Hydrogel Systems for Purification and Differentiation of Mid-Brain NPCs

用于中脑 NPC 纯化和分化的水凝胶系统

• 批准号: 8667930
• 负责人: CHRISTINE E SCHMIDT
• 金额: $ 18.02万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8667930
• 关键词: Hydrogel; Systems; Purification; Differentiation; Mid

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.

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。发展战略，以产生强大的文化DA神经元在合适的环境，以支持植入后的活力是必要的。此外，植入的细胞通常是处于不同发育阶段的NPC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，并可能作为车辆细胞移植。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DA前体。通过免疫染色进行蛋白质表达的定性分析和通过RT-PCR进行mRNA表达的定量分析将用于表征不同分化阶段的NPC。特别是，富集人口的DA前体将被确定。将使用荧光激活细胞分选（FACS）分析NPC群体的CD 44表达，并将建立不同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。如目的1所述，免疫染色和RT-PCR将用于表征3D培养物中DA前体的分化。纯化的NPC离体扩增和分化后，这些水凝胶有可能直接用作细胞移植的载体。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。

项目成果

Biomimetic hydrogels direct spinal progenitor cell differentiation and promote functional recovery after spinal cord injury.

• DOI: 10.1088/1741-2552/aaa55c
• 发表时间: 2018-04
• 期刊: Journal of neural engineering
• 影响因子: 4
• 作者: Geissler SA;Sabin AL;Besser RR;Gooden OM;Shirk BD;Nguyen QM;Khaing ZZ;Schmidt CE
• 通讯作者: Schmidt CE