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1.
Neural stem cells (NSCs) offer the potential to replace lost tissue after nervous system injury. This study investigated whether grafts of NSCs (mouse clone C17.2) could also specifically support host axonal regeneration after spinal cord injury and sought to identify mechanisms underlying such growth. In vitro, prior to grafting, C17.2 NSCs were found for the first time to naturally constitutively secrete significant quantities of several neurotrophic factors by specific ELISA, including nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor. When grafted to cystic dorsal column lesions in the cervical spinal cord of adult rats, C17.2 NSCs supported extensive growth of host axons of known sensitivity to these growth factors when examined 2 weeks later. Quantitative real-time RT-PCR confirmed that grafted stem cells expressed neurotrophic factor genes in vivo. In addition, NSCs were genetically modified to produce neurotrophin-3, which significantly expanded NSC effects on host axons. Notably, overexpression of one growth factor had a reciprocal effect on expression of another factor. Thus, stem cells can promote host neural repair in part by secreting growth factors, and their regeneration-promoting activities can be modified by gene delivery.  相似文献   

2.
In some parts of the CNS, depletion of a particular class of neuron might induce changes in the microenvironment that influence the differentiation of newly grafted neural precursor cells. This hypothesis was tested in the retina by inducing apoptotic retinal ganglion cell (RGC) death in neonatal and adult female mice and examining whether intravitreally grafted male neural precursor cells (C17.2), a neural stem cell (NSC)-like clonal line, become incorporated into these selectively depleted retinae. In neonates, rapid RGC death was induced by removal of the contralateral superior colliculus (SC), in adults, delayed RGC death was induced by unilateral optic nerve (ON) transection. Cells were injected intravitreally 6-48 h after SC ablation (neonates) or 0-7 days after ON injury (adults). Cells were also injected into non-RGC depleted neonatal and adult retinae. At 4 or 8 weeks, transplanted cells were identified using a Y-chromosome marker and in situ hybridisation or by their expression of the lacZ reporter gene product Escherichia coli beta-galactosidase (beta-gal). No C17.2 cells were identified in axotomised adult-injected eyes undergoing delayed RGC apoptosis (n = 16). Donor cells were however stably integrated within the retina in 29% (15/55) of mice that received C17.2 cell injections 24 h after neonatal SC ablation; 6-31% of surviving cells were found in the RGC layer (GCL). These NSC-like cells were also present in intact retinae, but on average, there were fewer cells in GCL. In SC-ablated mice, most grafted cells did not express retinal-specific markers, although occasional donor cells in the GCL were immunopositive for beta-III tubulin, a protein highly expressed by, but not specific to, developing RGCs. Targeted rapid RGC depletion thus increased cell incorporation into the GCL, but grafted C17.2 cells did not appear to differentiate into an RGC phenotype.  相似文献   

3.
Mi R  Luo Y  Cai J  Limke TL  Rao MS  Höke A 《Experimental neurology》2005,194(2):301-319
Pluripotent neural stem cells (NSCs) have been used as replacement cells in a variety of neurological disease models. Among the many different NSCs that have been used to date, most robust results have been obtained with the immortalized neural stem cell line (C17.2) isolated from postnatal cerebellum. However, it is unclear if other NSCs isolated from different brain regions are similar in their potency as replacement therapies. To assess the properties of NSC-like C17.2 cells, we compared the properties of these cells with those reported for other NSC populations identified by a variety of different investigators using biological assays, microarray analysis, RT-PCR, and immunocytochemistry. We show that C17.2 cells differ significantly from other NSCs and cerebellar granule cell precursors, from which they were derived. In particular, they secrete additional growth factors and cytokines, express markers that distinguish them from other progenitor populations, and do not maintain karyotypic stability. Our results provide a caution on extrapolating results from C17.2 to other nonimmortalized stem cell populations and provide an explanation for some of the dramatic effects that are seen with C17.2 transplants but not with other cells. We suggest that, while C17.2 cells can illustrate many fundamental aspects of neural biology and are useful in their own right, their unique properties cannot be generalized.  相似文献   

4.
5.
The fate of exogenous neural stem cells (NSCs) in the environment of the adult nervous system continues to be a matter of debate. In the present study, we report that cells of the murine NSC clone C17.2, when grafted into the lumbar segments of the spinal cord of adult rats, survive and undergo partial differentiation. C17.2 cells migrate avidly toward axonal tracts and nerve roots and differentiate into nonmyelinating ensheathing cells. Notably, C17.2 cells induce the de novo formation of host axon tracts aiming at graft innervation. Differentiation and inductive properties of C17.2 cells are independent of the presence of lesions in the spinal cord. The tropic/trophic interactions of C17.2 NSCs with host axons, the avid C17.2 cell-host axon contacts, and the ensheathing properties of these cells are related to their complex molecular profile, which includes the expression of trophic cytokines and neurotrophins such as glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor, glial growth factor receptors such as ErbB-2; and PASK, the mammalian homologue of the fray gene that is involved in axon ensheathment. These results show that NSCs might not only play a critical supportive role in repairing axonal injury in the adult spinal cord but also can be used as probes for exploring the molecular underpinnings of the regenerative potential of the mature nervous system after injury.  相似文献   

6.
Regeneration in the peripheral nervous system is impaired after prolonged periods of denervation. Currently, no interventions exist to alter the outcome after prolonged denervation. To examine the role of transplanted neural stem cells (NSC), we prepared chronically denervated distal tibial nerve segments. After 6 months of chronic denervation, we transplanted vehicle, C17.2 mouse NSCs, or C17.2 mouse NSCs engineered to overexpress GDNF to the distal tibial nerve and performed a peroneal nerve cross-suture. In animals transplanted with the NSCs, there was better regeneration of the peroneal axons into the tibial nerve as measured by counting the number of axons and by the emergence of compound motor action potentials in the tibial innervated foot muscles. Improved regeneration correlated with a reduction of chondroitin sulphate proteoglycan (CSPG) immunoreactivity in the extracellular matrix (ECM). In vitro, supernatant from C17.2 NSCs contained large quantities of secreted matrix metalloprotease-2 (MMP-2), degraded the CSPGs on chronically denervated tibial nerve sections, and reversed the CSPG-induced inhibition of neuritic outgrowth of DRG neurons. This reversal was inhibited by selective MMP-2 inhibitors. This is the first successful demonstration of regeneration through a chronically denervated nerve. These findings suggest that improved regeneration in the PNS can be accomplished by combining neurotrophic factor support and removal of axon growth inhibitory components in the extracellular matrix.  相似文献   

7.
背景:依达拉奉 (MCI-186)是一种新型自由基清除剂,已证实其能减轻急性脑梗死后的脑组织水肿、具有神经保护作用。 目的:探讨自由基清除剂MCI-186对大鼠缺血脑组织内源性神经干细胞的作用。 方法:Longa法构建SD大鼠大脑中动脉缺血2 h再灌注模型,分两组在动脉阻塞后立即开始予MCI-186或磷酸盐缓冲液治疗,在术后1,3 d和7 d,动态测定缺血周边脑组织丙二醛的含量以及脑源性神经生长因子蛋白和mRNA的表达,以及缺血脑区域Nestin阳性细胞Caspase-3阳性细胞表达,同时进行神经功能测定。 结果与结论:与假手术组相比,磷酸盐缓冲液组脑组织丙二醛水平明显升高,MCI-186治疗后明显降低(P均< 0.01);磷酸盐缓冲液组缺血后1 d,MCI-186组缺血后1,3 d脑源性神经生长因子 mRNA和蛋白的表达明显升高(P < 0.01)。缺血后 3 d和7 d MCI-186组Nestin阳性细胞明显高于磷酸盐缓冲液组(P < 0.05),Caspase-3阳性细胞显著低于磷酸盐缓冲液组 (P < 0.05)。缺血后7 d MCI-186组神经功能明显优于磷酸盐缓冲液组。结果提示,MCI-186能抑制脂质过氧化,增加缺血脑组织的脑源性神经生长因子分泌,保护神经干细胞,减少细胞凋亡。  相似文献   

8.
成人骨髓源性神经干细胞的致瘤性研究   总被引:8,自引:5,他引:3  
目的研究体外培养的成人骨髓源性神经干细胞的致瘤性。方法对骨髓源性神经干细胞分别进行细胞形态学观察、刀豆球蛋白A凝集试验和双层软琼脂培养以探明其是否具有恶性转化细胞的形态特征、表面结构及生长特性的变化:利用免疫细胞化学的方法检测骨髓源性神经干细胞的端粒酶和肿瘤相关基因的表达:将骨髓源性神经干细胞接种到裸鼠体内观察其成瘤性。结果骨髓源性神经干细胞不具有恶性转化细胞的形态特征,在不同刀豆球蛋白A浓度下均未见明显的凝集反应,在双层软琼脂不能形成细胞克隆;骨髓源性神经干细胞的c-myc、c-fos和p53基因均呈阴性表达,而端粒酶逆转录酶呈弱阳性表达:将骨髓源性神经干细胞接种于裸鼠皮下6个月未见肿瘤形成,亦未见其它组织形成。结论骨髓源性神经干细胞保持了正常细胞的生物学特征.体内和体外的各项指标均未提示其具有致瘤性.体外的培养条件没有使其发生恶性转化.从致瘤性方面证实了骨髓源性神经干细胞临床移植的安全性。  相似文献   

9.
Transplantation of human neural stem cells into the dentate gyrus or ventricle of rodents has been reportedly to enhance neurogenesis. In this study, we examined endogenous stem cell proliferation and angiogenesis in the ischemic rat brain after the transplantation of human neural stem cells. Focal cerebral ischemia in the rat brain was induced by middle cerebral artery occlusion. Human neural stem cells were transplanted into the subventricular zone. The behavioral performance of human neural stem cells-treated ischemic rats was significantly improved and cerebral infarct volumes were reduced compared to those in untreated animals. Numerous transplanted human neural stem cells were alive and preferentially localized to the ipsilateral ischemic hemisphere. Furthermore, 5-bromo-2′-deoxyuridine-labeled endogenous neural stem cells were observed in the subventricular zone and hippocampus, where they differentiated into cells immunoreactive for the neural markers doublecortin, neuronal nuclear antigen Neu N, and astrocyte marker glial fibrillary acidic protein in human neural stem cells-treated rats, but not in the untreated ischemic animals. The number of 5-bromo-2′-deoxyuridine-positive ? anti-von Willebrand factor-positive proliferating endothelial cells was higher in the ischemic boundary zone of human neural stem cells-treated rats than in controls. Finally, transplantation of human neural stem cells in the brains of rats with focal cerebral ischemia promoted the proliferation of endogenous neural stem cells and their differentiation into mature neural-like cells, and enhanced angiogenesis. This study provides valuable insights into the effect of human neural stem cell transplantation on focal cerebral ischemia, which can be applied to the development of an effective therapy for stroke.  相似文献   

10.
Generating neurons from human stem cells has potential for brain damage therapy and neurogenesis modeling, but current efficacy is limited by culture heterogeneity and the lack of markers. We have previously reported the heparan sulfate proteoglycans (HSPGs) glypican-1 (GPC1) and -4 (GPC4) as the markers of lineage-specific human neural stem cells (hNSCs) and mediators of hNSC lineage potential. Here, we further examined phenotypical characteristics and GPC1 and GPC4 during neural differentiation of hNSCs in the presence of two neurogenic growth factors reported to bind to heparan sulfate: brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor-B (PDGF-B). In hNSC neural cultures, GPC1 and GPC4 were expressed along neurites and cell bodies in long-term (40–60 days) neural differentiation cultures demonstrating the areas of differential localization—suggesting potentially different functions. Neural differentiation cultures in the presence of BDNF or PDGF-B generated phenotypically different neural cells with BDNF treatment associated with higher GPC4 versus GPC1 expression, increased heterogeneity, and differential neuron subtype marker expression to PDGF-B cultures. PDGF-B cultures exhibited higher levels of spontaneous activity and reduced heterogeneity over long-term culture associated with decreased GPC4. Untreated neural cultures were highly variable, supporting the use of neuroregulatory growth factors for guided differentiation. Targeted siRNA downregulation of GPC1/4 reduced neural differentiation markers and altered response to exogenous BDNF and PDGF-B. This work confirms GPC1 and GPC4 as regulators of human neural differentiation and supports their use as novel markers of neural cell characterization.  相似文献   

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