Characterization of stem cell-derived human dopaminergic neuron subtypes

加拿大在运动障碍的研究方面处于领先地位，尤其是帕金森氏症(PD)。作为第二常见的神经退行性疾病，帕金森病造成了巨大的经济、社会和个人负担。努力发现治疗PD患者的有效方法是当务之急，而帮助我们更好地了解PD患者大脑中神经元是如何丢失的研究对于实现治疗目标是至关重要的。多巴胺(DA)能神经元长期以来一直被认为是帕金森病中受影响的细胞类型；最近的研究发现，这一细胞群体比我们之前想象的要复杂得多，实际上有六种以上的DA神经元亚型。在帕金森病中，这些亚型中的一些比其他亚型更易受影响。这是一项具有临床意义的重要发现，但我们目前对DA神经元亚型的了解有限。令人难以置信的是，神经科学研究人员现在能够在实验室中从来自人类患者的干细胞中生成人类DA神经元，称为诱导多能干细胞(IPSCs)。IPSC来源的DA神经元已成为在大规模筛选实验中模拟帕金森病的发生和发展以及发现潜在治疗化合物的强大工具。然而，我们目前用于IPSC-DA神经元产生的协议有很大的局限性。这些问题包括分化效率低下(通常只有30%的神经元是DA神经元)，以及缺乏关于产生哪些DA神经元亚型的知识。在这项提案中，我们将使用高通量的复合筛选和成像方法来提高IPSC来源的DA神经元的生产效率，并允许对所产生的亚群进行可靠的分层。我们的目标是优化现有的方案，这样研究人员就可以直接从IPSCs中产生DA神经元的个别亚群。这一建议将利用以下方面的互补专业知识：1)Simon Fraser大学的Julian和vocadlo实验室，他们是神经科学、细胞命运生物调控和高通量分析方面的专家；2)Erin Huck博士和她在干细胞技术公司的神经科学团队，这是一家总部位于温哥华的国际知名公司，在IPSC神经元分化策略优化方面拥有长期的专业知识。我们的团队独一无二地准备开展这项工作，我们预计这项工作将对帕金森病和运动障碍研究社区产生重大影响。

Canada is a leader in research on movement disorders, especially Parkinson's Disease (PD). As the second most common neurodegenerative condition, PD is responsible for substantial economic, societal, and personal burden. Efforts to uncover effective approaches to treat PD patients are a high priority, and research to help us better understand how neurons are lost in the brains of PD patients is essential to reach therapeutic goals. Dopaminergic (DA) neurons have long been known as the cell type affected in PD; recent findings have revealed that this cell population is more complex than we previously thought and there are actually more than six subtypes of DA neurons. Some of these subtypes are more susceptible than others in PD. This is an important discovery with clinical implications, but our current understanding of DA neuron subtypes is limited.Incredibly, neuroscience researchers are now able to generate human DA neurons in the lab from easy to generate stem cells derived from human patients, termed induced pluripotent stem cells (iPSCs). iPSC-derived DA neurons have served as powerful tools to model the onset and progression of PD, and to uncover potential therapeutic compounds in large-scale screening experiments. There are substantial limitations, however, to our current protocols for iPSC-DA neuron generation. These include inefficient differentiation (typically, only ~30% of neurons generated are of DA identity) and a lack of knowledge as to which DA neuron subtypes are produced. In this proposal we will use high throughput compound screening and imaging methods to improve the efficiency of iPSC-derived DA neuron production and permit reliable stratification of the subgroups produced. Our goal is to optimize existing protocols so researchers can directly produce individual subgroups of DA neurons from iPSCs. This proposal will harness the complementary expertise of: 1) the Julian and Vocadlo labs at Simon Fraser University, experts in neuroscience, biological regulation of cell fate determination and high-throughput analysis; and 2) Dr. Erin Knock and her Neuroscience team at STEMCELL Technologies, an internationally renowned Vancouver-based company with long established expertise in optimization of iPSC neuronal differentiation strategies. Our team is uniquely poised to conduct this work that we anticipate will substantially impact the PD and movement disorder research communities.