Mammalian neural stem cells

Neural stem cells exist not only in the developing mammalian nervous system but also in the adult nervous system of all mammalian organisms, including humans. Neural stem cells can also be derived from more primitive embryonic stem cells. The location of the adult stem cells and the brain regions to which their progeny migrate in order to differentiate remain unresolved, although the number of viable locations is limited in the adult. The mechanisms that regulate endogenous stem cells are poorly understood. Potential uses of stem cells in repair include transplantation to repair missing cells and the activation of endogenous cells to provide "self-repair. " Before the full potential of neural stem cells can be realized, we need to learn what controls their proliferation, as well as the various pathways of differentiation available to their daughter cells.     

Generalized potential of adult neural stem cells

The differentiation potential of stem cells in tissues of the adult has been thought to be limited to cell lineages present in the organ from which they were derived, but there is evidence that some stem cells may have a broader differentiation repertoire. We show here that neural stem cells from the adult mouse brain can contribute to the formation of chimeric chick and mouse embryos and give rise to cells of all germ layers. This demonstrates that an adult neural stem cell has a very broad developmental capacity and may potentially be used to generate a variety of cell types for transplantation in different diseases.     

牛iPSCs向神经细胞的诱导分化

【目的】通过类胚体介导体外自由分化与诱导分化,验证所建立的牛诱导性多能干细胞(Bovine induced pluripotent stem cells,biPSCs)能否分化为外胚层的神经细胞。【方法】将biPSCs悬浮培养以制备类胚体,通过类胚体介导使biPSCs在添加全反式维甲酸(RA)和β巯基乙醇(β-Me)诱导体系中分化为神经细胞,比较其诱导效率;提取不同诱导体系的分化细胞与类胚体的总RNA,利用RT-PCR方法鉴定分化细胞中神经细胞特异性基因的表达。【结果】培养的biPSCs集落呈球形,中央隆起,周围界限清晰,与胚胎干细胞集落形态类似。biPSCs碱性磷酸酶(AP)染色为阳性,并表达SSEA-4干细胞特异性表面蛋白。biPSCs通过类胚体介导分化,在未添加化学诱导物的条件下,可自由分化为Nestin、GFAP与NSE阳性神经细胞,其阳性细胞率分别为(15.14±1.13)%,(6.25±0.35)%和(5.45±0.62)%;biPSCs在RA诱导组中分化的神经细胞的数量最多,诱导后表达Nestin、GFAP和NSE细胞的阳性率分别为(49.56±2.33)%,(16.58±1.28)%和(13.66±2.21)%;在β-Me诱导组中,表达Nestin的阳性细胞率为(42.23±1.25)%。2个诱导组的诱导效率与无化学诱导物组有显著差异(P0.05)。RT-PCR检测结果显示,不同诱导体系获得的分化细胞均能扩增到神经细胞特异性基因Nestin和βⅢ-Tubulin片段,产物长度与预期结果完全一致。【结论】biPSCs在体外具有向神经细胞发育的能力;RA是诱导biPSCs向神经细胞分化的良好诱导物。     

Mosaic organization of neural stem cells in the adult brain

The in vivo potential of neural stem cells in the postnatal mouse brain is not known, but because they produce many different types of neurons, they must be either very versatile or very diverse. By specifically targeting stem cells and following their progeny in vivo, we showed that postnatal stem cells in different regions produce different types of neurons, even when heterotopically grafted or grown in culture. This suggests that rather than being plastic and homogeneous, neural stem cells are a restricted and diverse population of progenitors.     

仔猪神经干细胞体外培养及鉴定

由仔猪脑部海马体齿状回分离得到神经干细胞,利用无血清培养基添加特定生长因子方式培养;采用RT-PCR鉴定神经干细胞和诱导分化后细胞生物特性;利用免疫荧光化学技术检测克隆的细胞抗原和分化后特异性成熟神经细胞抗原表达。由海马体齿状回分离的细胞群具有连续克隆能力;根据免疫荧光检测,脑神经干细胞表达巢蛋白、配对盒因子6和解整合素样金属蛋白酶10;体外培养下NSCs可被诱导分化为神经源性细胞,且表达微管相关蛋白2、胶质纤维酸性蛋白和髓鞘碱性蛋白。分离和培育NSCs细胞结果显示,其具有更新能力和分化为神经元和胶质细胞的潜能,可为干细胞移植提供基础数据。     

Segregation of human neural stem cells in the developing primate forebrain

Many central nervous system regions at all stages of life contain neural stem cells (NSCs). We explored how these disparate NSC pools might emerge. A traceable clone of human NSCs was implanted intraventricularly to allow its integration into cerebral germinal zones of Old World monkey fetuses. The NSCs distributed into two subpopulations: One contributed to corticogenesis by migrating along radial glia to temporally appropriate layers of the cortical plate and differentiating into lamina-appropriate neurons or glia; the other remained undifferentiated and contributed to a secondary germinal zone (the subventricular zone) with occasional members interspersed throughout brain parenchyma. An early neurogenetic program allocates the progeny of NSCs either immediately for organogenesis or to undifferentiated pools for later use in the "postdevelopmental" brain.     

3种培养体系下人胚胎干细胞神经诱导分化的比较

【目的】比较人胚胎干细胞在人胚胎成纤维细胞、鼠胚胎成纤维细胞饲养层和无饲养层3个诱导体系下形成神经上皮祖细胞的能力。【方法】将人胚胎干细胞在人、鼠胚胎成纤维细胞饲养层或无饲养层体系中诱导10 d后,检测其胚胎干细胞和神经上皮祖细胞相关基因(Oct4、Nestin、Pax6、Sox1、Sox2、Sox3、β-actin)的表达,用免疫荧光染色法检测神经上皮祖细胞抗原标记Pax6、Musashi、Nestin和胚胎干细胞抗原标记SSEA4的表达。【结果】3种诱导体系下均有神经上皮祖细胞形成,均表达神经上皮祖细胞抗原标记Pax6、Musashi、Nestin及Pax6、Nes-tin、Sox家族基因。无饲养层体系诱导后的细胞,Oct4基因的表达高于有饲养层体系;其SSEA4染色阳性细胞比例为(26.3±3.5)%,高于有饲养层体系;其Pax6和Musashi染色阳性细胞比例分别为(81.0±3.0)%,(79.0±4.4)%,均低于有饲养层细胞体系。【结论】人胚胎干细胞在有饲养层和无饲养层体系中均可诱导分化形成神经上皮祖细胞,但无饲养层体系的效率相对较低,且有未分化胚胎干细胞残余。     

Instructive role of Wnt/beta-catenin in sensory fate specification in neural crest stem cells

Wnt signaling has recently emerged as a key factor in controlling stem cell expansion. In contrast, we show here that Wnt/beta-catenin signal activation in emigrating neural crest stem cells (NCSCs) has little effect on the population size and instead regulates fate decisions. Sustained beta-catenin activity in neural crest cells promotes the formation of sensory neural cells in vivo at the expense of virtually all other neural crest derivatives. Moreover, Wnt1 is able to instruct early NCSCs (eNCSCs) to adopt a sensory neuronal fate in a beta-catenin-dependent manner. Thus, the role of Wnt/beta-catenin in stem cells is cell-type dependent.     

大鼠胎儿神经干细胞的分离、培养及诱导分化

为了探讨神经干细胞(Neural stem cells,NSCs)在体外培养中的生物学特性,研究不同细胞因子对NSCs分化的影响,试验对大鼠胎儿神经干细胞进行了分离培养,并研究了10,50和100 ng/mL的神经生长因子(nerve growth factor,NGF)、脑源性神经营养因子(brain derived neruotrophic factor,BDNF)和胶质源性神经生长因子(glial cell line-derived neurotrophic factor,GDNF)对NSCs的定向诱导分化作用。结果表明,大鼠胎儿NSCs可在体外增殖并形成典型的神经球,指数增长期在传代后的第4~5天;10和50 ng/mL的NGF、BDNF和GDNF对NSCs的诱导分化作用不明显,100 ng/mL的NGF、BDNF和GDNF可诱导NSCs分化为神经元或胶质细胞。说明NGF和BDNF可诱导NSCs向神经元方向分化,GDNF诱导其向胶质细胞分化。     

Failure of limiting antigen doses to selectively stimulate high-avidity memory cells

Mouse spleen cells secondarily stimulated with a small dose of antigen, which elicited few antibody-forming cells, produced antibodies of avidity no greater than that of antibodies synthesized by cells stimulated with an optimal antigen dose. This result is in conflict with the hypothesis that maturation of the immune response is based on competition for limiting amounts of antigen among cells with receptors of varying avidity.     

成体小鼠骨髓中神经干细胞的分离与培养

在无菌条件下抽取成体小鼠骨髓,在含体积分数1%B27,20ng/mLEGF和10ng/mLbFGF的F12-DMEM(1∶1)培养基中,于37℃、50mL/LCO2饱和湿度下常规培养,对培养的细胞进行nestin免疫荧光染色检测,并将pEGFP-N2基因在80V、80μs条件下采用电击法转化导入神经干细胞中。结果表明,培养7d后,有大部分细胞聚集成团,并大量增殖;Nestin免疫荧光染色后呈阳性;导入pEGFP-N2基因的神经干细胞培养后在荧光显微镜下可观察到绿色荧光。表明从成体小鼠获得的骨髓,在该试验培养条件下有神经干细胞生成,并且外源标记基因pEGFP-N2在其细胞中实现了表达。     

白消安处理消融猪内源精原干细胞的效果及外源精原干细胞移植

目的 研究猪睾丸组织注射白消安消融猪内源精原干细胞(Spermatogonial stem cell,SSC)的效果,以及猪SSC同种异体移植后对内源性SSC消融受体生殖能力恢复的影响。方法 采用3 mg/kg剂量的白消安对9头6周龄大白公猪进行睾丸注射,另外3头注射2 mL 二甲基亚砜作为对照。3周后,对试验组公猪以相同剂量进行第2次睾丸注射。第2次注射3周后,采集试验组和对照组公猪睾丸进行相关检测,评估内源性SSC消融情况。第2次白消安注射1个月后,以两步酶消化法处理5~7日龄大白仔猪睾丸分离得到睾丸单细胞悬液,并用明胶差速贴壁法进行纯化,纯化后以免疫荧光和流式细胞术分析SSC的纯度。异体移植SSC 4个月后,用微卫星标记检测受体公猪精液以及睾丸组织中供体来源SSC的存在。结果 以3 mg/kg剂量的白消安处理大白公猪2次后,睾丸组织苏木精−伊红染色以及免疫组织化学染色结果显示,试验组睾丸曲细精管中各级生精细胞消融但其支持细胞结构完好,可以支持外源性SSC的定植及发育。免疫荧光以及流式细胞术结果表明,分离得到的睾丸单细胞悬液经纯化后UCHL-1阳性细胞占比由差速贴壁前的16.3%提高到了50.8%。苏木精−伊红染色以及免疫组织化学染色结果显示,猪SSC移植4个月后,移植组睾丸组织生精细胞恢复,在受体睾丸曲细精管基底膜上可检测到UCHL-1阳性SSC。受体睾丸组织的微卫星标记分析显示了供体SSC的存在,表明移植进入受体睾丸中的供体SSC可以在受体睾丸中定植存活超过4个月;精液微卫星标记未检测到供体来源的精子。结论 以3 mg/kg剂量的白消安注射公猪睾丸能有效消融内源性SSC,可以用来制备SSC移植的受体猪。两步酶消化及明胶差速贴壁法可成功分离纯化猪SSC。猪SSC经同种异体移植后可以在受体睾丸中定植存活超过4个月。     

成体干细胞的可塑性

传统观念认为,成体干细胞只能分化成其自身来源组织的细胞类型。但近年大量的研究表明,成体干细胞可跨系甚至跨胚层分化成在发育上与之无关的其他组织的细胞类型,即具有可塑性。文章对成体干细胞可塑性的研究结果、分化机制、应用前景及需解决的问题等进行了综述。     

骨髓基质细胞诱导的神经干细胞移植对颞叶癫痫大鼠学习记忆障碍的改善作用

   探讨骨髓基质细胞诱导的神经干细胞移植至海人酸(kanic acid,KA)致痫大鼠海马后对学习记忆障碍的改善作用,从而为干细胞移植治疗癫痫提供理论依据.体外分离大鼠骨髓基质细胞,并在特定条件下培养、诱导分化为神经干细胞.在海马内注射KA建立大鼠癫痫模型,之后把诱导的神经干细胞移植至KA致痫鼠的海马内,用Morris水迷宫测试大鼠学习记忆能力,用PCR方法检测海马内NSE、c-fos和SEZ-6基因表达,采用Western blot技术检测海马内NSE、C-FOS和SEZ-6蛋白表达.结果显示:骨髓基质细胞成功诱导分化为神经干细胞,诱导分化的神经干细胞移植到脑内可明显改善大鼠的学习记忆障碍;移植组大鼠海马内NSE、c-fos和SEZ-6基因表达下调,NSE、C-FOS和SEZ-6蛋白表达亦下调,与阳性对照组及假移植组比较,差异显著(P<0 05).说明骨髓基质细胞诱导的神经干细胞移植至KA致痫大鼠海马后对学习记忆障碍有明显的改善作用,其机制可能与NSE、c-fos、SEZ-6基因和蛋白的表达下调相关.     

胚胎干细胞的分离克隆

从全能性或多能性细胞获取、分化抑制物选择、培养基、分离方法、鉴定、保存等６个方面，对胚胎干细胞的分离培养体系的建立进行了系统综述。     

哺乳动物ES细胞与EG细胞研究进展

综述了国内外对哺乳动物ES细胞和EG细胞的研究概况, 并着重讨论了近年来国内对于ES/EG细胞的饲养层制备、诱导分化、种系嵌合与成纤维细胞的冷冻保存等方面的研究进展.探讨了ES细胞与EG细胞的共同特征及其起源的差异,指出EG细胞与ES细胞相比,具有培养材料易获得、存在时间长、无种间差异等优点.     

Purification and characterization of mouse hematopoietic stem cells

Mouse bone marrow hematopoietic stem cells were isolated with the use of a variety of phenotypic markers. These cells can proliferate and differentiate with approximately unit efficiency into myelomonocytic cells, B cells, or T cells. Thirty of these cells are sufficient to save 50 percent of lethally irradiated mice, and to reconstitute all blood cell types in the survivors.