神经干细胞向少突胶质前体细胞的定向分化诱导

本研究采用神经胶质瘤细胞株(B104 neuroblatoma cells,B104 cells)培养上清(B104CM)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF),将冷冻复苏的大鼠胚胎脊髓神经干细胞(neural stem cells,NSCs)定向诱导为少突胶质前体细胞(oligodendrocyte precusor cells,OPCs)。形态学和免疫组化的结果显示,诱导后95％以上的细胞具有双极或多极突起的典型OPCs形态,并表达A285和血小板源生长因子受体-α(platelet derived growth factor receptor-α,PDGFR-α等0PCs标志,所有PDGFR-α阳性的OPCs均不表达β-Tublin Ⅲ,其中仅少量细胞表达胶质原纤维酸性蛋白(glia fibrillary acidic protein,GFAP)。在B104CM和bFGF共存的培养条件下,悬浮培养的OPCs可大量增殖形成少突胶质细胞球,该细胞球可通过传代继续扩增,且扩增的OPCs仍能维持其特有的形态和自我增殖的特性。撤去bFGF和B104CM后,OPCs能进一步分化为成熟的少突胶质细胞(oligodendrocytes,OLs)或Ⅱ型星形胶质细胞。实验表明,诱导NSCs产生的OPCs在形态、增殖以及分化格局等方面均与已报道的存在于胚胎脑区的O-2A前体细胞相类似。该培养系统可为实验性细胞移植的研究提供丰富的细胞来源。     

一些细胞因子对哺乳动物神经干细胞增殖分化的影响

神经干细胞(neural stem cells,NSCs)的增殖、分化与中枢神经系统(central nervous system,CNS)的自我更新、神经病理损伤的修复密切相关。细胞因子可以调节NSCs的增殖,诱导NSCs进行特定分化,这对于治疗CNS损伤疾病具有重要的临床意义。该文综述了生长因子(growth factor,GF)、白细胞介素(interleukin,IL)、干扰素(interferon,IFN)等常见细胞因子对NSCs增殖和分化的影响及其可能的作用机制。其中,神经生长因子(nerve growth factor,NGF)、碱性成纤维生长因子(base fi broblast growth factor,b FGF)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)促进NSCs增殖,IL-1β、IL-17、IFN-α抑制NSCs增殖。IL-1α、血管内皮生长因子(vascular endothelial growth factor,VEGF)、IFN-γ促进NSCs向神经细胞方向分化,TNF-α、IL-1β促进NSCs向神经胶质细胞分化。有些细胞因子仅对增殖或分化有影响,有些细胞因子对增殖和分化均有影响。此外,大多数细胞因子的不同亚型产生的效应大致相同,但少数亚型可能会产生不同甚至相反的效应。     

阿魏酸钠的神经保护和神经发生增强作用

作用靶点新颖而又无副作用的抗抑郁药正处于巨大需求之中。阿魏酸(ferulic acid,FA)是一种广泛存在的低毒酚酸,阿魏酸钠(sodium ferulate,SF)则是其钠盐。我们先前的研究已经表明,阿魏酸具有显著的抗兴奋性中毒和抗抑郁样作用,而我们现在则研究其神经保护和神经发生增强效果,并讨论其神经保护和神经发生增强效果与它的抗抑郁样作用的关系。MTT法检测阿魏酸钠对PC12细胞生长的影响和它的神经保护作用;形态学和免疫细胞化学方法检测其诱导分化作用;免疫组织化学和BrdU-掺入方法检测抑郁症样模型大鼠海马神经生长因子(nerve growth factor,NGF)和脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)的表达水平及神经干细胞/神经前体细胞的增殖状况。体外试验表明,阿魏酸钠促进PC12细胞的增殖,部分保护PC12细胞免受过氧化氢(H_2O_2)和地塞米松(dexamethasone,DXM)诱导的损伤,诱导PC12细胞分化为形态特征和分子标记与神经样细胞一致的细胞。体内试验表明,阿魏酸钠上调NGF和BDNF的表达,增强抑郁症样模型大鼠海马神经干细胞/神经前体细胞的增殖。上述研究提供的证据表明,阿魏酸钠具有神经保护和神经发生增强作用,而阿魏酸钠的抗抑郁样效果可能系其神经保护和神经发生增强作用所致。     

诱导小鼠胚胎干细胞在体外分化为骨骼肌细胞的实验研究

小鼠胚胎干细胞是从胚泡未分化的内部细胞团中得到的干细胞,它在体外培养的环境中具有无限增殖、自我更新以及多向分化的特性。将小鼠胚胎干细胞在体外诱导分化为肌肉细胞,并且利用这些分化得来的肌肉细胞治疗肌肉退行性疾病,是干细胞研究领域的热点。该实验的目的在于筛选小鼠胚胎干细胞向骨骼肌细胞定向分化的实验条件,有效地将体外单层贴壁培养的小鼠胚胎干细胞诱导分化成骨骼肌细胞。最终发现,10-8mol/L维甲酸(retinoid acid,RA)+0.5%二甲基亚砜(dimethyl sulfoxide,DMSO)组诱导小鼠胚胎干细胞在体外分化成骨骼肌前体细胞的效率最高,分化得到的骨骼肌前体细胞经进一步纯化,能分化为多核的肌管。该实验为治疗肌肉退行性疾病提供了细胞来源,也为研究小鼠胚胎干细胞分化为骨骼肌细胞的机制提供了有利的条件。     

中脑神经前体细胞的体外分离、培养和特性

为了解中脑神经前体细胞的体外培养特性和建立中脑神经前体细胞的体外分化调控机制提供细胞模型。本实验采用含有丝分裂源表皮生长因子(EGF)的无血清培养基培养来源于大鼠胚胎E14.5天的中脑神经前体细胞,应用免疫细胞化学方法了解其前体细胞特性。结果发现中脑神经前体细胞呈神经前体细胞特征性标记Nestin免疫染色阳性,无分化细胞标记;细胞克隆实验证实中脑神经前体细 胞有自我更新能力;在EGF刺激下增殖迅速;当撤去EGF后置于含胎牛血清的培养基和被覆多聚赖氨酸(PLL)的培养皿内,中脑神经前体细胞可分化成神经元和星形胶质细胞。本试验证明我们培养的中脑神经前体细胞具有增殖、自我更新能力和多向分化潜能特性。     

猕猴神经干细胞的诱导分化、干性维持及同种脑内移植

为探索猕猴神经干细胞分化及特性维持,推进神经干细胞临床应用研究,该实验以绿色荧光蛋白(green fluorescence protein,GFP)为标记探讨猕猴胚胎干细胞向玫瑰花环(rosettes)结构神经干细胞的分化及其碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和表皮生长因子(epidermal growth factor,EGF)的扩增培养。结果表明:1)建立了稳定高效的猕猴神经干细胞分化体系,在该分化体系下,GFP标记猕猴胚胎干细胞在分化的第12天时,95%以上的细胞分化为神经干细胞;2)分化得到的Rosettes结构神经干细胞经bFGF/EGF扩增后,能够较好地维持其Rosettes结构;3)经bFGF/EGF扩增后的rosettes结构神经干细胞移植到猕猴脑内后能够较好的存活并向神经元分化,即bFGF/EGF扩增培养能较好地维持Rosettes结构的神经干细胞,且移植到猕猴脑内的该细胞亦能够较好地存活并向神经元分化,该结果为神经干细胞应用于临床提供了基础理论依据。     

从小鼠大脑皮层制备高纯度胶质限制前体细胞

目的:探索一种简单易行的分离、制备高纯度胶质限制前体细胞(Glial restricted precursor,GRP)的方法.方法:利用胶质限制前体细胞与星形胶质细胞分层生长的特点,采用摇培的方法将吸附在星形胶质细胞上的GRP分离下来,再利用单克隆形成实验获得单细胞来源的细胞克隆,接着细胞免疫荧光和定向分化实验鉴定细胞种类.结果:经鉴定所形成的单克隆90％为胶质限制前体细胞,它们能够分化成星形胶质细胞和少突胶质细胞,而没有分化成神经元的潜能.结论:利用细胞分层生长的特点和单克隆形成可以简便的制备高纯度的胶质限制前体细胞.     

神经干细胞--治疗脑神经性疾病的希望(1)

神经干细胞是脑中多潜能的具有自我更新能力的细胞,在特定环境和因子的诱导下能定向分化成不同的神经细胞类型,它为脑损伤修复及神经性疾病的治疗提供了新的途径,因此具有巨大的潜在应用价值和重大理论研究意义。已经发现在成体鸟类、啮齿类、灵长类和人类脑中都存在能产生神经元的神经干细胞。祖先干细胞向不同细胞系的分化受内源性和外源性机制的调制,尽管目前对这种调制的分子机制并不清楚,然而关于对神经干细胞鉴定、分离及对其特性的研究已经获得了很大的进展。     

生长分化因子-5及碱性成纤维细胞生长因子诱导家兔椎间盘髓核细胞成骨作用的研究

目的建立家兔椎间盘髓核细胞的体外培养模型,研究重组人生长分化因子-5(recombinant human growth differentiation factor-5,rhGDF-5)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对髓核细胞成骨潜能的激发作用。方法将诱导剂rhGDF-5和bFGF分别及联合加入体外培养的髓核细胞中,观察髓核细胞的成骨表型表达和细胞学特性的变化。结果 rhGDF-5和bFGF均能促进钙盐沉积形成钙结节。rhGDF-5抑制髓核细胞增殖同时增加骨钙素表达;bFGF促进髓核细胞增殖及Ⅰ型胶原表达,但对骨钙素表达无显著影响。联合使用rhGDF-5和bFGF对髓核细胞成骨潜能(促进髓核细胞增殖、Ⅰ型胶原及骨钙素表达和钙盐沉积)的激发作用均优于单独使用其中任一细胞因子。结论 rhGDF-5诱导髓核细胞向成骨细胞分化,bFGF加强该诱导作用,联合使用rhGDF-5和bFGF能充分激发髓核细胞的成骨潜能。     

microRNA参与体细胞重编程诱导分化为神经细胞的研究进展

神经退行性疾病是临床上常见的疾病,目前其治疗只停留在药物治疗及手术治疗阶段。由于神经细胞是难以再生的一种细胞类型,寻找替代神经细胞对神经退行性疾病移植治疗具有重要意义。现有研究表明,MSC(mesenchymal stem cell)、ESC(embryonic stem cell)或i PSC(induced pluripotent stem cell)能在体外分化为神经细胞,干细胞治疗神经退行性疾病具有良好的临床前景,但目前受到神经分化效率低、免疫排斥等因素的限制。研究显示,micro RNA具有参与神经发育和分化等作用,且具有重编程神经干细胞并治疗神经退行性疾病的能力。因此,该文就micro RNA参与体细胞的重编程诱导分化为神经细胞机制与作用等作一简要综述,探讨micro RNA对体细胞重编程调控作用和临床应用前景。     

BHK-21-derived cell lines that produce basic fibroblast growth factor, but not parental BHK-21 cells, initiate neuronal differentiation of neural crest progenitors.

We present evidence that basic fibroblast growth factor (bFGF)-producing cells stimulate primary differentiation of neurons from neural crest progenitors. Baby hamster kidney (BHK-21) cells were stably cotransfected with plasmid pSV2/neo, which contains the gene conferring resistance to the neomycin analog G418 and expression vectors containing the human bFGF cDNA. Various clones, which differed in their bFGF production levels, were isolated. Homogeneous neural crest cells were cultured on monolayers of bFGF-producing, BHK-21-derived cell lines. While the parental BHK-21 cells, which do not produce detectable bFGF, had poor neurogenic ability, the various bFGF-producing clones promoted a 1.5- to 4-fold increase in neuronal cell number compared to the parental cells. This increase was correlated with the levels of bFGF produced by the different transfected clones, which ranged between 2.3 and 140 ng/mg protein. In contrast, no stimulation of neuronal differentiation was observed when neural crest cells were grown on monolayers of parental BHK cells transfected with plasmid pSV2/neo alone, or on a parental BHK-derived clone, which secretes high amounts of recombinant vascular endothelial growth factor (VEGF). Furthermore, the neuron-promoting ability of bFGF-producing cells could be mimicked by addition of exogenous bFGF to neural crest cells grown on the parental BHK line. A similar treatment of neural crest cells grown on laminin substrata, instead of BHK cells, resulted in increased survival of non-neuronal cells, but not of neurons (see also Kalcheim, C. 1989, Dev. Biol. 134, 1-10). Taken together, these results suggest that bFGF stimulates neuronal differentiation of neural crest cells by a cell-mediated signalling mechanism.     

Interaction of p53 and ASPPs regulates rhesus monkey embryonic stem cells conversion to neural fate concomitant with apoptosis

The tumor suppressor p53 is a key regulator of cell apoptosis and cell cycle arrest. Recent studies show that the delicate balance of p53 expression is important for neural tube defects, neuronal degeneration, embryonic lethality, as well as differentiation and dedifferentiation. Moreover, p53 showed different regulatory patterns between rodent and primate embryonic stem cells (ESCs). However, the role of p53 and apoptosis stimulating protein of p53 (ASPP) during neural differentiation (ND) from primate ESCs is still unknown. In this study, using an FGF-2 and/or HGF selectively containing ND culture systems for rhesus monkey ESCs (rESCs), the changes of p53 and ASPPs, and p53 targets, i.e. BAX and p21, were analyzed. Our results showed that the expression patterns of ASPP1/ASPP2 and iASPP were opposite in rESCs but similar in differentiated cells, and the expression of p53 was approximately consistent with BAX, but not p21. These findings indicate that the strong expression of iASPP in ESCs and weak expression of ASPP1/ASPP2 maintain the stability of stemness; and in ND niche, unimpaired iASPP may decrease its inhibition of ASPP1/ASPP2 expression, the interaction of p53 and ASPPs causing rESCs to convert towards a neural fate concomitant with apoptosis, but not to cell cycle arrest.     

Human endometrial stem cell neurogenesis in response to NGF and bFGF

The potential of cell therapy is promising in nerve regeneration, but is limited by ethical considerations about the proper and technically safe source of stem cells. We report the successful differentiation of human EnSCs (endometrial stem cells) as a rich source of renewable and safe progenitors into high-efficiency cholinergic neurons. The extracellular signals of NGF (nerve growth factor) and bFGF (basic fibroblast growth factor) could induce cholinergic neuron differentiation. ChAT (choline acetyltransferase), MAP2 (microtubule associated protein 2) and NF-l (neurofilament L) increased after administration of bFGF and NGF to the EnSC cultures. trkC and FGFR2 (fibroblast growth factor receptor 2), which belong to the NGF and bFGF receptors respectively, were determined in populations of EnSCs. NGF, bFGF and their combination differentially influenced human EnSCs high efficiency differentiation. By inducing cholinergic neurons from EnSCs in a chemically defined medium, we could produce human neural cells without resorting to primary culture of neurons. This in vitro method provides an unlimited source of human neural cells and facilitates clinical applications of EnSCs for neurological diseases.     

Effects of Nerve Growth Factor and Basic Fibroblast Growth Factor Promote Human Dental Pulp Stem Cells to Neural Differentiation

Dental pulp stem cells (DPSCs) were the most widely used seed cells in the field of neural regeneration and bone tissue engineering, due to their easily isolation, lack of ethical controversy, low immunogenicity and low rates of transplantation rejection. The purpose of this study was to investigate the role of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on neural differentiation of DPSCs in vitro. DPSCs were cultured in neural differentiation medium containing NGF and bFGF alone or combination for 7 days. Then neural genes and protein markers were analyzed using western blot and RT-PCR. Our study revealed that bFGF and NGF increased neural differentiation of DPSCs synergistically, compared with bFGF and NGF alone. The levels of Nestin, MAP-2, βIII-tubulin and GFAP were the most highest in the DPSCs?+?bFGF?+?NGF group. Our results suggested that bFGF and NGF signifiantly up-regulated the levels of Sirt1. After treatment with Sirt1 inhibitor, western blot, RT-PCR and immunofluorescence staining showed that neural genes and protein markers had markedly decreased. Additionally, the ERK and AKT signaling pathway played a key role in the neural differentiation of DPSCs stimulated with bFGF?+?NGF. These results suggested that manipulation of the ERK and AKT signaling pathway may be associated with the differentiation of bFGF and NGF treated DPSCs. Our date provided theoretical basis for DPSCs to treat neurological diseases and repair neuronal damage.     

Wnt 3a在神经干细胞向多巴胺能神经元分化过程中作用的研究

Wnt信号在中枢神经系统发育过程中起重要的作用,控制着细胞的生长及分化.Wnt3a是Wnt家族的成员之一,对神经干细胞的增殖及分化有一定的调控作用.将重组Wnt3a腺病毒转入神经干细胞中,研究Wnt3a在定向诱导神经干细胞向多巴胺能神经元分化过程中的作用.将神经干细胞分为4组,对照组(不加任何诱导因子组)、抗坏血酸诱导组(AA组)、Wnt3a重组腺病毒诱导组(Wnt3a组)以及Wnt3a重组腺病毒加抗坏血酸诱导组(Wnt3a AA组).结果显示,Wnt3a组细胞中的多巴胺能神经元前体细胞特异性标志Nurr1表达量显著增多,Wnt3a AA组多巴胺能神经元明显多于AA组,酪氨酸羟化酶(TH)在mRNA水平上的表达是AA组的1.86倍.蛋白质印迹及免疫细胞化学染色显示,各诱导组均有TH的表达,Wnt3a组和AA组多巴胺能神经元阳性细胞数比例分别为(5.76±3.34)%和(37.42±2.54)%,与Wnt3a AA组(73.96±2.61)%比较,差异有统计学意义(P<0.05).利用高效液相色谱法检测到诱导后的细胞可分泌多巴胺.结果表明,Wnt3a可促进神经干细胞向多巴胺能神经元前体细胞分化,再通过抗坏血酸的诱导作用,在体外可获得大量的多巴胺能神经元,这些神经元有分泌多巴胺的功能.     

Hepatocyte growth factor induces proliferation and differentiation of multipotent and erythroid hemopoietic progenitors

Hepatocyte growth factor (HGF) is a mesenchymal derived growth factor known to induce proliferation and "scattering" of epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c- MET protooncogene. Here we show that highly purified recombinant HGF stimulates hemopoietic progenitors to form colonies in vitro. In the presence of erythropoietin, picomolar concentrations of HGF induced the formation of erythroid burst-forming unit colonies from CD34-positive cells purified from human bone marrow, peripheral blood, or umbilical cord blood. The growth stimulatory activity was restricted to the erythroid lineage. HGF also stimulated the formation of multipotent CFU- GEMM colonies. This effect is synergized by stem cell factor, the ligand of the tyrosine kinase receptor encoded by the c-KIT protooncogene, which is active on early hemopoietic progenitors. By flow cytometry analysis, the receptor for HGF was found to be expressed on the cell surface in a fraction of CD34+ progenitors. Moreover, in situ hybridization experiments showed that HGF receptor mRNA is highly expressed in embryonic erythroid cells (megaloblasts). HGF mRNA was also found to be produced in the embryonal liver. These data show that HGF plays a direct role in the control of proliferation and differentiation of erythroid progenitors, and they suggest that it may be one of the long-sought mediators of paracrine interactions between stromal and hemopoietic cells within the hemopoietic microenvironment.     

Basic fibroblast growth factor promotes epidermal growth factor responsiveness and survival of mesencephalic neural precursor cells.

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) induce proliferation of neural precursor cells from several central nervous system regions in vitro. We have previously described two neural precursor cell populations from 13.5 days postcoitium (dpc) mesencephalon, one forming colonies in response to EGF, present in the ventral mesencephalon, and other forming colonies in response to EGF + bFGF, mainly present in the dorsal mesencephalon. In the present work, we show that 13.5 dpc dorsal mesencephalic cells required bFGF only for 1 h to form colonies in response to EGF alone, indicating that these two growth factors act in sequence rather than simultaneously. Absence of bFGF at the beginning of the culture gave rise to very few colonies, even after the addition of EGF + bFGF, suggesting that cells responsive to bFGF were very labile in the primary culture condition. This result is in contrast with cells pretreated with bFGF, which could survive for up to 5 days in the absence of bFGF or EGF, and then were capable of efficiently forming colonies in response to EGF. Basic FGF was also able to support survival of EGF-responsive neural precursors from both ventral and dorsal mesencephalon. The population requiring bFGF to form colonies in response to EGF was identified at different developmental stages (11.5-15.5 dpc), with higher contribution to the total number of neural precursors cells detected (EGF-responsive plus bFGF-responsive) at early stages and in the dorsal region. We show that the differentiation effect of bFGF resulted in the appearance of the mRNA coding for the EGF receptor. Our data suggest that bFGF-responsive neural precursors are the source of EGF-responsive neural precursors.     

Growth factor supplemented matrigel improves ectopic skeletal muscle formation--a cell therapy approach

Following damage to skeletal muscle, satellite cells become activated, migrate towards the injured area, proliferate, and fuse with each other to form myotubes which finally mature into myofibers. We tested a new approach to muscle regeneration by incorporating myoblasts, with or without the exogenous growth factors bFGF or HGF, into three-dimensional gels of reconstituted basement membrane (matrigel). In vitro, bFGF and HGF induced C2C12 myoblast proliferation and migration and were synergistic when used together. In vivo, C2C12 or primary i28 myoblasts were injected subcutaneously together with matrigel and growth factors in the flanks of nude mice. The inclusion of either bFGF or HGF increased the vascularization of the gels. Gels supplemented with bFGF showed myogenesis accompanied by massive mesenchymal cell recruitment and poor organization of the fascicles. Samples containing HGF showed delayed differentiation with respect to controls or bFGF, with increased myoblast proliferation and a significantly higher numbers of cells in myotubes at later time points. HGF samples showed limited mesenchymal cell infiltration and relatively good organization of fascicles. The use of both bFGF and HGF together showed increased numbers of nuclei in myotubes, but with bFGF-mediated fibroblast recruitment dominating. These studies suggest that an appropriate combination of basement membrane components and growth factors could represent a possible approach to enhance survival dispersion, proliferation, and differentiation of myogenic cells during muscle regeneration and/or myoblast transplantation. This model will help develop cell therapy of muscle diseases and open the future to gene therapy approaches.     

Hepatocyte growth factor affects satellite cell activation and differentiation in regenerating skeletal muscle

Hepatocyte growth factor (HGF) is the onlyknown growth factor that activates quiescent satellite cells inskeletal muscle. We hypothesized that local delivery of HGF may enhanceregeneration after trauma by increasing the number of myoblastsavailable for restoring normal tissue architecture. Injection of HGFinto muscle at the time of injury increases myoblast number but doesnot enhance tissue repair as determined using quantitative histologicalanalyses. Rather, depending on the dose and the timing of HGFadministration relative to the injury, regeneration can be inhibited.The greatest inhibitory effect is observed when HGF is administered onthe day of injury and continued for 3 days, corresponding to the time when satellite cell activation, proliferation, and earlydifferentiation normally occur. To establish a mechanism for thisinhibition, we show that HGF can act directly on primary muscle cellsto block differentiation. These results demonstrate that1) exogenous HGF synergizes withfactors in damaged muscle to increase myoblast number,2) regeneration is not regulatedsolely by myoblast number, and 3)HGF inhibits muscle differentiation both in vitro and in vivo.

     

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside⁺ (GalC) and O4⁺ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.