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来生成用于细胞治疗的丰富的多巴胺能 (DA) 神经元群体。 DA 神经元的特发性死亡会导致帕金森病 (PD) 的症状，因此，替换这些细胞是 PD 研究的主要目标。开发策略以在合适的环境中生成强大的 DA 神经元培养物以支持植入后的活力是势在必行的。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。在此，提出了一种在 3D 透明质酸 (HA) 水凝胶中离体扩增和分化之前从腹侧中脑神经祖细胞 (VM NPC) 中分离 DA 前体的方法，透明质酸 (HA) 水凝胶可作为仿生培养环境，并可能作为细胞的载体移植。具体目标 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 在胎儿大脑中过度表达，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 涂层表面将用于从 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，将用于表征不同分化阶段的 NPC。特别是，将鉴定丰富的 DA 前体群体。将使用荧光激活细胞分选术 (FACS) 分析 NPC 群体的 CD44 表达，并建立不同 NPC 群体的 CD44 表达阈值。在具体目标 2 中，预先分离的富含 DA 前体的 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。这些水凝胶旨在提供一个仿生环境，其中的机械和化学特性与天然胎儿大脑的机械和化学特性非常相似。与目标 1 一样，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 进行离体扩增和分化后，这些水凝胶有可能直接用作细胞移植的载体。该项目的创新在于将 DA 前体在离体扩增之前的预选与随后在 3D、HA 生物材料中的分化相结合。 PI 假设使用这种两步方法可以产生足以产生治疗效果的大量 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