川芎嗪对体外培养的胎鼠神经干细胞增殖和分化的影响

目的：探讨川芎嗪（TMP）在体外神经干细胞（NSCs）增殖与分化中的作用。方法：原代提取孕14 d雌性大鼠的胎鼠大脑皮层分离培养,并作免疫荧光染色鉴定,取传代培养第3代的NSCs进行实验。实验分为对照组、β-巯基乙醇阳性对照组、TMP诱导组和TMP+EGTA组（n=4）。采用BrdU法和MTT法观察川芎嗪对NSCs增殖数量的影响,采用蛋白免疫印迹法检测NSCs的分化表达情况。结果：实验成功分离纯化原代NSCs,培养3~5 d可见部分神经球形成,具备典型的NSCs形态并表达NSCs特异抗原巢蛋白;BrdU法和MTT法结果均显示,与对照组和β-巯基乙醇阳性对照组相比,TMP组NSCs增殖数量明显增多（P<0.05）;蛋白免疫印迹结果显示,TMP组和TMP+EGTA组NSCs的神经元分化率明显增高,TMP+EGTA组分化率增高更明显（P<0.05）。结论：TMP能显著增强NSCs的增殖和神经元分化率。减少细胞外Ca²⁺可促进TMP诱导NSCs向神经元分化,Ca²⁺信号在TMP诱导NSCs向神经元分化过程中起重要作用。     

胎胰源性Nestin阳性细胞向多巴胺能细胞元定向诱导分化的研究

目的：探讨胎儿胰岛源性Nestin（神经上皮干细胞蛋白）阳性干细胞分化为多巴胺能神经元的潜能。方法：用胶原酶消化法分离胎儿胰岛,贴壁培养后获得增殖力旺盛的细胞;用免疫组化法、免疫荧光法分别检测其增殖细胞核抗原（PCNA）及神经干细胞标志物Nestin的表达;用流式细胞术测定Nestin阳性细胞的比例;经N2培养液筛选后,分别用SHH（sonichedgehog）蛋白、成纤维细胞生长因子（FGF）8、胶质细胞源性神经营养因子（GDNF）和脑源性神经营养因子（BDNF）向多巴胺能神经元定向诱导,检测诱导细胞的多巴胺能神经元标志酪氨酸羟化酶（TH）和芳香左旋氨基酸脱羧酶（AADC）的表达情况。结果：免疫荧光显示,从胎儿胰岛分离的干细胞表达PCNA和Nestin;流式细胞术检测Nestin阳性率达13．74％;筛选后向神经细胞定向诱导分化,细胞表达TH和AADC。结论：从胎儿胰岛中可以分离出Nestin阳性的神经干细胞,该细胞具有向多巴胺能神经元定向分化的能力。     

溶血磷脂酸促进离体大鼠胚胎神经干细胞的增殖及其向胆碱能神经元分化

溶血磷脂酸（1ysophosphatidic acid,LPA）是一种细胞外磷脂信号。本研究用[^3H]-胸腺嘧啶掺入法、免疫细胞化学和Western blot等技术,观察了LPA对体外培养的大鼠胚胎神经干细胞（neural stem cells,NSCs）的增殖以及向MAF2标记的一般神经元和ChAT标记的胆碱能神经元的分化的影响。结果显示：（1）在特殊的无血清培养基中加入低浓度的LPA（0．01-1．0μmol/L）后,NSCs对【^3H】-胸腺嘧啶的摄入呈剂量依赖性增加,表明LPA对NSCs有显著的促增殖作用;（2）在培养基中加入胎牛血清以诱导NSCs的分化,发现低浓度的LPA增加MAF2阳性和ChAT阳性神经元的比例,0．1μmol／L LPA引起的增加达到峰值;（3）Western blot分析显示LPA促进了MAP2和ChAT的表达;（4）在诱导NSCs出现分化早期,用倒置显微镜观察到低浓度的LPA明显促进细胞突起的生长和细胞的迁移。以上结果表明,低浓度LPA在一定范围内可以促进NSCs的增殖、并分化为一般的MAP2阳性神经元和特殊的胆碱能神经元,而且LPA可以促进在分化早期出现的神经元或神经胶质细胞前体细胞的迁移和突起生长。     

大鼠BMSCs条件培养基对NSCs形态、生长及分化的影响

通过对SD大鼠骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)及新生鼠神经干细胞(neural stem cells,NSCs)进行体外分离、培养及鉴定后,观察BMSCs条件培养基对NSCs向神经细胞分化的影响。采用全贴壁培养法培养BMSCs,并采用流式细胞术鉴定其特异性表面抗原标记。无血清技术培养NSCs,采用免疫荧光技术鉴定其特异性抗原标记。BMSCs条件培养基(含10%胎牛血清的DMEM培养基)对NSCs诱导7 d后,镜下观察细胞形态和生长,并采用免疫荧光技术检测NSCs分化。BMSCs高表达CD90、CD29(90%),而CD45呈阴性表达。免疫荧光染色显示,NSCs标记蛋白Nestin、SOX2为阳性。NSCs经BMSCs培养液诱导分化7 d后经免疫荧光鉴定,MAP-2及GFAP呈阳性,阳性率分别为73.80%和50.47%;NSCs经胎牛血清(fetal bovine serum,FBS)诱导分化7 d后经免疫荧光鉴定,MAP-2及GFAP呈阳性,阳性率分别为42.14%和31.90%。BMSCs条件培养基可诱导NSCs分化为神经元及星形胶质细胞,其中BMSCs分泌的细胞因子在诱导分化中可能发挥重要作用。     

低糖低氧对神经干细胞增殖和代谢的影响

目的:本研究旨在探讨低糖低氧对大鼠神经干细胞增殖和代谢的影响。方法:实验采用不同葡萄糖浓度的培养基以及不同的氧浓度进行处理:高糖(4.5g/L)、低糖(1.4g/L);常氧(20%O2)、低氧(3%O2);神经干细胞(NSCs)来自孕13.5d的大鼠中脑,在不同糖浓度下培养至第三代进行低氧处理,分为低糖常氧(L+N)、低糖低氧(L+H)、高糖常氧(H+N)、高糖低氧(H+H)组。神经干细胞在上述四种条件下分别培养1、3、5d后,利用CCK-8检测神经干细胞的增殖情况;生化分析仪测定细胞培养上清液中葡萄糖、乳酸、丙酮酸浓度;RT-PCR方法检测葡萄糖转运蛋白4(GluT4)、葡萄糖激酶(GK)、丙酮酸激酶(PK)和乳酸脱氢酶(LDH)的表达变化。结果:在低糖低氧条件下培养3d时NSCs的数量增加最为明显;低糖低氧条件下,葡萄糖浓度降低最为显著;而丙酮酸浓度在低糖处理组均高于高糖处理组;同样地,在低糖低氧处理组培养上清中乳酸含量增加的幅度最大;此外,在低糖或低氧时Glut4和PK的表达也明显高于对照组。结论:低氧能促进NSCs的增殖,而以低氧和低糖共同作用时更为明显;在低氧低糖条件下,神经干细胞的代谢发生变化,葡萄糖的利用明显增加,主要通过糖酵解途径代谢产能。     

乳鼠海马神经干细胞的体外分离、扩增和分化研究

探讨海马神经干细胞（neuralstemcells,NSCs）在体外分离扩增和诱导分化的可行性。无菌条件下分离新生（24h）SD大鼠海马神经干细胞,采用无血清培养和胎牛血清诱导分化。免疫荧光染色技术分别检测诱导前细胞巢蛋白（Nestin）的表达,以及分化细胞的神经元特异性烯醇化酶（neuron specific enolase,NSE）、胶质纤维酸性蛋白（glialfibrillaryacidicprotein,GFAP）的表达,以鉴定细胞类型。流式细胞仪检测神经干细胞分化前后增殖能力的变化。结果显示：从乳鼠海马分离培养的细胞生长状态良好,具有克隆增殖能力,并呈Nestin表达阳性,分化后可出现NSE及GFAP表达阳性的细胞。流式细胞仪检测显示：诱导前,细胞增殖活跃,S＋G2／M期细胞为（36．27±1．99）％,而分化各阶段（3,7,10d）S＋G2／M期细胞比例与诱导前（Ctrl）相比则明显下降（尸〈0．05）,分别为（26．39±1．10）％、（26．33±1．33）％和（24．54±1．12）％。这些结果表明乳鼠海马存在神经干细胞,并具有自我更新和多向分化的潜能,可用于基础和临床的相关研究。     

体外诱导人骨髓间充质干细胞向多巴胺神经元分化的研究

通过体外诱导人骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)向多巴胺(dopamine, DA)神经元分化,探讨人BMSCs来源的DA神经元的功能特征及其分化机制,为临床上细胞移植替代治疗诸如帕金森氏病(parkinson's disease, PD)等神经精神性疾病提供一种理想的细胞来源。通过密度梯度离心获取人骨髓中的单个核细胞,贴壁培养纯化BMSCs。50μmol/L脑源性神经营养因子(brain derived neurotrophy factor, BDNF),10μmol/L forskolin(FSK)和10μmol/L DA联合对BMSCs进行诱导。电子显微镜观察诱导2周后细胞是否具有神经元的超微结构特点;免疫细胞化学染色和RT-PCR检测DA神经元分化过程中的标志物酪氨酸羟化酶(tyrosine hydroxylase, TH）的表达以及转录因子Nurr1、Ptx3和Lmx1b的表达;高效液相色谱（high performance liquid chromatogram, HPLC）检测诱导2周后的细胞多巴胺的释放水平。 结果表明,诱导2周后,电镜下细胞胞浆中有大量密集的呈扁平囊状的粗面内质网及其间的一些游离核糖体以及神经微丝的形成。RT-PCR结果显示NSE(neuron specific enolase)、Nurr1、Ptx3、Lmx1b和TH的mRNA均有表达;免疫细胞化学染色结果表明诱导2周后TH阳性细胞(24.80±3.36）%的表达较诱导3d后(3.77±1.77)%明显提高(P<0.01）;HPLC检测到诱导2周后的细胞DA释放水平［(1.22±0.36)μg/mL(n＝6)］高于未经诱导的细胞［(0.75±0.22)μg/mL(n＝6) (t＝－2.79,P＝0.038)］。 由此得出,BDNF、FSK和DA可以在体外诱导人BMSCs向DA神经元分化,并具有DA神经元的功能特征,是临床用于治疗神经精神性疾病的理想细胞来源。     

诱导人脐血间充质干细胞分化为多巴胺能神经元样细胞的研究

目的：探讨人羊膜上皮细胞条件培养液(ACM)及SHH,FGF8诱导人脐血间充质干细胞（CB-MSCs）分化为多巴胺( dopamine,DA )能神经元样细胞的作用及机制。方法：利用沉降红细胞、密度梯度离心和贴壁筛选法纯化CB-MSCs,加入ACM及SHH,FGF8分为CON,ACM,SHH+FGF8,ACM+SHH+FGF8四组诱导48h后,免疫细胞化学染色鉴定分化细胞的DA神经元表型。应用高压液相色谱技术测定细胞培养上清中DA含量,并应用Realtime PCR方法观察DA能神经元发育过程中的相关基因En-1,Foxa2,Lmx1b,Gli-1,Pitx3,Nurr1以及Ngn2的表达。结果：加诱导剂诱导48h后,各诱导组的TH和DAT的表达量均比对照组高。早期的转录因子En1,Foxa2 在各组中均有表达;而Pitx3,Lmx1b表达在各诱导组中;Lmx1b在ACM+SHH+FGF8组中表达最高;Pitx3在各诱导组中均有表达且无明显差异。Nurr1,Ngn2在SHH+FGF8组中表达很强。结论：ACM及SHH,FGF8可诱导脐血间充质干细胞分化为DA能神经元样细胞,其作用机制与多巴胺能神经元发育过程中的各种基因En-1,Foxa2,Lmx1b,Gli-1,Pitx3,Nurr1以及Ngn2相关。     

一氧化氮促进体外培养大鼠脑室下区神经干细胞的分化

目的：探讨一氧化氮（NO）对新生大鼠体外培养的神经干细胞（NSCs）分化的作用。方法：采用常规方法分离新生大鼠脑室下区（SVZ）组织,进行NSCs体外培养。用DETA／NO作为NO供体,用L-NAME作为一氧化氮合酶（NOS）抑制剂。免疫荧光法检测NSCs标志物-巢蛋白（nestin）、神经元标志物-8Ⅲ型微管蛋白（Tuj-1）和星型胶质细胞标志物-胶质原纤维酸性蛋白（GFAP）的表达,还检测了神经元型NOS的表达。用Greiss还原法检测培养液中总NO的浓度。结果：培养的神经球均为nestin阳性、BIdu阳性和nNOS阳性。NSCs和40μmol／L、50μmol／L、60μmol／LDEFA／N0共培养5d,实验组培养液中N0浓度较对照组显著增高（P〈0．01）,相应实验组分化的神经元数和星型胶质细胞数较对照组明显增加（P〈0．01和P〈0．05）。NSCs和100μmol／L、150μmol／L、200μmol／LL-NAME共培养5d,实验组培养液中NO浓度较对照组降低（P〈0．05）,相应实验组分化的神经元数和星型胶质细胞数也较对照组减少（P〈0．05）。结论：NO能直接促进大鼠SVZ体外培养的NSCs分化。     

Characteristics of neural stem cells expanded in lowered oxygen and the potential role of hypoxia-inducible factor-1Alpha

It has recently been reported that hypoxia promotes the survival and proliferation of neural stem cells (NSCs). In the present study, we examine the differentiation ability of neural precursors expanded under lowered oxygen conditions, and the potential role of hypoxia-inducible factor (HIF)-1alphain vitro, which is the key molecule in response to lowered oxygen. The NSCs were cultured in a 3% O(2) environment for 3 days, and differentiated with 1% fetal bovine serum (FBS) for another 5-7 days, and the cell lineage was evaluated by immunohistochemistry, flow cytometry and HPLC. Compared with the normal condition, the NSCs cultured in hypoxia (3% O(2)) displayed an increase in the percentage of neurons. Especially the percentage of TH-positive neurons differentiated from NSCs in lowered oxygen increased significantly; the dopamine (DA) content in the medium was higher than under normal conditions. These data indicate that lowered oxygen favors dopaminergic differentiation. We then examined the expression of HIF-1alpha during differentiation of NSCs. The levels of HIF-1alpha mRNA expression under 3% oxygen did not change as compared with those under normal conditions. However, HIF-1alpha protein expression was higher from 3 to 72 h during hypoxia than under normal conditions. Overexpression of HIF-1alpha significantly increased the number of TH-positive cells and the DA content in culture medium under normal conditions. These results suggest that HIF-1alpha is involved in the regulation of dopaminergic differentiation of NSCs in lowered oxygen. This study may also offer a new approach to yield DA neurons using a physical factor.     

Generation of dopamine neurons from human embryonic stem cells in vitro

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESCs) in vitro. It was shown that human ESCs can be differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by the successive application of noggin and bFGF growth factors and collagen and matrigel substrates for 3–4 weeks. The efficiency of differentiation was evaluated by the number of colonies with cells that express tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension estimated by flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. The lack of pluripotent cells in population at the final stage of differentiation is encouraging and shows that this protocol of human ESC differentiation may be applied to generate DA neurons for their transplantation into the animals modeling neurodegenative (Parkinson) disease without the risk of tumor growth.     

Generation of dopamine neurons from human embryonic stem cells in vitro

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth.     

胚胎大鼠脑和脊髓神经干细胞的分离和培养

研究采用显微解剖、无血清细胞培养和免疫荧光细胞化学染色等实验技术 ,成功地建立了胚胎大鼠脑和脊髓神经干细胞 (NSCs)的分离和培养方法。结果显示 ,( 1)在含成纤维细胞生长因子 2 (FGF 2 )和表皮生长因子(EGF)的无血清培养液中 ,两种来源的NSCs经体外培养 8- 10代后 ,其细胞数呈指数级增加 ,其中脑来源的NSCs数由原代培养时的 1× 10 6 增加至 1× 10 12 ,脊髓来源的NSCs数从 1× 10 6 增加至 1× 10 11。增殖的细胞表达神经上皮干细胞蛋白 (nestin) ;( 2 )在含 1%胎牛血清 (FBS)的培养条件下 ,它们都能被诱导分化为神经元、少突胶质细胞和星型胶质细胞。但其分化比例可随细胞传代次数的增加而改变 ,其中 ,大脑来源的NSCs分化为神经元的比例从第二代 (P2 )的 11 95± 2 5 %下降至第五代 (P5)的 1 97± 1 16% (P <0 0 1) ,而少突胶质细胞的分化比例则基本保持不变 ,这一分化格局同样可在脊髓来源的NSCs中发现。结果表明 ,我们所分离和培养的细胞在体外经多次传代后仍具有很强的增殖能力和多向分化潜能 ,它们都表达nestin ,属于中枢神经系统的干细胞     

体外诱导人骨髓间充质干细胞向多巴胺神经元分化的研究

通过体外诱导人骨髓间充质干细胞(bone marrow mesenchymal stemcells,BMSCs)向多巴胺(dopamine,DA)神经元分化,探讨人BMSCs来源的DA神经元的功能特征及其分化机制,为临床上细胞移植替代治疗诸如帕金森氏病(parkinson’sdisease,PD)等神经精神性疾病提供一种理想的细胞来源。通过密度梯度离心获取人骨髓中的单个核细胞,贴壁培养纯化BMSCs。50μmol/L脑源性神经营养因子(brain derivedneurotrophy factor,BDNF),10μmol/Lforskolin(FSK)和10μmol/LDA联合对BMSCs进行诱导。电子显微镜观察诱导2周后细胞是否具有神经元的超微结构特点;免疫细胞化学染色和RT-PCR检测DA神经元分化过程中的标志物酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达以及转录因子Nurr1、Ptx3和Lmx1b的表达;高效液相色谱(highperformance liquid chromatogram,HPLC)检测诱导2周后的细胞多巴胺的释放水平。结果表明,诱导2周后,电镜下细胞胞浆中有大量密集的呈扁平囊状的粗面内质网及其间的一些游离核糖体以及神经微丝的形成。RT-PCR结果显示NSE(neuron specificenolase)、Nurr1、Ptx3、Lmx1b和TH的mRNA均有表达;免疫细胞化学染色结果表明诱导2周后TH阳性细胞(24·80±3·36)%的表达较诱导3d后(3·77±1·77)%明显提高(P<0·01);HPLC检测到诱导2周后的细胞DA释放水平[(1·22±0·36)μg/mL(n=6)]高于未经诱导的细胞[(0·75±0·22)μg/mL(n=6)(t=-2·79,P=0·038)]。由此得出,BDNF、FSK和DA可以在体外诱导人BMSCs向DA神经元分化,并具有DA神经元的功能特征,是临床用于治疗神经精神性疾病的理想细胞来源。     

Effects of hypoxia on the proliferation and differentiation of NSCs

Oxygen is vital to nearly all forms of life on Earth via its role in energy homeostasis and other cell functions. Until recently, the effects of oxygen on the proliferation and differentiation of neural stem cells (NSCs) have been largely ignored. Some studies have been carried out on the basis of the fact that NSCs exists within a “physiological hypoxic” environment at 1 to 5% O₂ in both embryonic and adult brains. The results showed that hypoxia could promote the growth of NSCs and maintain its survival in vitro. In vivo studies also showed that ischemia/hypoxia increased the number of endogenous NSCs in the subventricular zone and dentate gyrus. In addition, hypoxia could influence the differentiation of NSCs. More neurons, especially more doparminergic neurons, were produced under hypoxic condition. The effects of hypoxia on the other kind of stem cell were briefly introduced as additional evidence. The mechanism of these responses might be primarily involved in the hypoxic inducible factor-1 (HIF-1) signal pathway. The present review summarizes recent works on the role of hypoxia in the proliferation and differentiation of NSCs both in vitro and in vivo, and the mechanism involved in HIF-1 signaling pathway behind this response was also discussed.     

Protoplasmic astrocytes enhance the ability of neural stem cells to differentiate into neurons in vitro

Protoplasmic astrocytes have been reported to exhibit neuroprotective effects on neurons, but there has been no direct evidence for a functional relationship between protoplasmic astrocytes and neural stem cells (NSCs). In this study, we examined neuronal differentiation of NSCs induced by protoplasmic astrocytes in a co-culture model. Protoplasmic astrocytes were isolated from new-born and NSCs from the E13-15 cortex of rats respectively. The differentiated cells labeled with neuron-specific marker β-tubulin III, were dramatically increased at 7 days in the co-culture condition. Blocking the effects of brain-derived neurotrophic factor (BDNF) with an anti-BDNF antibody reduced the number of neurons differentiated from NSCs when co-cultured with protoplasmic astrocytes. In fact, the content of BDNF in the supernatant obtained from protoplasmic astrocytes and NSCs co-culture media was significantly greater than that from control media conditions. These results indicate that protoplasmic astrocytes promote neuronal differentiation of NSCs, which is driven, at least in part, by BDNF.     

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.     

Derivation of neural precursor cells from human ES cells at 3% O(2) is efficient, enhances survival and presents no barrier to regional specification and functional differentiation

In vitro stem cell systems traditionally employ oxygen levels that are far removed from the in vivo situation. This study investigates whether an ambient environment containing a physiological oxygen level of 3% (normoxia) enables the generation of neural precursor cells (NPCs) from human embryonic stem cells (hESCs) and whether the resultant NPCs can undergo regional specification and functional maturation. We report robust and efficient neural conversion at 3% O(2), demonstration of tri-lineage potential of resultant NPCs and the subsequent electrophysiological maturation of neurons. We also show that NPCs derived under 3% O(2) can be differentiated long term in the absence of neurotrophins and can be readily specified into both spinal motor neurons and midbrain dopaminergic neurons. Finally, modelling the oxygen stress that occurs during transplantation, we demonstrate that in vitro transfer of NPCs from a 20 to 3% O(2) environment results in significant cell death, while maintenance in 3% O(2) is protective. Together these findings support 3% O(2) as a physiologically relevant system to study stem cell-derived neuronal differentiation and function as well as to model neuronal injury.