胚胎干细胞向造血细胞分化研究

胚胎干（embryonic stem,ES）细胞是来源于囊胚的内细胞团（inner cell mass,ICM）,具有发育的全能性或多能性,能嵌合到早期胚胎,在体内可以参与各种组织发育甚至包括生殖细胞;在体外分化培养条件下,可以顺序分化出各种组织细胞,与体内完整胚胎发育过程相符合,而且可以通过调节ES细胞某些基因的表达而调节其分化。因此,ES细胞是研究哺乳动物早期胚胎发育、细胞分化及其关键基因鉴定的理想模型。另外,胚胎生殖脊（embryonic germ,EG）细胞系也具有同样的生物学特性,它是由早期胚胎的原始生殖脊（primordial germ,PG）细胞建株而来。最近研究显示：ES细胞在体外不但可以分化为所有造血细胞系,而且还可以分化为具有长期增殖能力的造血干细胞。作者就胚胎干细胞向造血细胞和造血干细胞分化及其诱导因子和调控基因的表达作一综述。     

胚胎干细胞体外分化心肌细胞的研究

胚胎干细胞是从动物胚胎内细胞团分离的具有全能性的细胞.研究证明分离的小鼠胚胎干细胞在体外可以分化成心肌细胞,这一发现为小鼠胚胎干细胞向各种特化心脏细胞(动脉样细胞、血管样细胞、窦样细胞、心室样细胞、蒲肯野氏样细胞)分化提供了依据,使基因功能的研究在体外成为可能.1998年末人类ES细胞的成功培养奠定了心脏细胞的再生性治疗的战略基础.主要综述了目前ES细胞体外分化心肌细胞的进程,讨论了此进程对心脏基因研究的促进作用.     

人胚胎干细胞和拟胚体基因表达谱的初步分析

利用人类全基因组Affymetrix芯片检测人胚胎干细胞与其自发分化7d的拟胚体之间的差异表达基因.结果显示:与未分化的人胚胎干细胞相比.在分化7d的拟胚体中表达下调2倍及以上的已知和未知基因共有1100个,表达上调2倍及以上的已知或未知基因共有2283个.利用Gostat对这些差异表达基因进行功能分析,发现它们分别与细胞的生物代谢过程、信号传导通路、系统发育、细胞分化、分子功能及亚细胞组分相关.胚胎干细胞具有自我更新能力,是研究早期胚胎发育理想的细胞模型,因此对差异表达基因的功能研究有助于了解维持人胚胎干细胞自我更新的分子机制以及胚胎发育早期的分子事件.     

哺乳动物单倍体胚胎干细胞的建立与应用

哺乳动物胚胎干细胞通常为二倍体,限制了其在后基因组时代功能基因研究上的应用.近年来,随着不同物种单倍体胚胎干细胞的建立,科学家获得了一种可用于高通量正反向遗传筛选的细胞工具.单倍体胚胎干细胞具有类似于二倍体胚胎干细胞的无限增殖能力、基因表达模式、分化潜能等特点,可以在细胞水平开展遗传筛选研究;通过不同物种单倍体干细胞的融合形成异源二倍体细胞可以用于研究基因的演化.另外,小鼠单倍体胚胎干细胞可代替配子用于产生动物,这为研究基因印记在胚胎发育过程中的作用、快速构建携带复杂基因修饰的模式动物、进行个体水平遗传筛选等提供了一个新的遗传学工具.最近,单倍体胚胎干细胞的基因组标签计划的提出,以构建所有编码蛋白质基因的标签小鼠资源平台为目的,将促进蛋白质研究的标准化,并为揭开胚胎发育复杂而有序的蛋白质调控网络提供工具.     

干细胞生物学研究进展

自从1981年小鼠胚胎干细胞(Embryonicstemcell,ES细胞)分离培养成功以来,人们又致力于羊、猪、牛等大型动物胚胎干细胞研究,因为ES细胞在培养细胞与个体发育和体细胞与生殖细胞之间架起了桥梁。ES细胞可以作为遗传物质的载体来改造动物和研究基因对胚胎发育的影响。人体胚胎干细胞培养成功是干细胞生物学技术又一重大突破,它与动物体细胞克隆技术相结合,给组织器官工程带来美好前景。1　人体胚胎干细胞Thomson和Gearhart2个研究小组分别采用不同教材成功培养人体ES细胞,如图1示:图1　　根据鼠ES细胞研究结果,推测人体ES细胞基础和临床…     

人胚胎干细胞向神经上皮祖细胞的诱导分化

人胚胎干细胞具有自我更新和多向分化潜能,是研究早期胚胎发育和细胞替代治疗的重要细胞来源.采用一种与小鼠成纤维细胞共培养的方法进行人胚胎干细胞的神经诱导,可产生高纯度的神经上皮祖细胞,其神经上皮特异性基因的表达有一定的时空性;诱导生成的神经上皮祖细胞具有增殖潜能并可分化为神经元和星型胶质细胞,是潜在的神经干细胞.人胚胎干细胞来源的神经上皮祖细胞为研究神经发育和神经诱导提供了新材料,也为神经系统疾病的细胞替代治疗提供了新的细胞来源.     

不同人胚胎干细胞系向限定性内胚层分化能力存在差异

研究不同的人胚胎干细胞系向限定性内胚层细胞分化能力是否存在差异,并尝试寻找造成差异的原因.基于已建立的人胚胎干细胞库资源和限定性内胚层定向诱导分化体系,流式检测诱导分化3天后10株细胞系Sox17的阳性比率发现其值在60%到80%之间波动,而SSEA4的阳性比率在人胚胎干细胞系中不存在明显差异.基因表达谱结果显示像Sox17,Foxa2等内胚层标记在这些细胞之间不存在显著的差异,而像MEG3和SNORD114-3在差异细胞系之间和同株细胞早晚期代数之间存在表达差异.结果提示不同的人胚胎干细胞系向限定性内胚层细胞分化能力存在着差异,推测这些差异可能与MEG3和SNORD114-3的差异表达相关.     

敲除Pofut1不影响胚胎干细胞向神经元分化

蛋白O-连接岩藻糖基转移酶1 (Pofut1)基因缺失可导致Notch分子无法与配体结合并启动信号传递. 为研究Pofut1基因对哺乳动物胚胎干细胞（ESC）向神经分化的影响,利用Pofut1基因敲除的胚胎干细胞与野生型胚胎干细胞,经体外培养诱导拟胚体（EB）分化为神经细胞,计数分化为神经细胞的比例,采用细胞免疫组化染色和real-time PCR等方法,分析神经细胞特异性标志分子的表达. 结果显示,Pofut1基因缺失后,对EBC生长没有明显影响,分化过程中形成的拟胚体数量明显增多,分化的神经样细胞以及神经标志物分子的表达也明显多于对照组;Notch信号缺失对小鼠胚胎干细胞生长无明显影响,但可以促进ES细胞向神经细胞分化.     

一种新的人胚胎干细胞自身来源的滋养层支持其体外培养

摘要: 通过人胚胎干细胞(Human embryonic stem cells, hESCs)经体内分化获取间充质干细胞(Mesenchymal stem cells, MSCs)为人胚胎干细胞提供一种新的滋养层。将约5×106个hESCs注射入重症免疫联合缺陷小鼠形成畸胎瘤, 8周后再从畸胎瘤中分离MSCs并鉴定, 将MSCs作为hESCs的滋养层细胞, 并检测和观察hESCs的生长情况、细胞特性和分化能力。从畸胎瘤中获得了纯度较高的具有类似骨髓来源的MSC特性的细胞群, 其形态相似、表面抗原标志相似(CD34和CD45阴性, CD29、CD49b、CD105、CD73和CD90阳性), 经诱导可以向成骨细胞和成脂细胞分化。将hESCs在MSCs滋养层细胞上传代培养10代以上, hESCs依然具有正常的细胞形态, 反转录PCR证实其特异转录因子Oct4、Nanog的表达, 干细胞表面标记SSEA-1显示为阴性, SSEA-4、TRA-1-60、TRA-1-81显示为阳性, 碱性磷酸酶染色显示为阳性, 并且核型正常。体外EB形成和体内畸胎瘤形成证明了其全能性。因此来源于hESCs本身的MSCs可以被用来作为支持胚胎干细胞生长并维持其未分化状态的滋养层细胞。     

干细胞定向分化的基础和临床应用研究

胚胎干细胞具有多向性分化的潜能,可以分化成为内、中、外三个胚层的所有细胞,存在于组织器官中的成体干细胞（包括心脏等的前体细胞）也能分化成为某些细胞,用来修复、补充体内受损、死亡的细胞.目前干细胞研究的重点是：干细胞未分化和多向性机制的基础研究;干细胞向特定细胞群体分化的调控和分化细胞的应用研究,而后者是连接基础研究和临床研究的必经之路.干细胞的基础和临床应用研究不但可以了解正常的胚胎发育过程,而且利用掌握的知识通过体外诱导或体内激活的方法针对性地治疗某些疾病.目前我们的研究集中在神经细胞（包括视网膜细胞和内耳前体细胞）、脂肪细胞和心肌细胞定向分化的分子机理,并通过疾病动物模型验证这些定向分化的细胞的功能.希望通过建立人胚胎干细胞以及成体干细胞向外胚层的特种神经元（包括前脑神经上皮细胞、GABA和胆碱能神经元、视觉细胞、听觉细胞、多巴胺能神经元）和中胚层的脂肪细胞、骨细胞以及心肌细胞定向分化的模型,继而采用蛋白质组学和基因组学最新技术分析这些建立的模型,研究相关因子通过哪条信号传导通路导致这些细胞的定向分化或者通过改变哪个目的基因的表达,或改变目的蛋白的修饰导致干细胞定向成神经细胞、脂肪细胞和心肌细胞;研究成年脑内源性干细胞定向诱导成这些功能性神经元的机理,并进行比较研究.用Lentivirus转染干细胞高表达、或用RNA干扰抑制上述研究得到的目的基因,在细胞模型和动物体内验证这些信号通路和目的基因在干细胞定向分化中的作用.     

Combinatorial signals of activin/nodal and bone morphogenic protein regulate the early lineage segregation of human embryonic stem cells

Cell fate commitment of pre-implantation blastocysts, to either the inner cell mass or trophoblast, is the first step in cell lineage segregation of the developing human embryo. However, the intercellular signals that control fate determination of these cells remain obscure. Human embryonic stem cells (hESCs) provide a unique model for studying human early embryonic development. We have previously shown that Activin/Nodal signaling contributes to maintaining pluripotency of hESCs, which are derivatives of the inner cell mass. Here we further demonstrate that the inhibition of Activin/Nodal signaling results in the loss of hESC pluripotency and trophoblast differentiation, similar to BMP4-induced trophoblast differentiation from hESCs. We also show that the trophoblast induction effect of BMP4 correlates with and depends on the inhibition of Activin/Nodal signaling. However, the activation of BMP signaling is still required for trophoblast differentiation when Activin/Nodal signaling is inhibited. These data reveal that the early lineage segregation of hESCs is determined by the combinatorial signals of Activin/Nodal and BMP.     

Erk2 signaling and early embryo stem cell self-renewal

Differentiation of the mammalian blastocyst generates two distinct cell lineages: the trophectoderm, which contributes to the trophoblast layers of the placenta, and the inner cell mass, which forms the embryo. We and others recently demonstrated that the MAP kinase ERK2 is essential for trophoblast development. Erk2 mutant embryos fail to form extra-embryonic ectoderm and the ectoplacental cone, suggesting a role for ERK2 activation in the proliferation of trophoblast stem (TS) cells. Previous studies have documented that ERK1/2 activity is dispensable for proliferation of embryonic stem (ES) cells and rather interferes with self-renewal. Thus, signaling by the ERK1/2 MAP kinase pathway appears to be critical for the regulation of self-renewal and propagation of early embryo stem cell populations.     

Rat blastocyst-derived stem cells are precursors of embryonic and extraembryonic lineages

Despite recent advances in the derivation of rat embryonic stem cells, clear comprehension of the timing and mechanisms underlying rat early embryo lineage selection is lacking. We have previously shown the in vivo contribution of rat embryonic stem-like cells exclusively to developing extraembryonic tissues. To elucidate possible mechanisms governing the in vitro and in vivo behaviors of these rat blastocyst-derived stem cells, we evaluated their developmental capacity by using several approaches. Molecular marker analysis demonstrated the expression profile of genes characterizing not only pluripotency but also extraembryonic endoderm and trophoblast. In vitro differentiation through embryoid body formation showed in vitro pluripotent capacity through differentiation into derivatives of all three embryonic germ layers. Following either blastocyst injection, diploid or tetraploid aggregation, and embryo transfer, these rat blastocyst-derived stem cells also demonstrated in vivo multipotency through contribution to multiple developmentally distinct extraembryonic lineages. Features of phenotypic heterogeneity were revealed following examination of cell line morphology and culture behavior, as well as quantitative analysis of marker expression in discrete undifferentiated and differentiated populations of cells by flow cytometry. We demonstrate for the first time that stem cells derived from the rat blastocyst have the ability to contribute to the embryonic and extraembryonic lineages. Together, these results provide a valuable new model for rat stem cell biology and for the elucidation of early lineage selection in the embryo.     

BMP4 initiates human embryonic stem cell differentiation to trophoblast

The excitement and controversy surrounding the potential role of human embryonic stem (ES) cells in transplantation therapy have often overshadowed their potentially more important use as a basic research tool for understanding the development and function of human tissues. Human ES cells can proliferate without a known limit and can form advanced derivatives of all three embryonic germ layers. What is less widely appreciated is that human ES cells can also form the extra-embryonic tissues that differentiate from the embryo before gastrulation. The use of human ES cells to derive early human trophoblast is particularly valuable, because it is difficult to obtain from other sources and is significantly different from mouse trophoblast. Here we show that bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor-beta (TGF-beta) superfamily, induces the differentiation of human ES cells to trophoblast. DNA microarray, RT-PCR, and immunoassay analyses demonstrate that the differentiated cells express a range of trophoblast markers and secrete placental hormones. When plated at low density, the BMP4-treated cells form syncytia that express chorionic gonadotrophin (CG). These results underscore fundamental differences between human and mouse ES cells, which differentiate poorly, if at all, to trophoblast. Human ES cells thus provide a tool for studying the differentiation and function of early human trophoblast and could provide a new understanding of some of the earliest differentiation events of human postimplantation development.     

SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation

Suppressor of cytokine signaling 3 (SOCS3) binds cytokine receptors and thereby suppresses cytokine signaling. Deletion of SOCS3 causes an embryonic lethality that is rescued by a tetraploid rescue approach, demonstrating an essential role in placental development and a non-essential role in embryo development. Rescued SOCS3-deficient mice show a perinatal lethality with cardiac hypertrophy. SOCS3-deficient placentas have reduced spongiotrophoblasts and increased trophoblast secondary giant cells. Enforced expression of SOCS3 in a trophoblast stem cell line (Rcho-1) suppresses giant cell differentiation. Conversely, SOCS3-deficient trophoblast stem cells differentiate more readily to giant cells in culture, demonstrating that SOCS3 negatively regulates trophoblast giant cell differentiation. Leukemia inhibitory factor (LIF) promotes giant cell differentiation in vitro, and LIF receptor (LIFR) deficiency results in loss of giant cell differentiation in vivo. Finally, LIFR deficiency rescues the SOCS3-deficient placental defect and embryonic lethality. The results establish SOCS3 as an essential regulator of LIFR signaling in trophoblast differentiation.     

Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts

The extra-embryonic endoderm lineage plays a major role in the nutritive support of the embryo and is required for several inductive events, such as anterior patterning and blood island formation. Blastocyst-derived embryonic stem (ES) and trophoblast stem (TS) cell lines provide good models with which to study the development of the epiblast and trophoblast lineages, respectively. We describe the derivation and characterization of cell lines that are representative of the third lineage of the blastocyst -extra-embryonic endoderm. Extra-embryonic endoderm (XEN) cell lines can be reproducibly derived from mouse blastocysts and passaged without any evidence of senescence. XEN cells express markers typical of extra-embryonic endoderm derivatives, but not those of the epiblast or trophoblast. Chimeras generated by injection of XEN cells into blastocysts showed exclusive contribution to extra-embryonic endoderm cell types. We used female XEN cells to investigate the mechanism of X chromosome inactivation in this lineage. We observed paternally imprinted X-inactivation, consistent with observations in vivo. Based on gene expression analysis, chimera studies and imprinted X-inactivation, XEN cell lines are representative of extra-embryonic endoderm and provide a new cell culture model of an early mammalian lineage.     

Diploid,but not haploid,human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells.     

人胚胎干细胞建系和鉴定

人胚胎干细胞是一种取自人囊胚内细胞团且具有形成所有三个胚层细胞能力的全能细胞。建立一个理想的人胚胎干细胞培养系统是研究和利用这种具有巨大潜力细胞的首要条件。本文讨论了目前建立的人胚胎干细胞培养系统,阐述了其有利的和不利的一面,并着重讨论其体外培养方法和鉴定策略。