Development of forebrain organoid platform for modelling human cortical neurogenesis

 描述（由申请人提供）：在过去十年中，干细胞领域的一项重大突破是开发了将人类体细胞重编程为诱导多能干细胞或iPSC的技术。除了在培养皿中从人类iPSC中获得特定细胞类型作为2D培养物的能力之外，该领域的快速进展已经使得从类似于整个发育器官的iPSC产生3D培养物或类器官成为可能，包括肠，肾，视网膜和大脑皮层。人类类器官提供了一个独特的机会，在培养系统中模拟器官发育，这类似于体内的人类器官发生。此外，类器官培养物提供了对影响多种细胞类型的疾病建模和研究非细胞自主效应的机会。我实验室的工作重点是使用小鼠模型和iPSC模型进行神经发育;我们的长期目标是了解正常大脑发育、神经发育疾病的机制，并帮助制定合理的治疗策略。尽管脑类器官在模拟脑发生和脑疾病方面具有巨大的前景，但目前可用的技术存在几个主要限制，包括高成本、低再现性和高可变性，这限制了我们定量分析和广泛应用脑类器官的能力。 技术.我们最近开发了一种新方法，通过将用于产生脑类器官的关键组分进行纯化，从而大幅减少材料，细胞培养基，空间和成本。在这个探索性的项目中，我们建议进一步标准化前脑特异性类器官的生产，并优化细胞培养条件，以实现定向和持续生长。作为一个证明的原则，我们将使用这个系统来测试的假设，即15q11.2微缺失，一个突出的遗传风险因素癫痫，导致癫痫发作的易感性异常皮层神经发生。我们相信，我们的方法将改变器官发生建模，并促进识别疾病相关的生物过程，这是很难概括的二维单层培养。

 DESCRIPTION (provided by applicant): A major breakthrough in the stem cell field over the last decade has been the development of technology to reprogram human somatic cells into induced pluripotent stem cells or iPSCs. In addition to the ability to derive specific cell types from human iPSCs in dish as 2D cultures, rapid progress in the field has made it possible to generate 3D cultures, or organoids, from iPSCs resembling whole developing organs, including intestinal, kidney, retinal, and cerebral cortex. Human organoids provide a unique opportunity to model organ development in a culture system that is similar to human organogenesis in vivo. Furthermore, organoid cultures provide the opportunity to model diseases that affect multiple cell types and to investigate non-cell- autonomous effects. The work in my laboratory focuses on neural development using both mouse models and iPSC models; and our long term goal is to understand mechanisms underlying normal brain development, neurodevelopmental diseases and to aid in the development of rational therapeutic strategies. Despite the tremendous promise of cerebral organoids to model brain genesis and brain diseases, there are several major limitations of the currently available technology, including high cost, low reproducibility and high variability, that limit our ability for quantitative analyses and broad application of the technology. We have recently developed a new approach by miniaturizing the critical components used to generate cerebral organoids, which allows for a dramatic reduction in materials, cell culture media, space and costs. In this exploratory project, we propose to further standardize forebrain specific organoid production and optimize cell culture conditions for directed and sustained growth. As a proof-of-principle, we will use this system to test the hypothesis that 15q11.2 microdeletion, a prominent genetic risk factor for epilepsy, leads to aberrant cortical neurogenesis for seizure susceptibility. We believe that our approach will transform organogenesis modeling and facilitate the identification of disease-relevant biological processes that are difficult to recapitulate in 2D monolayer cultures.