Directing Fate, Subtype Identity and Survival in Human Pluripotent-Derived Midbrain Dopamine Neurons

指导人类多能源性中脑多巴胺神经元的命运、亚型识别和生存

• 批准号: 10596583
• 负责人: Doron Betel
• 金额: $ 63.69万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10596583
• 关键词: Acceleration; Address; Adult; Affect; Agreement; Automobile Driving; Behavior; Biological Models; Bradykinesia; Brain; CRISPR screen; Cell Line; Cell Separation; Cell Survival; Cell Therapy; Cell Transplantation; Cells; Chromatin; Clinical; Clinical Trials; Communities; Corpus striatum structure; Data; Derivation procedure; Development; Disease; Disease model; Dopamine; Dose; Embryo; Enteral; Environment; Floor; Future; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Generations; Genetic; Human; In Vitro; Knowledge; Laboratories; Location; Maps; Methods; Microglia; Midbrain structure; Modeling; Molecular; Movement Disorders; Mus; Neuronal Differentiation; Neurons; Noise; Parkinson Disease; Pathway interactions; Patients; Phenotype; Production; Property; Protocols documentation; Reporting; Role; SOX6 gene; Signal Transduction; Sorting; Specific qualifier value; Substantia nigra structure; Surface; Techniques; Technology; Testing; Transplantation; Tremor; Variant; Ventral Tegmental Area; Work; candidate identification; cell type; clinical translation; disease-in-a-dish; disorder subtype; dopaminergic neuron; drug discovery; fetal; fibroblast growth factor 18; first-in-human; genetic selection; human disease; human pluripotent stem cell; human stem cells; improved; in vivo; induced pluripotent stem cell; insight; motor symptom; neural; neuronal survival; novel; postnatal; research clinical testing

Project Summary Parkinson's disease (PD) is a movement disorder that involves the selective loss of midbrain dopamine (mDA) neurons in the substantia nigra. Human stem cells, such as embryonic (hESCs) and induced pluripotent (hiPSCs), represent a powerful technology to study and potentially treat PD. Methods to generate mDA neurons from human stem cells have been pioneered by our group. Such work enabled applications of mDA neurons for modeling PD in a dish and for the development of cell-based therapies. In fact, based on our work, the transplantation of human mDA neurons is at the verge of clinical testing in PD. Despite such progress, current strategies for generating mDA neurons are suboptimal and the resulting cells do not match all the molecular features of mDA neurons in the brain. In addition, there are no reliable purification methods to specifically enrich for mDA neurons. The lack of such methods is a problem, particularly in disease modeling, where mDA neurons are compared across cell lines from many PD patients and where variability in yield can be a major confounding factor. Furthermore, the use of purified mDA neurons will allow more precise transplantation studies to define optimal graft composition. Another important challenge is the limited survival of mDA neurons after transplantation (~10% of grafted cells), a problem that remains unresolved, and that can cause variability in cell dosing and complicate the routine application of this technology. A final challenge is the lack of knowledge how to preferentially generate mDA neurons of either A9 (substantia nigra) or A10 (ventral tegmental area) identity. Both A9 and A10 are mDA neurons, but they represent subtypes with different molecular and functional properties, and with A9 being the desired subtype for disease modeling and cell therapy in PD. Here, we propose three specific aims to address these outstanding questions. In Aim1, based on exciting preliminary data, we will refine our mDA neuron differentiation strategy to obtain mDA neurons with improved molecular and functional properties and a sorting method that will enable routine purification of mDA neurons. We propose the use of single cell gene expression analysis to assess whether mDA neurons under such improved conditions more fully match mDA neurons in the developing or adult brain. In Aim 2, we will define the factors that limit survival of mDA neurons upon cell transplantation. We have developed a very promising, CRISPR-based screening technology to define survival factors, and already identified candidates acting either directly within mDA neurons or via the host environment. Finally, in Aim 3, we will use single cell gene expression and chromatin accessibility studies to map A9/A10 subtype diversity of mDA neurons from human stem cells. The results from those in-depth single cell profiling studies will be used to identify and test factors that are functionally important in subtype specification. Each of the three aims addresses a critical and complementary challenge in the mDA field towards unlocking the full potential of human stem cell-derived mDA neurons for cell therapy and human disease modeling.

项目摘要 帕金森氏病（PD）是一种运动障碍，涉及中脑多巴胺（MDA）的选择性丧失 黑质的神经元。人类干细胞，例如胚胎（HESC）和诱导多能 （HIPSC）代表了一种强大的技术，可以研究和治疗PD。生成MDA神经元的方法 从人类干细胞中，我们的组已开创了我们的小组。此类工作启用了MDA神经元的应用 在菜肴中对PD进行建模，以开发基于细胞的疗法。实际上，根据我们的工作 人类MDA神经元的移植处于PD中临床测试的边缘。尽管取得了这样的进展，但 产生MDA神经元的策略是次优的，所得细胞不匹配所有分子 大脑中MDA神经元的特征。另外，没有可靠的纯化方法 富含MDA神经元。缺乏这种方法是一个问题，尤其是在疾病建模中，其中MDA 比较来自许多PD患者细胞系的神经元，而产率的可变性可能是主要的 混杂因素。此外，使用纯化的MDA神经元将允许更精确的移植 定义最佳移植物组成的研究。另一个重要的挑战是MDA神经元的生存有限 移植后（约10％的移植细胞），这个问题仍未解决，可能会导致可变性 在细胞剂量中，并使该技术的常规应用复杂化。最后一个挑战是缺乏 知识如何优先生成A9（底底）或A10的MDA神经元（腹侧段） 区域）身份。 A9和A10都是MDA神经元，但它们代表具有不同分子和不同的亚型 功能性能，A9是PD中疾病建模和细胞疗法的所需亚型。 在这里，我们提出了三个具体目标，以解决这些杰出的问题。在AIM1中，基于令人兴奋的 初步数据，我们将完善我们的MDA神经元分化策略，以获得改进的MDA神经元 分子和功能特性以及一种可以使MDA神经元常规纯化的分类方法。 我们建议使用单细胞基因表达分析来评估在这种情况下MDA神经元是否 改善的条件更完全匹配发育中或成人大脑中的MDA神经元。在AIM 2中，我们将定义 限制细胞移植后MDA神经元存活的因素。我们发展了一个非常有前途的 基于CRISPR的筛查技术来定义生存因素，并且已经确定了行动的候选人 直接在MDA神经元内或通过主机环境。最后，在AIM 3中，我们将使用单细胞基因表达 和染色质可及性研究以绘制人类干细胞中MDA神经元的A9/A10亚型多样性。 这些深入的单细胞分析研究的结果将用于识别和测试因素 功能在亚型规范中很重要。三个目标中的每个目标都解决了一个关键和互补的 MDA领域的挑战，以解锁人类干细胞衍生的MDA神经元对细胞的全部潜力 治疗和人类疾病建模。