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）：中脑多巴胺能（mDA）神经元位于中脑腹侧，对自主运动、奖励和情绪相关行为具有重要控制作用，其退化/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。为了深入研究mda相关疾病的分子和细胞机制，促进新疗法的发展，建立可靠、高效的疾病模型系统至关重要。最近建立的诱导多能干细胞技术是一个很有前途的模型系统，它可能提供无限的细胞来源，具有正常和疾病表型。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分化后代，这可能会模糊分析结果和比较。因此，必须在使用前纯化mDA细胞，以保证进一步应用的可靠和特异性细胞来源。在早期大脑发育过程中，关键信号和转录因子介导的调节级联反应协调了复杂的分化途径，在这些途径中，特定的神经前体（NPs）在不同区域产生，从而导致最终的神经元亚型分化，如mDA神经元。鉴定和分离这种mDA指定的NPs将提供可扩展的细胞来源，可以很容易地产生成熟的mDA神经元。尽管没有已知的单一标记物可用于纯化mDA NPs，但我们假设基于mDA神经元发育研究获得的知识，可以鉴定和纯化mDA NPs。mDA神经元来源于底板，通过细胞表面标记物Corin的表达进行特异性鉴定。此外，Wnt5a的受体frizzled5 （Fzd5）在发育中的中枢神经系统的前脑和中脑心室区表达。因此，这两个基因的表达只在发育胚胎的mDA结构域重叠。基于这些发现，我们建议通过从人类esc来源的NPs和人类ipsc来源的NPs中共同表达这两个表面标记来纯化人类mDA NPs。纯化的人mDA NPs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。我们提出的实验将识别和表征人类mDA NPs，这将允许前所未有的mDA生物学解剖，并作为开发新治疗方法的平台。