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

基本信息

批准号：
10378152
负责人：
Doron Betel
金额：
$ 63.71万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
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/10378152
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

项目摘要

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.

项目概要帕金森病 (PD) 是一种运动障碍，涉及中脑多巴胺 (mDA) 的选择性丧失黑质中的神经元。人类干细胞，例如胚胎干细胞 (hESC) 和诱导多能干细胞（hiPSC）代表了研究和潜在治疗帕金森病的强大技术。生成 mDA 神经元的方法我们的团队率先从人类干细胞中提取。这些工作使得 mDA 神经元的应用成为可能在培养皿中对 PD 进行建模并用于开发基于细胞的疗法。事实上，根据我们的工作，人类 mDA 神经元移植即将进入 PD 临床测试阶段。尽管取得了这些进展，但目前生成 mDA 神经元的策略不是最理想的，并且产生的细胞与所有分子不匹配大脑中 mDA 神经元的特征。此外，目前还没有可靠的纯化方法来专门丰富 mDA 神经元。缺乏此类方法是一个问题，特别是在疾病建模中，mDA 对来自许多帕金森病患者的细胞系中的神经元进行比较，其中产量的变异性可能是一个主要因素混杂因素。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移植物组成。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），这个问题仍未解决，并且可能导致变异细胞剂量的变化并使该技术的常规应用变得复杂。最后一个挑战是缺乏了解如何优先生成 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，A9 是 PD 疾病建模和细胞治疗所需的亚型。在这里，我们提出三个具体目标来解决这些悬而未决的问题。在Aim1的基础上，令人兴奋初步数据，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一种能够常规纯化 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 mDA 神经元是否在这种情况下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。我们开发了一种非常有前途的、基于 CRISPR 的筛选技术来定义生存因素，并且已经确定了候选者直接在 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以及染色质可及性研究，以绘制人类干细胞 mDA 神经元 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以下因素：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且互补的目标 mDA 领域的挑战是释放人类干细胞衍生的 mDA 神经元对细胞的全部潜力治疗和人类疾病建模。