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; 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，以生成用于细胞疗法的多巴胺能（DA）神经元的富集群体。 DA神经元的特发性死亡会导致帕金森氏病（PD）的症状，因此，取代这些细胞是PD研究的主要目标。对于细胞替代疗法，目前胎儿衍生的细胞在实验模型和临床试验中显示出最有希望的结果。但是，在临床研究中，一些患者患有移植引起的运动障碍（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，制定协议以获取更统一的NPC进行未来的移植研究，将非常有价值。在这里，提出了一种方法，将DA前体与腹脑神经祖细胞（VM NPC）分离，然后在3D，透明质酸（HA）水凝胶中离体膨胀和分化，这些水凝胶是生物映射培养环境，可能是细胞移植车。具体的目标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。在发育过程中，HA和CD44在胎儿大脑中都过表达。 PI的实验室的初步结果表明，与混合培养物相结合的HA涂层表面结合了未成熟的NPC。不同浓度的HA涂层表面将用于用于从VM NPC的主要分离的神经元前体。使用免疫染色和RT-PCR，将分离并分离粘附的细胞和非粘附细胞，并分析DA先驱，增殖和分化电位。同时，将使用流式细胞仪和不同NPC群体的CD44表达的阈值分析NPC的CD44表达。在特定的目标2中，将在3D中培养富含DA前体的预先分离的NPC，PI先前开发的HA水凝胶以增强VM NPC的分化为神经元。这些水凝胶旨在提供一个仿生环境，其中的机械和化学性质与天然胎儿大脑的特性非常相似。另外，将将天然ECM蛋白（例如层粘连蛋白，纤连蛋白）添加到水凝胶中，以更好地细胞粘附和分化。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离体扩张和随后的3D生物材料的分化之前，将DA前体进行预选的结合。 PI假设可以使用这种两步方法来产生大量足以用于治疗益处的DA神经元。 公共卫生相关性：该项目的目的是制定策略，以扩展，区分和净化神经祖细胞（NPC），以足够的量用于再生医学。例如，这对于治疗中枢神经系统（CNS）疾病（例如帕金森氏病）的疗法最终可能很有用。通常，NPC的有效扩展和功能分化构成了重大挑战。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平键二级分离株来增强NPC培养物的纯度。第二个目标是通过提供适当的矩阵和可溶性提示来提高神经元分化与这些纯化的NPC种群的效率。净化原发性干细胞并随后控制其培养物的分化潜力的技术的发展将是一个巨大的干细胞生物学发展的机会。