Regulation of adult human oligodendrocyte progenitors

描述（由申请人提供）：在该基金的前4年，我们评估了成人少突胶质细胞祖细胞的细胞生物学和移植特性。我们使用CNP 2：GFP和基于A2 B5的荧光激活细胞分选（FACS）建立了用于它们的特异性分离的方法，然后评估它们的谱系潜力。我们发现，当移到低密度培养时，祖细胞是多能的，并产生神经元和神经胶质细胞。因此，缺乏自分泌和旁分泌的影响，其分化，成人WMPC不限于少突胶质细胞的命运。为了评估组织环境在调节祖细胞命运中的作用，我们研究了成年人WMPC的基因表达。我们专注于确定WMPC相对于其局部组织环境差异表达的那些转录本。这一过程使我们能够预测维持祖细胞状态的配体-受体相互作用，以及决定给定细胞是否发育成星形胶质细胞、少突胶质细胞或神经元的配体-受体相互作用。该分析确定了一组平行的途径，这些途径共同调节实质祖细胞的维持、动员和分化命运。特别是，我们注意到以下因素的复杂相互作用：1）受体酪氨酸磷酸酶β/zeta信号传导，由其配体多效因子和NrCAM调节; 2）syndecan 3通过CASK和tbr 1调节信号传导的平行途径; 3）FGFR 3依赖性信号传导，可能通过syndecan 3蛋白水解和CASK释放介导;和4）神经素和BAMBI对BMP信号的抑制。似乎这些途径的净输出使成年祖细胞偏向于自我更新或分化。在本申请中，我们建议使用蛋白质递送、腺病毒过表达和慢病毒RNAi敲低的组合来评估这些候选系统的各个元件。通过这种方法，我们打算更好地定义成人白色物质中胶质细胞生成的生态位，并通过这样做来建立必要和足够的遗传靶点，用于指导由常驻祖细胞产生的表型。

DESCRIPTION (provided by applicant): In the first 4 years of this grant, we assessed the cell biology and transplantation characteristics of adult human oligodendrocyte progenitor cells. We established means for their specific isolation, using CNP2:GFP and A2B5 based fluorescence-activated cell sorting (FACS), and then assessed their lineage potential. We discovered that when removed to low density culture, the progenitors were multipotential, and gave rise to neurons as well as to gila. Thus, absent autocrine and paracrine influences on their differentiation, adult WMPCs were not restricted to oligodendrocytic fate. To assess the role of the tissue environment in regulating progenitor fate, we then investigated gene expression by adult human WMPCs. We focused on identifying those transcripts that are differentially expressed by the WMPC, relative to its local tissue environment. This process enabled us to predict ligand-receptor interactions that maintain the progenitor state, as well as those that determine whether a given cell develops into an astrocyte, oligodendrocyte, or neuron. This analysis identified a set of parallel pathways that together appear to regulate the maintenance, mobilization and differentiated fate of parenchymal progenitor cells. In particular, we noted a complex interaction of: 1) receptor tyrosine phosphatase beta/zeta signaling, as regulated by its ligands pleiotrophin and NrCAM; 2) a parallel avenue of syndecan3 regulated signaling through CASK and tbr1; 3) FGFR3-dependent signaling, likely mediated through syndecan3 proteolysis and release of CASK; and 4) a neuralin and BAMBI-suppression of BMP signals. It appears that the net output of these pathways biases adult progenitors to either self-renewal or differentiation. In this application, we propose to use a combination of protein delivery, adenoviral overexpression and lentiviral RNAi knock-down to evaluate the individual elements of these candidate systems. By this means, we intend to better define the niche for gliogenesis in the adult human white matter, and by so doing to establish both necessary and sufficient genetic targets for directing the phenotypes generated by resident progenitor cells.