Isolation and characterization of midbrain dopaminergic neuronal precursors

描述(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/功能障碍与帕金森氏病(PD)和精神分裂症等主要大脑疾病有关。为了剖析丙二醛相关疾病的分子和细胞机制，推动新的治疗方法的发展，建立可靠、高效的疾病模型系统至关重要。一个有希望的模型系统是最近建立的诱导多能干细胞技术，该技术可能提供无限的细胞来源，具有正常和疾病的表型。多能干细胞虽然能够被诱导分化为丙二醛表型，但往往会产生随机和异质的分化后代，这可能会模糊分析结果和比较。因此，在使用前对丙二醛细胞进行纯化是至关重要的，以便为进一步的应用提供可靠和特定的细胞源。在大脑发育的早期，关键信号和转录因子的调控级联调控复杂的分化途径，在不同的区域产生特定的神经前体(NPs)，导致最终分化为最终的神经元亚型，如丙二醛(MDA)神经元。这种丙二醛特异的NPs的鉴定和分离将提供容易产生成熟的丙二醛神经元的可扩展的细胞来源。尽管目前还没有已知的单一标记物可用于纯化丙二醛NPs，但我们假设可以基于对丙二醛神经元发育研究获得的知识来鉴定和纯化丙二醛NPs。丙二醛神经元被证明来自底板，通过细胞表面标记Corin的表达来特异性地识别底板。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的脑室区有表达。因此，这两个基因的表达仅在发育中的胚胎中的丙二醛结构域重叠。基于这些发现，我们建议通过从人ESC来源的NPs和人IPSC来源的NPs中共表达这两个表面标志来提纯人的MDA NPs。纯化的人丙二醛纳米粒将在体外和体内进一步鉴定其增殖和发育潜力，以及将其移植到帕金森病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丙二醛纳米粒，这将允许前所未有的丙二醛生物学剖析，并作为开发新的治疗方法的平台。 公共卫生相关性：中脑多巴胺(MDA)神经元在调节自愿运动、情绪和奖赏相关行为方面发挥关键作用；它们的退化和/或功能障碍与帕金森氏病(PD)、精神分裂症和药物成瘾等主要大脑疾病有关。本研究旨在分离人多能干细胞体外分化后的丙二醛神经前体细胞，并对其在体内和体外的增殖、发育和功能进行研究。 潜力。这些研究不仅将有助于进一步了解MDA NP生物学，而且还将为PD和其他DA相关疾病的新治疗方法打开大门。

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.