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

基本信息

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
项目类别：
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

项目摘要

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）：中脑中脑中脑中脑中脑中脑多巴胺能（MDA）神经元严重控制自愿运动，奖励和与情绪相关的行为，其变性/功能障碍与主要脑疾病（如帕金森氏病（PD）和精神分裂症等重大脑疾病有关。为了剖析与MDA相关疾病的分子和细胞机制，并推进新型治疗剂的发展，开发可靠有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，它可能会提供具有正常疾病表型的无限细胞来源。多能干细胞虽然能够被诱导与MDA表型分化，但倾向于产生随机和异质分化的后代，这可能会掩盖测定结果和比较。因此，必须在使用前纯化MDA细胞，以确保可靠的特定细胞来源以进行进一步应用。在早期大脑发育过程中，通过关键信号和转录因子进行的调节级联序列策划了复杂的分化途径，其中在不同区域产生了特定的神经前体（NP），从而导致与MDA神经元等神经元的最终亚型相应的分化。这种MDA指定的NP的识别和隔离将提供可扩展的细胞来源，可以容易产生成熟的MDA神经元。即使没有已知的单个标记可以净化MDA NP，我们假设可以根据MDA神经元的发展研究获得的知识来识别和纯化MDA NP。 MDA神经元显示出源自地板板，该板的表面表面标记Corin的表达是特异性识别的。此外，WNT5A的受体Frizzled-5（FZD5）在发育中的CNS的前脑和中脑的心室区域表达。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，我们建议通过从人类ESC衍生和人类IPSC衍生的NP中共表达这两个表面标记来净化人类MDA NP。纯化的人类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。我们提出的实验将识别和表征人类MDA NP，这将允许对MDA生物学的前所未有的解剖，并用作开发新型治疗方法的平台。公共卫生相关性：中脑多巴胺（MDA）神经元在调节自愿运动，情感和与奖励相关的行为中起关键作用；它们的退化和/或功能障碍与诸如帕金森氏病（PD），精神分裂症和药物成瘾等主要脑部疾病有关。拟议的研究旨在分离人类多能干细胞的体外分化后纯MDA神经元前体（NP），并表征其体内和体外增殖，发育和功能潜在的。这些研究不仅将促进对MDA NP生物学的进一步了解，而且还为PD和其他DA相关疾病的新型治疗方法打开了大门。