Directing fate, subtype identity and survival in human pluripotent-derived midbrain dopamine neurons

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 黑质内的神经元。人类干细胞，如胚胎干细胞(HESCs)和诱导多能性 (HiPSCs)，代表了一种研究和潜在治疗帕金森病的强大技术。丙二醛神经元的产生方法 从人类干细胞中分离出来是我们团队的先驱。这项工作使丙二醛神经元的应用成为可能 在培养皿中模拟帕金森病，并用于细胞疗法的开发。事实上，根据我们的工作， 人类丙二醛神经元移植治疗帕金森病即将进行临床试验。尽管取得了这样的进展，但目前 生成丙二醛神经元的策略不是最优的，所得到的细胞并不与所有分子匹配 脑内丙二醛神经元的特征。此外，目前还没有可靠的提纯方法来具体地 丰富丙二醛神经元。缺乏这样的方法是一个问题，特别是在疾病建模中，其中 对来自许多帕金森病患者的细胞系的神经元进行比较，其中产量的差异可能是主要的 混杂因素。此外，使用纯化的丙二醛神经元将允许更精确的移植 确定最佳接枝成分的研究。另一个重要的挑战是丙二醛神经元的有限存活 移植后(约10%的移植细胞)，这是一个仍未解决的问题，可能会导致变异 在细胞给药方面，这项技术的常规应用变得复杂起来。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)的丙二醛神经元 区域)身份。A9和A10都是MDA神经元，但它们代表的亚型具有不同的分子和 A9是帕金森病疾病模型和细胞治疗的理想亚型。 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中，基于令人兴奋的 初步数据，我们将完善我们的丙二醛神经元分化策略，以获得具有改进的丙二醛神经元 分子和功能特性以及一种能够进行常规纯化的丙二醛神经元的分选方法。 我们建议使用单细胞基因表达分析来评估在这种情况下丙二醛神经元 改善的条件与发育中的或成年大脑中的丙二醛神经元更加匹配。在目标2中，我们将定义 限制细胞移植后丙二醛神经元存活的因素。我们已经开发出一种非常有前途的， 基于CRISPR的筛查技术，以定义生存因素，并已确定的候选人 直接在丙二醛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，以定位来自人类干细胞的丙二醛神经元的A9/A10亚型多样性。 这些深入的单细胞图谱研究的结果将被用来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解决了一个关键和互补的问题 丙二醛领域对释放人干细胞来源的丙二醛神经元对细胞的全部潜力的挑战 治疗和人类疾病模型。

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.