Isolation and characterization of midbrain dopaminergic neuronal precursors

中脑多巴胺能神经元前体的分离和表征

• 批准号: 8356550
• 负责人: SANGMI CHUNG
• 金额: $ 19.75万
• 依托单位: MCLEAN HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8356550
• 关键词: Address; Advanced Development; Animals; Area; Behavior; Biological Assay; Biological Models; Biological Neural Networks; Biology; Brain; Brain Diseases; Cell Line; Cell surface; Cells; Characteristics; Corpus striatum structure; Development; Disease; Disease model; Dissection; Dopamine; Dopaminergic Cell; Drug Addiction; Embryo; Embryonic Development; Emotions; Etiology; Fiber; Floor; Functional disorder; Gene Expression; Genetic; Genomics; Growth; Human; Immunohistochemistry; In Vitro; Intervention; Knowledge; Laboratories; Maintenance; Methods; Midbrain structure; Modification; Molecular; Moods; Movement; Neuronal Differentiation; Neurons; Neurotransmitters; Overlapping Genes; Parkinson Disease; Pathway interactions; Pattern; Phenotype; Play; Pluripotent Stem Cells; Population; Population Heterogeneity; Preclinical Drug Evaluation; Property; Prosencephalon; Rattus; Regulation; Reporter; Resources; Rewards; Rodent Model; Role; Schizophrenia; Scientist; Signal Transduction; Signaling Molecule; Source; Specific qualifier value; Staging; Surface; Testing; Therapeutic; Transplantation; Ventricular; base; behavior test; calbindin; design; disease phenotype; dopaminergic differentiation; dopaminergic neuron; human embryonic stem cell; human tissue; immunocytochemistry; in vivo; induced pluripotent stem cell; migration; nerve stem cell; nerve supply; novel therapeutic intervention; novel therapeutics; receptor; research study; reuptake; stem cell technology; success; synaptogenesis; therapeutic development; tool; transcription factor

DESCRIPTION (provided by applicant): Midbrain dopaminergic (mDA) neurons residing in the ventral midbrain critically control voluntary movement, reward, and mood-related behaviors, and their degeneration/dysfunction is associated with major brain disorders such as Parkinson's disease (PD) and schizophrenia. In order to dissect the molecular and cellular mechanisms of mDA-related diseases and advance the development of novel therapeutics, it is critical to develop reliable and efficient disease model systems. A promising model system is the recently established induced pluripotent stem cell technology that potentially can provide unlimited cell sources with normal vs. disease phenotypes. Pluripotent stem cells, though able to be induced to differentiate to the mDA phenotype, tend to generate stochastic and heterogeneous differentiated progenies, which can obscure assay results and comparisons. Thus, it is essential to purify mDA cells prior to usage in order to guarantee a reliable and specific cell source for further application. During early brain development, regulatory cascades by key signals and transcription factors orchestrate intricate differentiation pathways in which specific neural precursors (NPs) are generated in different areas, leading to consequential differentiation to final subtype of neurons such as mDA neurons. Identification and isolation of such mDA-specified NPs will provide expandable cell sources that can readily generate mature mDA neurons. Even though no known single marker is available to purify mDA NPs, we hypothesize that mDA NPs can be identified and purified based on the knowledge gained from developmental studies of mDA neurons. mDA neurons were shown to be derived from the floor plate, which is specifically identified by the expression of the cell surface marker Corin. In addition, Frizzled-5 (Fzd5), the receptor for Wnt5a, is expressed in the forebrain and the ventricular zone of the midbrain in the developing CNS. Thus, the expression of these two genes overlaps only in the mDA domains in developing embryo. Based on these findings, we propose to purify human mDA NPs by the co-expression of these two surface markers from human ESC-derived and human iPSC-derived NPs. Purified human mDA NPs will be further characterized in vitro and in vivo for their proliferative and developmental potentials as well as their functionality following transplantation into a rodent model of PD. Our proposed experiments will identify and characterize human mDA NPs that will allow unprecedented dissection of mDA biology as well as serve as a platform to develop novel therapeutic approaches. PUBLIC HEALTH RELEVANCE: Midbrain dopamine (mDA) neurons play critical roles in the regulation of voluntary movement, emotion, and reward-related behavior; their degeneration and/or dysfunction is associated with major brain disorders such as Parkinson's disease (PD), schizophrenia, and drug addiction. The proposed studies are aimed at isolating a pure population of mDA neuronal precursors (NPs) after in vitro differentiation of human pluripotent stem cells, and characterize their in vivo and in vitro proliferative, developmental and functional potential. These studies will not only facilitate further understanding of mDA NP biology but also open the door to novel therapeutic approaches for PD and other DA-related disorders.

描述（由申请人提供）：位于中脑腹侧的中脑多巴胺能（mDA）神经元关键控制自主运动、奖励和情绪相关行为，它们的退化/功能障碍与帕金森病（PD）和精神分裂症等主要脑部疾病有关。为了剖析 mDA 相关疾病的分子和细胞机制并推进新疗法的开发，开发可靠、高效的疾病模型系统至关重要。一个有前途的模型系统是最近建立的诱导多能干细胞技术，它有可能提供无限的具有正常与疾病表型的细胞来源。多能干细胞虽然能够被诱导分化为 mDA 表型，但往往会产生随机和异质的分化后代，这可能会掩盖测定结果和比较。因此，在使用前纯化 mDA 细胞至关重要，以保证进一步应用的可靠且特定的细胞来源。在早期大脑发育过程中，关键信号和转录因子的级联调节协调了复杂的分化途径，其中不同区域产生特定的神经前体 (NP)，从而导致最终分化为神经元的最终亚型，例如 mDA 神经元。鉴定和分离此类 mDA 特异性 NP 将提供可扩展的细胞来源，可轻松生成成熟的 mDA 神经元。尽管没有已知的单一标记可用于纯化 mDA NP，但我们假设可以根据 mDA 神经元发育研究获得的知识来鉴定和纯化 mDA NP。 mDA 神经元被证明源自底板，这是通过细胞表面标记 Corin 的表达来明确识别的。此外，Frizzled-5 (Fzd5)（Wnt5a 的受体）在发育中的 CNS 的前脑和中脑脑室区表达。因此，这两个基因的表达仅在发育胚胎的 mDA 结构域中重叠。基于这些发现，我们建议通过人 ESC 衍生的 NP 和人 iPSC 衍生的 NP 的这两种表面标记的共表达来纯化人 mDA NP。纯化的人 mDA NP 将在体外和体内进一步表征其增殖和发育潜力以及移植到 PD 啮齿动物模型后的功能。我们提出的实验将鉴定和表征人类 mDA NP，这将允许对 mDA 生物学进行前所未有的剖析，并作为开发新治疗方法的平台。 公共卫生相关性：中脑多巴胺 (mDA) 神经元在随意运动、情绪和奖励相关行为的调节中发挥着关键作用；它们的退化和/或功能障碍与帕金森病（PD）、精神分裂症和毒瘾等主要脑部疾病有关。拟议的研究旨在在人多能干细胞体外分化后分离纯 mDA 神经元前体 (NP) 群体，并表征其体内和体外增殖、发育和功能。 潜在的。这些研究不仅有助于进一步了解 mDA NP 生物学，而且为 PD 和其他 DA 相关疾病的新治疗方法打开了大门。