人胚脊髓神经干细胞的分离培养和鉴定

目的:探讨人胚脊髓神经干细胞的体外培养和分化的方法,观察其增殖和分化特点。方法:利用无血清培养和单细胞克隆技术从人胚脊髓组织中分离培养出神经干细胞并用血清诱导其分化,应用免疫荧光细胞化学技术对培养细胞及其分化细胞进行鉴定。结果:从人胚脊髓组织分离的细胞在EGF单独存在时无法形成神经球,在bFGF单独存在时只形成少量神经球,在EGF和bFGF共同存在时形成大量具有连续增殖能力的神经球,表达神经干细胞的标志物Nestin,经血清诱导后分化为神经元、星形胶质细胞和少突胶质细胞并表达特异性抗原NSE、GFAP和CNP。结论:在体外培养条件下可从人胚脊髓组织中培养出神经干细胞,它可为神经干细胞的基础研究和临床应用提供材料。     

脊髓神经干细胞培养及体外诱导分化实验研究

目的探讨胚胎大鼠脊髓神经干细胞(SNSCs)的分离培养及体外诱导分化的特点和规律。方法利用无血清培养技术和细胞免疫化学方法。结果在表皮生长因子(EGF),碱性成纤维细胞生长因子(bFGF)的作用下,SNSCs快速增殖,形成多细胞组成的细胞球,将这些细胞球分离成单细胞重新培养,单个的细胞又很快变成细胞球。虽然白血病抑制因子(LIF)不是SNSCs培养所必需的,但是它可明显促进SNSCs增殖。诱导分化实验显示:SNSCs可分化为神经元、星型胶质细胞和少突胶质细胞,而LIF组则分化很少。结论从胚鼠脊髓分离神经干细胞是可行的,这些细胞在体外经多次传代后仍具有较强的增殖能力和多向分化潜能;LIF可以促进SNSCs增殖并抑制其分化。     

胚鼠室管膜神经干细胞体外诱导分化实验研究

目的探寻胚鼠室管膜分离培养及诱导分化为神经干细胞(NSCs)的可行性及规律性,为进一步寻找非神经组织性NSCs种子源提供阳性参照。方法将分离的胚脑室管膜组织以神经干细胞培养液孵育,确定神经干细胞的最佳体外生存环境,细胞培养所涉及的细胞因子主要有:EGF、bFGF和LIF(分别为20 ng/ml)。以细胞克隆方法判断神经干细胞增殖;以镜下细胞形态初步判断神经干细胞分化。结果胚鼠室管膜源性神经干细胞在相应培养条件下呈现出神经干细胞快速增殖,形成由多细胞组成的细胞球(神经球);进一步将这些细胞球分离成单细胞并重新以克隆密度培养,单个的细胞又很快变成岛屿状神经球。神经干细胞连续培养可进一步有小芽形成并发育成突起状结构、建立神经纤维联系,其中有的胞体增大,逐渐发育为较成熟的长突起细胞,长突起相互连接、交织成网。结论由胚鼠室管膜分离培养神经干细胞是可行的;神经干细胞在发育分化过程,基本上遵循着游离神经干细胞、神经细胞球、分化神经元/胶质细胞的生长规律,显现出神经干细胞有别于一般神经细胞的增殖及多分化特性。     

丝裂原在海马神经干细胞培养中的作用

目的 研究表皮生长因子(epidemal growth factor，EGF)和碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)在海马神经干细胞培养中的作用，确定适合海马神经干细胞体外培养的丝裂原。方法 应用EGF和bFGF刺激海马神经干细胞增殖，观察神经干细胞生长、增殖和分化等特性。结果 EGF和bFGF都能刺激神经干细胞扩增，bFGF反应性神经干细胞团增殖缓慢，细胞团中部分细胞迁出，迁出的细胞形成新的细胞团或分化成神经细胞或神经胶质细胞，而且bFGF反应性神经干细胞贴壁很紧，不易传代；相反，EGF反应性神经干细胞快速增殖，易于传代，较少迁移和分化。结论 EGF可促使海马神经干细胞快速增殖，是体外培养海马神经干细胞比较合适的丝裂原。     

人小龄胚胎神经干细胞的分离培养、扩增及鉴定

目的 探索人胚胎神经干细胞的体外分离培养条件，从而在体外大量扩增神经干细胞；并观察神经干细胞增殖、分化的特点。方法 从人胚胎脑分离神经干细胞，部分细胞冻存，另一部分细胞进行体外培养。采用无血清培养液，加入表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF)刺激细胞增殖，进行体外扩增、传代培养。采用免疫荧光法鉴定神经干细胞和分化的神经元及胶质细胞。结果 从人胚胎脑分离的细胞在含有EGF和bFGF的无血清培养液中能形成大量的神经干细胞球，这些神经干细胞球可在体外扩增及传代培养。免疫荧光法鉴定神经干细胞球中大部分为神经上皮干细胞蛋白(nestin)表达阳性细胞。贴壁后可以分化出神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)表达阳性的细胞。经冻存后的胎脑细胞也能培养出具有同样特征的神经干细胞。结论 在含有EGF和bFGF的无血清培养液中，从人胚胎脑能分离培养出神经干细胞，并能在体外大量扩增。这为人类神经干细胞的进一步研究和应用提供了材料。     

bFGF和BDNF对小鼠神经干细胞体外增殖分化的影响

目的观察碱性成纤维生长因子（bFGF）和脑源性神经生长因子（BDNF）对小鼠源性神经干细胞（NSCs）体外增殖及分化的影响。方法用无血清培养与单克隆技术对小鼠胚胎脑组织进行分离、培养,根据培养液中所加营养因子的不同将传代的NSCs分为bFGF组、BDNF组、bFGF＋BDNF组,观察不同组别对NSCs的定向分化作用。结果与bFGF和BDNF组相比,bFGF＋BDNF组细胞分化为神经元的比例较高（P〈0.05）。结论 bFGF可以提高BDNF诱导小鼠NSCs向神经元分化的比例。     

碱性成纤维生长因子和表皮生长因子对脊髓神经干细胞增殖与分化的影响

目的观察碱性成纤维生长因子(bFGF)和表皮生长因子(EGF)对胚胎脊髓神经干细胞(NSC)增殖与分化的影响。方法从14 d胚胎大鼠的脊髓组织中分离培养脊髓NSC,并随机分为3组:EGF组、bFGF组和bFGF+EGF组。通过光镜观察不同时间点各组脊髓NSC克隆细胞团数量及直径大小,并采用免疫荧光染色检测各组脊髓NSC向神经元和星形胶质细胞分化的情况。结果①EGF组培养1、3、7 d后NSC克隆细胞团数量和直径均少于bFGF和bFGF+EGF组,差异有统计学意义(P0.05)。而bFGF+EGF组仅在培养1 d时克隆细胞团数量多于bFGF组,在培养3、7 d时差异无统计学意义。②EGF组分化细胞中神经元比例显著少于bFGF和bFGF+EGF组,星形胶质细胞数量明显大于bFGF和bFGF+EGF组,差异有统计学意义(P0.05)。而bFGF和bFGF+EGF组组间差异无统计学意义。结论 EGF对脊髓NSC克隆形成有一定作用,而bFGF能较好地促进克隆细胞团的形成及生长,两者联合应用在培养早期可显著促进克隆细胞团形成。EGF可诱导脊髓NSC更多分化为星形胶质细胞,而bFGF则可促进脊髓NSC向神经元分化。     

EGF、bFGF、NGF、RA对小鼠神经干细胞定向分化的调控

目的 观察EGF、bFGF、NGF、RA对小鼠神经十细胞(NSCs)增殖及定向分化的影响。方法 用无血清培养与单细胞克隆技术对小鼠胚胎脑组织进行分离、培养，随后分别加人EGF、bFGF、NGF、RA等因子观察其对NSCs定向分化的影响。结果 EGF培养的小鼠NSCs，2d后见细胞集落形成(典型克隆球)；克隆球中的细胞成圆形，形态规则，未见细胞突起，呈桑椹状聚集；经血清诱导分化后，则主要为星形胶质细胞，仅有个别的神经元。而bFGF培养的NSCs，生长良好，但克隆球数目较EGF组少，克隆球体积略小。3bFGF与EGF共同培养的NSCs，除诱导分化后，分化的细胞性质与EGF组的不同外，其余同EGF组。NGF对神经球的形成无明显的影响。RA组的克隆球数目量很少，形态以球形为主，部分细胞贴壁并出现突起。结论 EGF，bFGF对NSCs的增殖及分化种类均有一定的影响。而NGF与RA则主要影响其细胞的分化，且两者的共同点都是促使其向神经元方向分化。     

人类神经干细胞的长期培养和传代

目的 探讨人类神经干细胞的体外培养条件及其传代的方法。方法 采用机械方法从胎脑中分离神经细胞，应用N2培养基进行培养，bFGF和EGF刺激细胞扩增；传统方法和对神经球切割的方法进行传代培养；应用免疫组织化学染色对培养的细胞及其分化的细胞进行鉴定。结果 从胎脑当中成功培养出人类的神经干细胞，培养条件下呈悬浮状态生长，形成神经球，绝大多数的细胞表达波形蛋白和Musashil两种神经干细胞的标志物；这种细胞可分化为神经元和星型胶质细胞，早期的培养有少量的少突胶质细胞；在这种培养条件下，神经干细胞生长速度较慢，而采用切割神经球的方法保持了细胞间的，神经干细胞可获得较大的扩增速度。结论 在体外的培养条件下，可从胎脑组织中培养出神经干细胞，它可做为中枢神经系统疾病移植治疗的潜在细胞来源。     

人胚神经干细胞的体外培养及鉴定

目的 探讨人胚神经干细胞的体外培养和诱导分化的条件。方法 从药物流产的12周到16周的人胚胎海马组织中分离神经干细胞，在EGF、bFGF和LIF联合作用下使其稳定增殖，并用10％的胎牛血清诱导其贴壁分化，应用免疫荧光染色方法行Nestin、NSE、MAP-2、GFAP和GalC免疫荧光染色，对神经干细胞及其分化的细胞进行鉴定。结果 体外培养的神经干细胞增殖成神经干细胞球并传代，鉴定为Nestin染色阳性细胞，并可诱导分化为神经细胞、星形胶质细胞和少突胶质细胞。结论 利用无血清培养技术和特定生长因子，可培养出在体外稳定增殖并有多向分化潜能的人胚神经干细胞。     

Cellular composition of long-term human spinal cord- and forebrain-derived neurosphere cultures

In vitro expanded neural precursor cells (NPCs) may provide a stable source for cell therapy. In search of the optimal cell source for spinal cord repair, we investigated influences of gestational age, regional heterogeneity, and long-term in vitro propagation. The cellular content of neurosphere cultures prior to and after in vitro differentiation was studied by immunocytochemistry and flow cytometry. Human forebrain and spinal cord NPCs deriving from first-trimester tissue were cultured as neurospheres in the presence of epidermal growth factor, basic fibroblast growth factor, and ciliary neurotrophic factor. Proteins characteristic for embryonic stem cells, i.e., Tra-1-60, Tra-1-81, and SSEA-4, were present in approximately 0.5% of the cells in donor tissues and neurospheres. The proportions of nestin- and proliferating cell nuclear antigen-immunoreactive (IR) cells were also maintained, whereas the CD133-IR population increased in vitro. Glial fibrillary acidic protein-IR cells increased in number, and in contrast the fraction of beta-tubulin III-IR cells decreased, at and beyond passage 5 in spinal cord but not forebrain cultures. However, dissociated and in vitro-differentiated forebrain- and spinal cord-derived neurospheres generated similar proportions of neurons, astrocytes, and oligodendrocytes. Gestational age of the donor tissue, which ranged from 4.5 to 12 weeks for forebrain and from 4.5 to 9.5 weeks for spinal cord, did not affect the proportion of cells with different phenotypes in culture. Thus, cellular composition of human neurosphere cultures differs as a result of long-term in vitro propagation and regional heterogeneity of source tissue, despite expansion under equal culture conditions. This could in turn imply that human spinal cord and forebrain NPCs present different repair potentials in in vivo settings.     

miR-124 and miR-128 differential expression in bone marrow stromal cells and spinal cord-derived neural stem cells

BACKGROUND: MicroRNA (miRNA) expression in stem cells provides important clues for the molecular mechanisms of stem cell proliferation and differentiation. Bone marrow stromal cells and spinal cord-derived neural stem cells exhibit potential for neural regeneration. However, miRNA expression in these cells has been rarely reported. OBJECTIVE: To explore differential expression of two nervous system-specific miRNAs, miR-124 and miR-128, in bone marrow stromal cells and spinal cord-derived neural stem cells.DESIGN, TIME AND SETTING: An In vitro, cell biology experiment was performed at the Department of Biotechnology, Shanxi Medical University from June 2008 to June 2009.MATERIALS: TaqMan miRNA assays were purchased from Applied Biosystems. METHODS: Rat bone marrow stromal cells were isolated and cultured using the whole-bone marrow method, and rat spinal cord-derived neural stem cells were obtained through neurosphere formation. TaqMan miRNA assays were used to measure miR-124 and miR-128 expression in bone marrow stromal cells and spinal cord-derived neural stem cells.MAIN OUTCOME MEASURES: Morphology of bone marrow stromal cells and spinal cord-derived neural stem cells were observed by inverted microscopy. Expression of the neural stem cell-specific marker, nestin, the bone marrow stromal cell surface marker, CD71, and expression of miR-124 and miR-128, were detected by real-time polymerase chain reaction. RESULTS: Cultured bone marrow stromal cells displayed a short fusiform shape. Flow cytometry revealed a large number of CD71-positive cells (> 95%). Cultured spinal cord-derived neural stem cells formed nestin-positive neurospheres, and quantitative detection of miRNA demonstrated that less miR-124 and miR-128 was expressed in bone marrow stromal cells compared to spinal cord-derived neural stem cells (P < 0.05). CONCLUSION: Bone marrow stromal cells and spinal cord-derived neural stem cells exhibited differential expression of miR-124 and miR-128, which suggested different characteristics in miRNA expression.     

Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord

Recent advances in the isolation and characterization of neural precursor cells suggest that they have properties that would make them useful transplants for the treatment of central nervous system disorders. We demonstrate here that spinal cord cells isolated from embryonic day 14 Sprague-Dawley and Fischer 344 rats possess characteristics of precursor cells. They proliferate as undifferentiated neurospheres in the presence of EGF and bFGF and can be maintained in vitro or frozen, expanded and induced to differentiate into both neurons and glia. Exposure of these cells to serum in the absence of EGF and bFGF promotes differentiation into astrocytes; treatment with retinoic acid promotes differentiation into neurons. Spinal cord cells labeled with a nuclear dye or a recombinant adenovirus vector carrying the lacZ gene survive grafting into the injured spinal cord of immunosuppressed Sprague-Dawley rats and non-immunosuppressed Fischer 344 rats for up to 4 months following transplantation. In the presence of exogenously supplied BDNF, the grafted cells differentiate into both neurons and glia. These spinal cord cell grafts are permissive for growth by several populations of host axons, especially when combined with exogenous BDNF administration, as demonstrated by penetration into the graft of axons immunopositive for 5-HT and CGRP. Thus, precursor cells isolated from the embryonic spinal cord of rats, expanded in culture and genetically modified, are a promising type of transplant for repair of the injured spinal cord.     

Comparative analysis of in vitro conditions for rat adult neural progenitor cells

Various protocols have been published for in vitro expansion and maintenance of adult neural progenitor cells (ANPC). However, there are only few data comparing these protocols regarding their influence on proliferation, migration and differentiation. Freshly isolated ANPC from olfactory bulb (BO) and dentate gyrus (DG) of adult rat brains forming neurospheres and expressing the neural stem cell markers nestin and Sox-2 were used in a comparative analysis of five different medium combinations. Medium containing N2 and fetal calf serum (FCS), but no additional cytokines was unsuitable for an effective long-term expansion of ANPC due to a significantly reduced proliferation rate. Media containing BIT, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet-derived growth factor AB (PDGF-AB) and leukemia inhibitory factor (LIF) or B27, bFGF and EGF are recommendable for the cultivation of DG-derived ANPC as neurospheres only. Unlike, culture media containing BIT, bFGF, EGF and PDGF-AB or N2, bFGF and EGF were suitable for all applications tested as they responded similarly regarding proliferation, migration and expression of differentiation markers. The results of the present study might help to improve the effective in vitro expansion of ANPC derived from rare human tissue samples.     

Adult human spinal cord harbors neural precursor cells that generate neurons and glial cells in vitro

Adult human and rodent brains contain neural stem and progenitor cells, and the presence of neural stem cells in the adult rodent spinal cord has also been described. Here, using electron microscopy, expression of neural precursor cell markers, and cell culture, we investigated whether neural precursor cells are also present in adult human spinal cord. In well-preserved nonpathological post-mortem human adult spinal cord, nestin, Sox2, GFAP, CD15, Nkx6.1, and PSA-NCAM were found to be expressed heterogeneously by cells located around the central canal. Ultrastructural analysis revealed the existence of immature cells close to the ependymal cells, which display characteristics of type B and C cells found in the adult rodent brain subventricular region, which are considered to be stem and progenitor cells, respectively. Completely dissociated spinal cord cells reproducibly formed Sox2(+) nestin(+) neurospheres containing proliferative precursor cells. On differentiation, these generate glial cells and gamma-aminobutyric acid (GABA)-ergic neurons. These results provide the first evidence for the existence in the adult human spinal cord of neural precursors with the potential to differentiate into neurons and glia. They represent a major interest for endogenous regeneration of spinal cord after trauma and in degenerative diseases.     

Adult neural progenitor cell grafts survive after acute spinal cord injury and integrate along axonal pathways

The main rationale for cell-based therapies following spinal cord injury are: (i) replacement of degenerated spinal cord parenchyma by an axon growth supporting scaffold; (ii) remyelination of regenerating axons; and (iii), local delivery of growth promoting molecules. A potential source to meet these requirements is adult neural progenitor cells, which were examined in the present study. Fibroblast growth factor 2-responsive adult spinal cord-derived syngenic neural progenitor cells were either genetically modified in vitro to express green fluorescent protein (GFP) using retroviral vectors or prelabelled with bromodeoxyuridine (BrdU). Neural progenitor cells revealed antigenic properties of neurons and glial cells in vitro confirming their multipotency. This differentiation pattern was unaffected by retroviral transduction. GFP-expressing or BrdU-prelabelled neural progenitor cells were grafted as neurospheres directly into the acutely injured rat cervical spinal cord. Animals with lesions only served as controls. Three weeks postoperatively, grafted neural progenitor cells integrated along axonal profiles surrounding the lesion site. In contrast to observations in culture, grafted neural progenitor cells differentiated only into astro- and oligodendroglial lineages, supporting the notion that the adult spinal cord provides molecular cues for glial, but not for neuronal, differentiation. This study demonstrates that adult neural progenitor cells will survive after transplantation into the acutely injured spinal cord. The observed oligodendroglial and astroglial differentiation and integration along axonal pathways represent important prerequisites for potential remyelination and support of axonal regrowth.