方法:将在饲养细胞培养体系下培养27代的人类胚胎干细胞转入到化学限定性培养基体系中培养56代,然后再将其转回到饲养细胞培养体系中,将3种培养条件下的人类胚胎干细胞(饲养细胞培养体系培养70代、化学限定性培养体系培养56代、化学限定性培养体系下培养70代后转回饲养细胞培养体系下培养13代和20代)进行多能性分子标记SSEA4流式分析等检测分析,同时对3种培养条件下人类胚胎干细胞经拟胚体诱导分化后分别检测多能性基因和三胚层分化基因的表达。结果与结论:人类胚胎干细胞在饲养细胞和化学限定性培养体系下表现出不同的诱导分化倾向,在化学限定性培养体系下表现出向神经诱导分化抑制,这种不同的诱导分化倾向可发生可逆性转换,当人类胚胎干细胞由化学限定性培养体系转回到饲养细胞培养体系时,诱导分化倾向表现出与其在饲养细胞下诱导分化一致的模式。在拟胚体分化中,多能性基因Nanog高可能对诱导分化倾向起着重要作用。与此同时,人类胚胎干细胞SSEA4细胞亚群发生相应的变化,人类胚胎干细胞在饲养细胞和化学限定下培养体系下表现的分化倾向与人类胚胎干细胞亚群所占的比例存在关联。     

人胚胎干细胞接种在鼠尾胶原包被培养板上定向分化为血管内皮细胞

背景：胚胎干细胞在体外可诱导分化为血管内皮细胞,进而形成血管,因而成为血管组织工程理想的种子细胞来源之一。 目的：探讨建立定向诱导人胚胎干细胞向血管内皮细胞分化的方法。 方法：在小鼠胚胎成纤维细胞饲养层上培养人胚胎干细胞H1,将H1细胞团块转移到鼠尾胶原包被的培养板,贴壁24- 48 h后,培养基换为含不同辅助因子和内皮细胞生长添加剂的EGM-2内皮细胞培养基。 结果与结论：①在EGM-2内皮细胞培养基诱导下,细胞逐步表达内皮细胞特异性标记基因VEGFR-2、PECAM1、vWF、CD34、VE-cadherin、GATA-2。②分化细胞表达内皮细胞特异性标记VE-cadherin、CD31。③分化细胞具有吞噬低密度脂蛋白功能。说明将人胚胎干细胞接种在鼠尾胶原包被培养板上,通过EGM-2内皮培养体系诱导,可直接分化为功能性血管内皮细胞。为研究胞外基质对诱导胚胎干细胞血管内皮细胞分化的作用以及相关信号分子机制打下了基础。     

诱导多能干细胞及其在神经再生医学的应用前景

诱导多能干细胞 （induced pluripotent stem cells, iPS）最初由日本学者山中申弥（Shinya Yamanaka）于2006年利用病毒载体将四个转录因子（Oct4、Sox2、Klf4和c．Myc）的组合转入分化的体细胞中，使其重编程而得到的类似胚胎干细胞（Embryonic stem cells）的一种细胞类型。该类细胞在体内具有分化为任何一种细胞类型的潜能。更重要的是，诱导多能干细胞来源于自体，相比胚胎干细胞，诱导多能干细胞来源的细胞移植不需要免疫抑制治疗。此外，诱导多能干细胞的出现还省却了胚胎干细胞研究中涉及到的政治及伦理因素的困惑。这项细胞可被重塑的重要发现在医学研究领域具有旅程碑的作用。本文总结了目前医学研究领域里可用的的干细胞平台，并重点阐述了细胞的重编程及诱导多能干细胞在疾病模型及神经退行性疾病细胞移植治疗领域的应用前景。     

胚胎干细胞向胰岛细胞诱导分化的研究

胚胎干细胞(embryonicstemcell熏ESC)因具有全能分化潜能而有望成为组织工程和细胞治疗中的重要种子细胞来源。如能将其诱导分化为能分泌胰岛素的细胞,将有助于解决胰岛素绝对或相对缺乏引起的糖尿病代谢失调状态。目前已有研究者诱导分化出能分泌胰岛素的细胞,有的将之应用于糖尿病鼠模型并取得了可喜的效果。本文对这一领域的研究现状进行综述。     

Microsphere-based tracing and molecular delivery in embryonic stem cells

Embryonic stem (ES) cells are in vitro cell lines that can differentiate into all lineages of the fetus and the adult. Despite the versatility of genetic manipulation in murine ES cells, these approaches are time-consuming and rely on inefficient transient cellular delivery systems that can only be applied to undifferentiated ES cell cultures. Here we describe a polystyrene microsphere-based system designed to efficiently deliver biological materials into both undifferentiated and differentiating ES cells. Our results demonstrate that these microspheres can be successfully employed for simultaneous cellular labeling and controlled transfer of various cargos such as fluorophores, proteins and nucleic acids into ES cells without any significant toxicity or loss of pluripotency. This versatile delivery system is also effective in other stem cell lines derived from early embryos, trophoblast and neural stem cells.     

Derivation and spontaneous differentiation of human embryonic stem cells

Embryonic stem (ES) cells are unique cells derived from the inner cell mass of the mammalian blastocyst. These cells are immortal and pluripotent, retain their developmental potential after prolonged culture, and can be continuously cultured in an undifferentiated state. Many in vitro differentiation systems have been developed for mouse ES cells, including reproducible methods for mouse ES cell differentiation into haematopoietic and neural precursors, cardiomyocytes, insulin‐secreting cells, endothelial cells and various other cell types. The derivation of new human ES cell lines provides the opportunity to develop unique models for developmental research and for cell therapies. In this review we consider the derivation and spontaneous differentiation of human ES cells.     

RNAi in embryonic stem cells

Embryonic stem (ES) cells are pluripotent cells that can be isolated and grown in vitro from the inner cell mass of blastocysts. Their potential to differentiate into any cell of the body makes them a promising starting material for cell therapy Much progress has been made in recent years to develop ES cell differentiation protocols employing cocktails of certain growth factors or by using cell-type-restricted promoters driving the expression of selection markers or fluorescent proteins. However, little is known about the molecular details underlying the earliest processes of mammalian development. Genetic tools that provide novel insight into these processes would be very helpful to gain a better molecular understanding and to design better differentiation protocols. Recently, RNAi has emerged as a powerful technology to perform loss-of-function studies in mammalian cells. This technology should be ideal to identify and study genes required for ES cell self-renewal and differentiation. Here, we review the recent advances and challenges of RNAi research in ES cells and we provide a perspective on possible applications to enhance our understanding of ES cell self-renewal and early differentiation.     

组织工程中的新型种子细胞--胚胎干细胞

种子细胞的老化是组织工程中的一大难题.胚胎干细胞因其具有全能性和无限增殖的能力而有望成为组织工程中的种子细胞新来源.本文介绍了胚胎干细胞的建系和分化研究,并展望胚胎干细胞作为组织工程的种子细胞的前景和面临的困难.     

胚胎干细胞向表皮样干细胞分化体外条件的研究

目的　探索体外诱导胚胎干细胞分化为表皮样干细胞的最佳条件 ,为研究其分化的调控机理及寻找新的皮肤组织工程种子细胞奠定基础。方法　将小鼠胚胎干细胞与人羊膜共培养 ,以不同的羊膜铺布方式 ,分 4个实验组 :(1)羊膜上皮面向上铺布全孔底 ,(2 )羊膜基底面向上铺放全孔底 ,(3)羊膜上皮面向上铺布半孔底 ,(4)羊膜基底面向上铺布半孔底 ,以未加羊膜为对照组 ,倒置显微镜下观察细胞的生长和形态分化 ,用抗 β1整合素单克隆抗体免疫组化检测胚胎干细胞向表皮样干细胞的分化。结果　培养 4～ 5d后 ,实验组 (3)中的ES细胞分化为表皮样干细胞 ,细胞呈多边形 ,体积增大 ,排列紧密 ,形成大面积细胞单层 ,而其他各实验组 ,ES细胞虽可分化为表皮样细胞 ,但呈集落生长 ,不形成细胞单层 ;对照组大量细胞死亡 ,形态各异。各实验组的细胞绝大多数呈现 β1整合素阳性 ,而对照组未见 β1整合素阳性细胞。结论　与羊膜共培养可诱导胚胎干细胞分化为表皮样干细胞 ,羊膜上皮面向上铺布半孔底的效果最佳     

AFP(+), ESC-derived cells engraft and differentiate into hepatocytes in vivo

A major problem in gene therapy and tissue replacement is accessibility of tissue-specific stem cells. One solution is to isolate tissue-specific stem cells from differentiating embryonic stem (ES) cells. Here, we show that liver progenitor cells can be purified from differentiated ES cells using alpha-fetoprotein (AFP) as a marker. By knocking the green fluorescent protein (GFP) gene into the AFP locus of ES cells and differentiating the modified ES cells in vitro, a subpopulation of GFP(+) and AFP-expressing cells was generated. When transplanted into partially hepatectomized lacZ-positive ROSA26 mice, GFP(+) cells engrafted and differentiated into lacZ-negative and albumin-positive hepatocytes. Differentiation into hepatocytes also occurred after transplantation of GFP(+) cells in apolipoprotein-E- (ApoE) or haptoglobin-deficient mice as demonstrated by the presence of ApoE-positive hepatocytes and ApoE mRNA in the liver of ApoE-deficient mice or by haptoglobin in the serum and haptoglobin mRNA in the liver of haptoglobin-deficient mice. This study describes the first isolation of ES-cell-derived liver progenitor cells that are viable mediators of liver-specific functions in vivo.     

Extensive contribution of embryonic stem cells to the development of an evolutionarily divergent host

The full potential of embryonic stem (ES) cells to generate precise cell lineages and complex tissues can be best realized when they are differentiated in vivo-i.e. in developing blastocysts. Owing to various practical and ethical constraints, however, it is impossible to introduce ES cells of certain species into blastocysts of the same species. One solution is to introduce ES cells into blastocysts of a different species. However, it is not known whether ES cells can contribute extensively to chimerism when placed into blastocysts of a distantly related species. Here, we address this question using two divergent species, Apodemus sylvaticus and Mus musculus, whose genome sequence differs by approximately 18% from each other. Despite this considerable evolutionary distance, injection of Apodemus ES cells into Mus blastocysts led to viable chimeras bearing extensive Apodemus contributions to all major organs, including the germline, with Apodemus contribution reaching approximately 40% in some tissues. Immunostaining showed that Apodemus ES cells have differentiated into a wide range of cell types in the chimeras. Our results thus provide a proof of principle for the feasibility of differentiating ES cells into a wide range of cell types and perhaps even complex tissues by allowing them to develop in vivo in an evolutionarily divergent host-a strategy that may have important applications in research and therapy. Furthermore, our study demonstrates that mammalian evolution can proceed at two starkly contrasting levels: significant divergence in genome and proteome sequence, yet striking conservation in developmental programs.     

Genetically manipulated human embryonic stem cell-derived dendritic cells with immune regulatory function

Genetically manipulated dendritic cells (DC) are considered to be a promising means for antigen-specific immune therapy. This study reports the generation, characterization, and genetic modification of DC derived from human embryonic stem (ES) cells. The human ES cell-derived DC (ES-DC) expressed surface molecules typically expressed by DC and had the capacities to stimulate allogeneic T lymphocytes and to process and present protein antigen in the context of histocompatibility leukocyte antigen (HLA) class II molecule. Genetic modification of human ES-DC can be accomplished without the use of viral vectors, by the introduction of expression vector plasmids into undifferentiated ES cells by electroporation and subsequent induction of differentiation of the transfectant ES cell clones to ES-DC. ES-DC introduced with invariant chain-based antigen-presenting vectors by this procedure stimulated HLA-DR-restricted antigen-specific T cells in the absence of exogenous antigen. Forced expression of programmed death-1-ligand-1 in ES-DC resulted in the reduction of the proliferative response of allogeneic T cells cocultured with the ES-DC. Generation and genetic modification of ES-DC from nonhuman primate (cynomolgus monkey) ES cells was also achieved by the currently established method. ES-DC technology is therefore considered to be a novel means for immune therapy.     

Embryonic stem cells

Stem cells are unique cell populations with the ability to choose between self-renewal and differentiation. Embryonic stem (ES) cells have the ability to form any fully differentiated cell of the body. To date, only three species of mammals have yielded long-term cultures of self-renewing ES cells- mice, monkeys, and humans. These cells have some special requirements to maintain their undifferentiated state in culture, e.g., presence of feeder cells, serum, or cytokines. Many scientific studies have tried to manipulate the growth and differentiation conditions with varied success. Studies of development in model systems, such as mice help our efforts to manipulate human stem cells in vitro. Data are now emerging that ES cells can be directed toward lineage-specific differentiation programs. On the basis of this property, it is likely that human ES cells will provide a useful differentiation culture system to study the mechanisms of human development. Recent advances in culturing ES cells and success in exploiting their pluripotency brings great hope for using human ES cell-based reparative therapy in future.     

Neural commitment of embryonic stem cells: molecules, pathways and potential for cell therapy

The study of neuronal differentiation of embryonic stem cells has raised major interest over recent years. It allows a better understanding of fundamental aspects of neurogenesis and, at the same time, the generation of neurons as tools for various applications ranging from drug testing to cell therapy and regenerative medicine. Since the first report of human embryonic stem (ES) cells derivation, many studies have shown the possibility of directing their differentiation towards neurons. However, there are still many challenges ahead, including gaining a better understanding of the mechanisms involved and developing techniques to allow the generation of homogeneous neuronal and glial subtypes. We review the current state of knowledge of embryonic neurogenesis which has been acquired from animal models and discuss its translation into in vitro strategies of neuronal differentiation of ES cells. We also highlight several aspects of current protocols which need to be optimized to generate high-quality embryonic stem cell-derived neuronal precursors suitable for clinical applications. Finally, we discuss the potential of embryonic stem cell-derived neurons for cell replacement therapy in several central nervous system diseases.