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: 8240871
• 负责人: CHRISTINE E SCHMIDT
• 金额: $ 21.92万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 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 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8240871
• 关键词: Adherence; Adult; Astrocytes; Behavioral; Binding; Biocompatible Materials; Biomimetics; Brain; Bromodeoxyuridine; CD44 gene; Cell Culture Techniques; Cell Differentiation process; Cell Separation; Cell Surface Receptors; Cell Therapy; Cell Transplantation; Cell Transplants; Cell-Cell Adhesion; Cells; Central Nervous System Diseases; Cessation of life; Clinical Research; Clinical Trials; Controlled Environment; Corpus striatum structure; Cues; Development; Dyskinetic syndrome; Environment; Experimental Models; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblast Growth Factor 2; Fibronectins; Flow Cytometry; Fluorescence-Activated Cell Sorting; Future; Glycosaminoglycans; Goals; Growth Factor; Heterogeneity; Hyaluronic Acid; Hydrogels; Implant; Laminin; Mechanics; Methods; Midbrain structure; Modeling; Multipotent Stem Cells; Neuraxis; Neuronal Differentiation; Neurons; Oligodendroglia; Outcome; Parkinson Disease; Patients; Phenotype; Physiological; Population; Proliferating; Property; Protocols documentation; Receptor Cell; Regenerative Medicine; Replacement Therapy; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Sorting - Cell Movement; Staging; Stem cells; Substantia nigra structure; Symptoms; Synapses; System; Systems Development; Therapeutic; Transplantation; base; cell type; chemical property; clinically relevant; design; dopamine transporter; dopaminergic differentiation; dopaminergic neuron; fetal; implantation; in vivo; innovation; interest; mRNA Expression; nerve stem cell; nervous system development; nervous system disorder; overexpression; protocol development; scaffold; stem cell biology; surface coating; technology development; tool; two-dimensional

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.

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。对于细胞替代疗法，目前胎儿来源的细胞在实验模型和临床试验中显示出最有希望的结果。然而，在临床研究中，一些患者出现了移植物诱发的运动障碍（GID）。这些负面行为结果被认为部分归因于植入细胞的异质性。因此，制定方案以获得更均匀的 NPC 群体以用于未来的移植研究将非常有价值。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，并可能作为细胞移植载体。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神经元前体。将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 (Nurr1+)、增殖和分化潜力。同时，将使用流式细胞术分析 NPC 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。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，以实现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。 公共健康相关性：该项目的目标是制定策略来扩增、分化和纯化足够数量的神经祖细胞 (NPC)，以用于再生医学。例如，这最终可能有助于治疗帕金森病等中枢神经系统（CNS）疾病。一般来说，NPC 离体的有效扩增和功能分化提出了重大挑战。该提案的第一个目标是通过使用选择性透明质酸基质相互作用淘选初级分离株来提高 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这些纯化的 NPC 群体神经元分化的效率。纯化原代分离干细胞并随后控制其在培养物中的分化潜力的技术的发展将为干细胞生物学的进步提供巨大的机会。