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、亚型识别和存活

• 批准号: 10211441
• 负责人: Doron Betel
• 金额: $ 67.57万
• 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/10211441
• 关键词: Address; Adult; Affect; Agreement; Automobile Driving; Behavior; Biological Models; Brain; CRISPR screen; Cell Line; Cell Separation; Cell Survival; Cell Therapy; Cell Transplantation; Cells; Chromatin; Clinical; Clinical Trials; Communities; Corpus striatum structure; Culture Techniques; 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; Neurons; Noise; Parkinson Disease; Pathway interactions; Patients; Phenotype; Production; Property; Protocols documentation; Reporting; Role; SOX6 gene; Signal Transduction; Sorting - Cell Movement; Substantia nigra structure; Surface; Techniques; Technology; Testing; Transplantation; Tremor; Variant; Ventral Tegmental Area; Work; base; 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; novel; patient variability; postnatal; relating to nervous system; 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 帕金森病（PD）是一种运动障碍，涉及中脑多巴胺（mDA）的选择性丢失 黑质的神经元人干细胞，如胚胎干细胞（hESC）和诱导多能干细胞（诱导多能干细胞） hiPSC（hiPSC）代表了研究和潜在治疗PD的强大技术。产生mDA神经元的方法 人类干细胞的研究是我们团队的先驱。这些工作使mDA神经元的应用成为可能， 在培养皿中建立PD模型和开发基于细胞的疗法。事实上，根据我们的工作， 人mDA神经元的移植处于PD临床试验的边缘。尽管取得了这些进展， 产生mDA神经元的策略是次优的，并且所得细胞不匹配所有的分子， mDA神经元的特征。此外，没有可靠的纯化方法来特异性地 富集mDA神经元。缺乏这样的方法是一个问题，特别是在疾病建模中，其中mDA 在来自许多PD患者的细胞系中比较神经元，其中产量的变化可能是主要的 混杂因素此外，使用纯化的mDA神经元将允许更精确的移植 研究以确定最佳的移植物成分。另一个重要的挑战是mDA神经元的存活有限 移植后（约10%的移植细胞），这是一个尚未解决的问题，可能会导致变异性 并使该技术的常规应用复杂化。最后一个挑战是缺乏 了解如何优先产生A9（黑质）或A10（腹侧被盖）的mDA神经元 区）身份。A9和A10都是mDA神经元，但它们代表具有不同分子和结构的亚型。 功能特性，并且A9是PD中疾病建模和细胞治疗的期望亚型。 在此，我们提出三个具体目标，以解决这些悬而未决的问题。在Aim 1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，以获得具有改进的mDA神经元。 分子和功能特性以及能够常规纯化mDA神经元的分选方法。 我们建议使用单细胞基因表达分析来评估这种情况下mDA神经元是否 改善的条件更完全地匹配发育中或成年大脑中的mDA神经元。在目标2中，我们将定义 细胞移植后限制mDA神经元存活的因素。我们开发了一个非常有前途的， 基于CRISPR的筛选技术来定义生存因素，并且已经确定了候选人， 直接在mDA神经元内或通过宿主环境。最后，在目标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来自人干细胞的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 在子类型规范中具有重要的功能。这三个目标中的每一个都涉及一个关键和互补的问题， mDA领域的挑战是解锁人类干细胞衍生的mDA神经元的全部潜力， 治疗和人类疾病建模。