Human Mesenchymal Stem Cells Signals Regulate Neural Stem Cell Fate

Neural stem cells (NSCs) differentiate into neurons, astrocytes and oligodendrocytes depending on their location within the central nervous system (CNS). The cellular and molecular cues mediating end-stage cell fate choices are not completely understood. The retention of multipotent NSCs in the adult CNS raises the possibility that selective recruitment of their progeny to specific lineages may facilitate repair in a spectrum of neuropathological conditions. Previous studies suggest that adult human bone marrow derived mesenchymal stem cells (hMSCs) improve functional outcome after a wide range of CNS insults, probably through their trophic influence. In the context of such trophic activity, here we demonstrate that hMSCs in culture provide humoral signals that selectively promote the genesis of neurons and oligodendrocytes from NSCs. Cell–cell contacts were less effective and the proportion of hMSCs that could be induced to express neural characteristics was very small. We propose that the selective promotion of neuronal and oligodendroglial fates in neural stem cell progeny is responsible for the ability of MSCs to enhance recovery after a wide range of CNS injuries. Special issue dedicated to Anthony Campagnoni.     

BRCA-1 基因与神经干细胞

乳腺癌易感基因(Breast cancer susceptibility gene,Brca-1)是肿瘤抑制基因家族中的一员,它是乳腺癌特异性抑癌基因,1994年Miki等[1]采用定位克隆方法首次将Brca-1分离出来。Brca-1能防止细胞过快地或失去控制地生长和分化,在调节细胞进程、DNA损伤修复、细胞生长与凋亡及转录活化和抑制等多种生物学途径都发挥重要作用,Korhonen等2003年报道Brca-1基因可促进体外培养的大鼠来源的神经干细胞的增殖。     

猪神经干细胞分离培养研究进展

神经干细胞用于神经学临床修复和基础理论研究的前提是首先完成神经干细胞的体外分离、培养、纯化并大量扩增。鼠、人、猪中都已成功分离出神经干细胞并已尝试用于动物神经系统损伤等疾病的治疗,尽管在鼠和人上的研究很多,相对于鼠神经干细胞在神经学临床应用上的局限和人神经干细胞在材料来源上的不便,猪作为神经干细胞临床应用和基础研究的模式动物有很大的潜力。但关于猪神经干细胞体外分离培养的研究非常少,本文对这方面的研究进展做一综述。     

Neural regeneration by regionally induced stem cells within poststroke brains: Novel therapy perspectives for stroke patients

Ischemic stroke is a critical disease which causes serious neurological functional loss such as paresis. Hope for novel therapies is based on the increasing evidence of the presence of stem cell populations in the central nervous system(CNS) and the development of stem-cell-based therapies for stroke patients. Although mesenchymal stem cells(MSCs) represented initially a promising cell source,only a few transplanted MSCs were present near the injured areas of the CNS.Thus, regional stem cells that are present and/or induced in the CNS may be ideal when considering a treatment following ischemic stroke. In this context, we have recently showed that injury/ischemia-induced neural stem/progenitor cells(i NSPCs) and injury/ischemia-induced multipotent stem cells(i SCs) are present within post-stroke human brains and post-stroke mouse brains. This indicates that i NSPCs/i SCs could be developed for clinical applications treating patients with stroke. The present study introduces the traits of mouse and human i NSPCs,with a focus on the future perspective for CNS regenerative therapies using novel i NSPCs/i SCs.     

Identification of a neuron-specific human gene, KIAA1110, that is a guanine nucleotide exchange factor for ARF1

To identify neuron-specific genes, we performed gene expression profiling, cDNA microarray and in silico ESTs (expressed sequence tags) analyses. We identified a human neuron-specific gene, KIAA1110 (homologue of rat synArfGEF (Po)), that is a member of the guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF). RT-PCR analysis showed that the KIAA1110 gene was expressed specifically in the brain among adult human tissues, whereas no apparent expression was observed in immature neural tissues/cells, such as fetal brain, glioma tissues/cells, and neural stem/precursor cells (NSPCs). The KIAA1110 protein was shown to be expressed in mature neurons but not in undifferentiated NSPCs. Immunohistochemical analysis also showed that KIAA1110 was expressed in neurons of the human adult cerebral cortex. Furthermore, the pull-down assay revealed that KIAA1110 has a GEF activity toward ARF1 that regulates transport along the secretion pathway. These results suggest that KIAA1110 is expressed specifically in mature neurons and may play an important role in the secretion pathway as a GEF for ARF1.     

神经干细胞三维空间壳聚糖生物材料中培养

目的:体外培养神经干细胞,并将其种植在三维空间壳聚糖材料中,体外培养一段时间,使壳聚糖材料内尽量分布足够多的细胞.方法:将NSCs种植在4不同孔径直径16通道壳聚糖材料中,分别培养7d和14d.DAPI标记细胞.荧光镜下观察细胞在不同孔径直径材料中的分布.MTT法检测不同孔径直径壳聚糖材料内细胞的活性.结果:DAPI荧光显示,培养7d时.细胞仍然成团贴附在材料的通道内,少有细胞迁移至壳聚糖材料内,而培养14d可见细胞较均匀的分布在材料内,同时观察到,孔径直径为0-75μm和75-125μ m两种壳聚糖材料,容纳细胞数较孔径直径为125-200μ m和200-300μm少.MTT结果显示,200-300μ m孔径直径的壳聚糖材料内细胞活性为各组最高,间接提示其内所含细胞数最多,而培养7d和14d两种培养方式对同种孔径直径材料内所含细胞教并无影响.结论:壳聚糖可降解生物材料能显示出良好生物相容性;体外培养NSCs于孔径为200-300μm的壳聚糖材料内14d,其存活细胞多且分布较均匀.     

神经干细胞分离培养方法的介绍

在成体的许多组织中发现了多能干细胞,这些干细胞可以进行自我复制,参与组织的正常修复。神经干细胞在体外能分化为神经元、星形胶质细胞和少突胶质细胞,并具有多向分化潜能。成体神经干细胞和胚胎干细胞都能分化成成体神经系统中的各种神经细胞。神经干细胞具有自我更新能力,因此神经干细胞可以应用于神经损伤或者神经疾病的修复。本文概述了神经干细胞体外分离培养的方法及其生长影响因子。     

新生大鼠脊髓神经干细胞的分离培养及鉴定

目的　从新生大鼠的脊髓中分离培养神经干细胞并观察其增殖和分化能力。方法　采用细胞培养技术结合间接免疫荧光细胞化学法。结果　分离的细胞生长旺盛 ,单克隆化生成的细胞团 ,BrdU掺入呈强阳性。分离培养获得的细胞团呈Nestin强阳性 ,至今已在体外连续传代 8个月。培养的细胞团经 1%小牛血清诱导可分化为神经元和星形胶质细胞。结论　成功分离培养了新生大鼠脊髓神经干细胞     

Endothelial stress induces the release of vitamin D-binding protein, a novel growth factor

Endothelial cells (EC) under stress release paracrine mediators that facilitate accumulation of vascular smooth muscle cells (VSCM) at sites of vascular injury. We found that medium conditioned by serum-starved EC increase proliferation and migration of VSCM in vitro. Fractionation of the conditioned medium followed by mass spectral analysis identified one bioactive component as vitamin D-binding protein (DBP). DBP induced both proliferation and migration of VSMC in vitro in association with increased phosphorylation of ERK 1/2. PD 98059, a biochemical inhibitor of ERK 1/2, abrogated these proliferative and migratory responses in VSMC. DBP is an important carrier for the vitamin-D sterols, 25-hydroxyvitamin-D, and 1alpha,25-dihydroxyvitamin-D. Both sterols inhibited the activity of DBP on VSMC, suggesting that vitamin D binding sites are important for initiating the activities of DBP on VSMC. Release of DBP at sites of endothelial injury represents a novel pathway favoring accumulation of VSMC at sites of vascular injury.     

异氟烷对小鼠神经干细胞BDNF/Caspase3 及Notch信号相关基因表达的影响

目的:探讨异氟烷对小鼠神经干细胞的BDNF、Caspase3及Notch信号相关基因表达的影响。方法:给予体外培养的新生小鼠海马神经干细胞不同浓度异氟烷处理,实验分为对照组和异氟烷处理组(ISO1.0,ISO1.5),其中异氟烷组细胞分别给予1.0MAC和1.5 MAC两个浓度的异氟烷处理2小时,对照组给予O_2处理2小时,随后置于培养箱正常培养24小时后收集细胞,提取细胞RNA检测BDNF,Caspase3及Notch相关基因(Notch2、Notch 3和Hes5)的m RNA水平变化。结果:与对照组相比,(1)异氟烷组小鼠神经干细胞的功能基因BDNF m RNA水平下调,凋亡相关基因Caspase3的m RNA水平上调;(2)异氟烷组神经干细胞的Notch2和Notch3受体m RNA表达下调,Notch信号通路靶基因Hes5的m RNA水平也明显下调;(3)异氟烷对神经干细胞的作用具有剂量依赖性,浓度越高对神经干细胞BDNF、Caspase3及Notch信号相关基因表达的影响越大。结论:异氟烷可能通过抑制小鼠神经干细胞的Notch信号通路,下调BDNF的m RNA表达,上调Caspase3的m RNA水平,影响神经干细胞的正常功能。     

The novel steroidal alkaloids dendrogenin A and B promote proliferation of adult neural stem cells

Dendrogenin A (DDA) and dendrogenin B (DDB) are new aminoalkyl oxysterols which display re-differentiation of tumor cells of neuronal origin at nanomolar concentrations. We analyzed the influence of dendrogenins on adult mice neural stem cell proliferation, sphere formation and differentiation. DDA and DDB were found to have potent proliferative effects in neural stem cells. Additionally, they induce neuronal outgrowth from neurospheres during in vitro cultivation. Taken together, our results demonstrate a novel role for dendrogenins A and B in neural stem cell proliferation and differentiation which further increases their likely importance to compensate for neuronal cell loss in the brain.     

Melatonin Regulates the Viability and Differentiation of Rat Midbrain Neural Stem Cells

(1) Neurogenesis driven by neural stem cells (NSCs) is regulated by physiological and pathological factors. Melatonin (MT) has profound neurotrophic and neuroprotective effects. Hence, we studied the role of MT in regulating the viability and differentiation of NSCs derived from rat ventral midbrain. (2) NSCs were isolated from the rat ventral midbrain. The viability of NSCs was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-ulfophenyl)-2H-tetrazolium assay. The differentiation of NSCs was examined by analyzing the expression of the neural markers, MT receptors, brain derived neurotropic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) with semi-quantitative RT-PCR, immunofluorescence cytochemistry, and Western blot. (3) Our results showed that MT could promote the viability of NSCs. In addition, MT could significantly elevate the mRNA and protein levels of tyroxine hydroxylase (TH), a marker of dopaminergic neurons, and decrease the expression of the astrocytes maker glial fibrillary acidic protein (GFAP). MT also increased the production of BDNF and GDNF in the cultured NSCs. Meanwhile, we first found that two subtypes of MT receptors, MT1 and MT2, were expressed in the ventral midbrain NSCs. (4) These results demonstrated that MT could induce NSCs to differentiate into dopaminergic neurons and decrease astrocyte production. These findings also suggest that MT could offer a beneficial tool in guiding directional differentiation of NSCs.     

Neural stem cell transplantation therapy for brain ischemic stroke: Review and perspectives

Brain ischemic stroke is one of the most common causes of death and disability, currently has no efficient therapeutic strategy in clinic. Due to irreversible functional neurons loss and neural tissue injury, stem cell transplantation may be the most promising treatment approach. Neural stem cells (NSCs) as the special type of stem cells only exist in the nervous system, can differentiate into neurons, astrocytes, and oligodendrocytes, and have the abilities to compensate insufficient endogenous nerve cells and improve the inflammatory microenvironment of cell survival. In this review, we focused on the important role of NSCs therapy for brain ischemic stroke, mainly introduced the methods of optimizing the therapeutic efficacy of NSC transplantation, such as transfection and overexpression of specific genes, pretreatment of NSCs with inflammatory factors, and co-transplantation with cytokines. Next, we discussed the potential problems of NSC transplantation which seriously limited their rapid clinical transformation and application. Finally, we expected a new research topic in the field of stem cell research. Based on the bystander effect, exosomes derived from NSCs can overcome many of the risks and difficulties associated with cell therapy. Thus, as natural seed resource of nervous system, NSCs-based cell-free treatment is a newly therapy strategy, will play more important role in treating ischemic stroke in the future.     

脑挫裂伤致miR-9 升高促进神经干细胞分化

目的:探讨颅脑损伤后miR-9表达的变化和对神经干细胞分化和增值的影响,为颅脑损伤后神经功能修复治疗提出新的思路。方法:通过RT-PCR技术检测miR-9在挫裂伤脑组织中的表达情况;培养胚胎来源神经干细胞,并通过免疫荧光鉴定神经干细胞及其分化;转染miR-9后,通过MTT测定神经干细胞的增殖情况,和流式细胞仪检测分化神经元所占比例。结果:miR-9在挫裂伤脑组织中表达显著上升。对神经干细胞过表达miR-9可显著促进细胞增殖,并诱导分化成神经元。结论:脑挫裂伤时miR-9显著升高,并具有着促进神经干细胞增值和诱导分化的作用,可为伤后神经功能修复提供新的治疗方法。     

调节TREK1对小鼠海马神经干细胞增殖和BDNF表达的影响

目的:观察调节TWIK相关的K+通道1(TREK1)对小鼠海马神经干细胞增殖和脑源性神经营养因子(brain derived neurotrophic factor,BDNF)表达的影响。方法:从孕10.5 d C57bl/6J小鼠胚胎海马中分离出神经干细胞并培养,待细胞达到70%～80%融合后,对细胞进行慢病毒干预,细胞分为sham组,Ctrl组(转染对照病毒),Plenti-TREK-1组(转染携带TREK1高表达载体病毒)和sh-TREK-1组(转染携带TREK1-shRNA病毒)。采用CCK-8法检测病毒干预后3天和7天各组神经干细胞的细胞活力,采用q RT-PCR检测病毒干预后7天各组神经干细胞TREK-1基因表达情况,并通过Elisa检测各组神经干细胞上清液BDNF表达水平。结果:与sham组相比较,(1)Plenti-TREK-1组TREK1基因表达上调,神经干细胞的神经球体积减小,细胞活力降低,细胞增殖减少,细胞上清BDNF水平下降;(2)sh-TREK-1组TREK1基因表达下调,神经干细胞的神经球体积增加,细胞活力增高,细胞增殖增加,细胞上清BDNF水平上调;(3)上述各项指标Ctrl组与sham组之间无统计学差异。结论:神经干细胞的增殖与TREK1通道密切相关,上调TREK-1可以抑制神经干细胞增殖及其BDNF的表达水平;下调TREK-1则可以促进神经干细胞增殖并上调其BDNF的表达水平。