Hydrogel Systems for Purification and Differentiation of Mid-Brain NPCs

描述（由申请人提供）：PI提出了一种创新方法，以产生用于细胞治疗的多巴胺能（DA）神经元富集群。DA神经元的特发性死亡导致帕金森病（PD）的症状，因此，这些细胞的替代是PD研究的主要目标。对于细胞替代疗法，目前胎儿来源的细胞在实验模型以及临床试验中显示出最有希望的结果。然而，在临床研究中，一些患者发生了移植物诱导的运动障碍（GID）。这些负面的行为结果被认为部分是由于植入细胞的异质性。因此，开发方案以获得更均匀的NPC群体用于未来的移植研究将是极其有价值的。在这里，提出了一种方法，用于分离DA前体从腹侧中脑神经祖细胞（VM NPC）的体外扩增和分化之前的3D，透明质酸（HA）水凝胶，作为仿生培养环境，并可能作为细胞移植车辆。特异性目标1将利用细胞表面受体CD 44和糖胺聚糖HA之间的特异性相互作用。HA和CD 44在胎儿脑中过表达，并在发育过程中下调。PI实验室的初步结果表明，接种混合培养物的HA涂层表面选择性结合未成熟的NPC。不同浓度的HA涂覆表面将用于从VM NPC的初级分离物中淘选神经元前体。分离粘附和非粘附细胞，并使用免疫染色和RT-PCR分析DA前体（Nurr 1+）、增殖和分化潜力。同时，将使用流式细胞术分析NPC的CD 44表达，并建立不同NPC群体的CD 44表达阈值。在特定目标2中，富集DA前体的预分离NPC将在PI先前开发的3D HA水凝胶中培养，以增强VM NPC向神经元的分化。这些水凝胶被设计成提供仿生环境，其中机械和化学性质非常类似于天然胎儿大脑的那些。此外，天然ECM蛋白（例如，层粘连蛋白、纤连蛋白）将被添加到水凝胶中以获得更好的细胞粘附和分化。免疫染色和RT-PCR将用于表征3D培养物中DA前体的分化。该项目的创新在于在体外扩增之前对DA前体进行预选和随后在3D、HA生物材料中分化的组合。PI假设，使用这种两步法可以产生足够用于治疗益处的大量DA神经元。 公共卫生相关性：该项目的目标是开发策略，以扩增，分化和纯化足够数量的神经祖细胞（NPC）用于再生医学。例如，这最终可用于治疗中枢神经系统（CNS）疾病如帕金森病的疗法。一般来说，离体NPC的有效扩增和功能分化构成了重大挑战。该提案的第一个目的是通过使用选择性透明质酸基质相互作用淘选原代分离物来提高NPC培养物的纯度。第二个目标是通过提供适当的基质和可溶性线索来提高从这些纯化的NPC群体分化神经元的效率。开发纯化原代分离干细胞并随后控制其在培养中的分化潜能的技术将是干细胞生物学进步的巨大机会。

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. For cell replacement therapies, currently fetal derived cells show the most promising results in experimental models as well as in clinical trials. However, in clinical studies some patients developed graft induced dyskinesia (GID). These negative behavioral outcomes are thought to be due in part to the heterogeneity of the implanted cells. Therefore, developing protocols to obtain a more uniform population of NPCs for future transplantation studies will be extremely valuable. 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 cell transplantation vehicles. Specific Aim 1 will exploit the specific interactions between the cell surface receptor CD44 and the glycosaminoglycan HA. Both HA and CD44 are overexpressed 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 neuronal precursors from primary isolations of VM NPCs. Adhered and non-adhered cells will be separated and analyzed for DA precursor (Nurr1+), proliferation and differentiation potentials using immunostaining and RT-PCR. In parallel, CD44 expression of NPCs will be analyzed using flow cytometry 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. Additionally, native ECM proteins (e.g., laminin, fibronectin) will be added into the hydrogels for better cell adhesion and differentiation. Immunostaining and RT-PCR will be used to characterize differentiation of DA precursors in 3D cultures. 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. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop strategies to expand, differentiate and purify neural progenitor cells (NPCs) in sufficient quantities for use in regenerative medicine. For example, this could be useful ultimately for therapies to treat central nervous system (CNS) disorders such as Parkinson's disease. In general, efficient expansion and functional differentiation of NPCs ex vivo poses a significant challenge. The first objective of this proposal is to enhance the purity of NPC cultures by panning primary isolates using selective hyaluronic acid matrix interactions. The second objective is to enhance the efficiency of neuronal differentiation from these purified NPC populations by providing appropriate matrix and soluble cues. The development of technologies to purify primary isolated stem cells and to subsequently control their differentiation potential in culture would be a vast opportunity for advancements in stem cell biology.