IMMORTALIZATION & GRAFTING OF NEURAL PROGENITORS

在神经移植领域， 正在寻找组织作为移植材料。以前，通过逆转录病毒- 介导的基因转移，我们产生了永生化，克隆，多能 小鼠神经祖细胞系。审查其区别 在体外的潜力表明巨大的可塑性的水平， 个体祖先移植后，这些永生的 在细胞结构和功能上 适当的方式，概括其体内多能性和表达 逆转录病毒转导的外源基因， 在脑实质中长时间地扩散这些祖先可以 嫁接并正常参与多种结构的发展 沿着神经轴&在从胚胎到 成年人了它们分化成多种细胞类型， 在特定的发展阶段的相应区域的信号。 虽然最初有丝分裂时最容易植入的祖细胞， 迁移性，可塑性，快速分化，下调其 永生基因产物，从未形成肿瘤中枢神经系统细胞结构 从未被破坏，血脑屏障保持完整，接受者 动物表现正常。当移植到各种动物模型中时， 神经退化，祖细胞整合到细胞结构中 通常假设缺失细胞类型的表型。此外，委员会认为， 祖细胞成功地表达了宿主中缺失的基因产物 移植到各种动物模型的中枢神经系统中， 已经定义了基因（例如，某些神经内脏储存疾病）。 这些数据表明，使用永生化祖细胞， 提供具有治疗或发展意义的外源性因素 （组成性地或通过基因工程），或实现修复， 中枢神经系统细胞结构的组成部分，可能是可行的。生成 具有类似潜能的人类胚胎神经组织的祖细胞系 是下一步从原代人类胎儿神经组织开始， 拟议的研究将试图建立一个神经祖细胞的范例， 移植作为一种治疗发育，退行性和获得性 通过以下4个目的对CNS损伤进行评估：（1）确定 在体外维持人胎儿神经组织，这将优化 存在适合永生化的祖细胞。(2)生成& 表征体外克隆、永生化、多能性人神经 祖先系(3)将候选品系移植到小鼠宿主中 （环孢菌素处理的或SClD小鼠）以评估移植能力， 在体内分化，并表达报告基因转基因。(4)尝试 将可移植祖系移植到小鼠模型中 神经退化

In the field of neural transplantation, alternatives to primary fetal tissue are being sought as graft material. Previously, through retrovirus- mediated gene transfer, we generated immortalized, clonal, multipotent neural progenitor lines from mouse. Examination of their differentiation potential in vitro suggested enormous plasticity at the level of the individual progenitor. Upon transplantation, these immortalized progenitors engrafted in a cytoarchitecturally and functionally appropriate manner, recapitulating their multipotency in vivo & expressing retrovirally-transduced exogenous genes in a robust, stable fashion throughout brain parenchyma for prolonged periods. These progenitors could engraft & participate normally in the development of multiple structures along the neuraxis & at multiple stages spanning from embryonic to the adult. They differentiated into multiple cell types, presumably responding to signals of the respective region at the particular developmental stage. The progenitors, though most engraftable when initially mitotic, migratory, & plastic, quickly differentiated, down-regulated their immortalizing gene product, & never formed tumors. CNS cytoarchitecture was never disrupted, the blood-brain barrier remained intact, & recipient animals behaved normally. When transplanted into various animal models of neural degeneration, the progenitors integrated into cytoarchitecture often assuming the phenotype of the missing cell type. Furthermore, progenitors expressing gene products missing in the host successfully engrafted into the CNS of various animal models in which defects in those genes had been defined (e.g., certain neurovisceral storage diseases). These data suggest the feasibility of using immortalized progenitors to provide exogenous factors of therapeutic or developmental interest (constitutiveiy or through genetic engineering), or to effect repair as integral members of CNS cytoarchitecture, may be feasible. The generation of progenitor lines with similar potential from human fetal neural tissue is the next step. Starting with primary human fetal neural tissue, the proposed studies will attempt to establish a paradigm of neural progenitor transplantation as a therapy for developmental, degenerative, & acquired injury to CNS through the following 4 aims: (1) Determine conditions for maintaining human fetal neural tissue in vitro which would optimize the presence of proper progenitors for immortalization. (2) Generate & characterize in vitro clonal, immortalized, multipotent human neural progenitor lines. (3) Transplant candidate lines into mouse hosts (cyclosporin-treated or SClD mice) to assess ability to engraft, differentiate in vivo, & express a reporter transgene. (4) Attempt transplantation of engraftable progenitor lines into mouse models of neural degeneration.