Hydrogel Systems for Purification and Differentiation of Mid-Brain NPCs

描述(由申请人提供)：PI提出了一种创新的方法来产生丰富的多巴胺(DA)神经元群体，用于细胞治疗。DA神经元的特发性死亡导致帕金森病(PD)的症状，因此，这些细胞的替换是PD研究的主要目标。发展策略，在合适的环境中产生强健的DA神经元培养，以支持植入后的生存能力是势在必行的。此外，移植细胞通常是处于不同发育阶段的神经干细胞的混合群体，据报道，这会对治疗效果产生负面影响，并导致副作用。在此，提出了一种从腹侧中脑神经前体细胞(VM-NPC)中分离DA前体细胞的方法，然后在3D透明质酸(HA)水凝胶中进行体外扩增和分化，作为仿生培养环境，并可能作为细胞移植的载体。特定目标1将利用细胞表面受体CD44和糖胺聚糖HA之间的特定相互作用。HA和CD44在胎脑中过度表达，在发育过程中表达下调。PI实验室的初步结果表明，种植了混合培养物的HA涂层表面选择性地结合了未成熟的NPC。不同浓度的HA涂层表面将用于从VM NPC的初级分离中PAN寻找DA前体。通过免疫染色对蛋白质表达进行定性分析，通过RT-PCR对mRNA表达进行定量分析，将用于鉴定不同分化阶段的鼻咽癌。特别是，将确定DA前体的丰富种群。采用流式细胞仪分析鼻咽癌组织中CD44的表达，并建立不同鼻咽癌组织中CD44表达的阈值。在特定目标2中，将富含DA前体的预分离的神经前体细胞在PI先前开发的HA水凝胶中进行3D培养，以促进VM神经干细胞向神经元的分化。这些水凝胶被设计成提供一个仿生环境，在这个环境中，机械和化学特性非常类似于天然胎儿的大脑。与目标1一样，免疫染色和RT-PCR将用于鉴定3D培养中DA前体细胞的分化。在体外扩增和分化纯化的神经干细胞后，这些水凝胶有可能直接用作细胞移植的载体。本项目的创新之处在于结合了在体外扩增前对DA前体进行预选和随后在3D、HA生物材料中进行分化。PI假设，使用这种两步法可以产生大量的DA神经元，足够用于治疗。

DESCRIPTION (provided by applicant): The PI proposes an innovative approach to generate enriched populations of dopaminergic (DA) neurons for cell therapies. Idiopathic death of DA neurons causes the symptoms of Parkinson's disease (PD), thus, replacement of these cells is a primary goal of PD research. Development of strategies to generate robust cultures of DA neurons within suitable environments to support viability after implantation is imperative. In addition, implanted cells are generally a mixed population of NPCs at various stages of development, which has been reported to negatively impact therapeutic efficacy and result in side effects. Here, a method is proposed for separation of DA precursors from ventral mesencephalic neural progenitor cells (VM NPCs) prior to ex vivo expansion and differentiation in 3D, hyaluronic acid (HA) hydrogels, which serve as biomimetic culture environments and possibly as vehicles for cell transplantation. Specific Aim 1 will exploit the specific interactions between the cell surface receptor CD44 and the glycosaminoglycan HA. Both HA and CD44 are over expressed in fetal brain and down regulated during development. Preliminary results in the PI's lab demonstrate that HA-coated surfaces seeded with mixed cultures selectivity bind immature NPCs. HA-coated surfaces of varying concentrations will be used to pan for DA precursors from primary isolations of VM NPCs. Qualitative analysis of protein expression via immunostaining and quantitative analysis of mRNA expression via RT-PCR will be used to characterize NPCs at different stages of differentiation. In particular, enriched population of DA precursors will be identified. CD44 expression of NPC populations will be analyzed using fluorescence-activated cell sorting (FACS) and threshold values of CD44 expression for different NPC populations will be established. In Specific Aim 2, pre-separated NPCs enriched in DA precursors will be cultured in 3D, HA hydrogels previously developed by the PI to enhance differentiation of VM NPCs into neurons. These hydrogels have been designed to provide a biomimetic environment in which the mechanical and chemical properties closely resemble those of native fetal brain. As in Aim 1, immunostaining and RT-PCR will be used to characterize differentiation of DA precursors in 3D cultures. After ex vivo expansion and differentiation of purified NPCs, these hydrogels could potentially be used directly as vehicles for cell transplantation. The innovation of this project lies in the combination of pre-selection for DA precursors prior to ex vivo expansion and subsequent differentiation in 3D, HA biomaterials. The PI hypothesizes that large numbers of DA neurons, sufficient for therapeutic benefit, can be generated using this two-step approach.