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）：位于腹侧中脑中的中脑多巴胺能（mDA）神经元关键性地控制自主运动、奖励和情绪相关行为，并且它们的变性/功能障碍与主要脑部疾病（如帕金森病（PD）和精神分裂症）相关。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，开发可靠且有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，它可能提供无限的细胞来源与正常与疾病的表型。多能干细胞虽然能够被诱导分化成mDA表型，但倾向于产生随机和异质分化的后代，这可能使测定结果和比较模糊。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。在早期脑发育过程中，关键信号和转录因子的调控级联协调了复杂的分化途径，其中在不同区域产生特定的神经前体（NP），导致随后分化为最终的神经元亚型，如mDA神经元。这种mDA特异性NP的鉴定和分离将提供可容易地产生成熟mDA神经元的可扩展的细胞来源。尽管没有已知的单一标记可用于纯化mDA NP，但我们假设可以基于从mDA神经元的发育研究中获得的知识来鉴定和纯化mDA NP。显示mDA神经元来源于底板，其通过细胞表面标记Corin的表达特异性鉴定。此外，Wnt 5a的受体Frizzled-5（Fzd 5）在发育中的CNS的前脑和中脑的脑室区中表达。因此，这两个基因的表达仅在发育胚胎中的mDA结构域中重叠。基于这些发现，我们提出通过从人ESC衍生的NP和人iPSC衍生的NP共表达这两种表面标志物来纯化人mDA NP。将在体外和体内进一步表征纯化的人mDA NP的增殖和发育潜力以及移植到啮齿动物PD模型后的功能。我们提出的实验将识别和表征人类mDA NP，这将允许前所未有的mDA生物学解剖，并作为开发新的治疗方法的平台。 公共卫生关系：中脑多巴胺（mDA）神经元在自主运动、情绪和奖励相关行为的调节中起关键作用;它们的变性和/或功能障碍与主要的脑疾病如帕金森病（PD）、精神分裂症和药物成瘾相关。拟开展的研究旨在分离人多能干细胞体外分化后的纯mDA神经元前体（NPs）群体，并表征其体内和体外增殖、发育和功能 潜力这些研究不仅有助于进一步了解mDA NP生物学，还为PD和其他DA相关疾病的新治疗方法打开了大门。