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。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)。这些负面的行为结果被认为部分是由于植入的细胞的异质性。因此，为将来的移植研究开发获得更统一的神经干细胞群体的方案将是非常有价值的。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，作为仿生培养环境和可能的细胞移植载体。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PAN从VM NPC的初级分离中寻找神经元前体。分离贴壁和未贴壁细胞，用免疫组织化学染色和RT-PCR分析DA前体(Nurr1+)、增殖和分化潜能。同时，应用流式细胞术分析鼻咽癌组织中CD44的表达，建立不同鼻咽癌人群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。此外，天然的细胞外基质蛋白(如层粘连蛋白、纤维连接蛋白)将被添加到水凝胶中，以实现更好的细胞黏附和分化。免疫染色和RT-PCR将用于鉴定3D培养中DA前体细胞的分化。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。 公共卫生相关性：该项目的目标是制定策略，以扩增、分化和纯化足够数量的神经前体细胞(NPC)，以用于再生医学。例如，这最终可能对治疗中枢神经系统(CNS)疾病(如帕金森氏症)有用。总的来说，神经干细胞的体外高效扩增和功能分化是一个巨大的挑战。这项建议的第一个目标是通过使用选择性透明质酸基质相互作用来淘洗原代分离株来提高鼻咽癌培养的纯度。第二个目标是通过提供适当的基质和可溶的线索来提高从这些纯化的鼻咽癌细胞中分化出神经元的效率。纯化原代分离干细胞并随后控制其在培养中的分化潜力的技术的发展，将是干细胞生物学进步的巨大机遇。