Perfusable brain organoids: a long-term culture approach

神经精神障碍主要是从死后大脑样本中研究的，因为没有能力 从活的受影响的个体中分离出神经元。不幸的是，尸检样本只能提供有限的视角。 障碍，因为障碍的发病时间通常比大脑样本早几十年。此外，蜂窝 生理学很难在死后组织中进行分析，这限制了实验设计。因此，进步 对神经精神障碍背后的细胞和分子机制的了解一直很差。 我们将利用最新的发现，这些发现已经证明iPSC源自3D大脑 器官培养允许模仿人脑发育的多种神经元规格。这些 由于代谢不良，3D球体中心附近的坏死极大地限制了人们的努力 与最内部的细胞交换，并整体限制细胞培养存活。 我们建议将有机化合物移植到小鼠体内，作为一种实现长期培养条件的方法，通过活跃的 通过宿主血管系统的血液灌流。我们希望宿主的血管系统能使 移植的器官。我们建议通过发展血管系统来减少血管形成的时间。 使用人内皮细胞和间充质干细胞的有机化合物。这还有一个额外的好处，即限制 人类器官中的小鼠细胞的数量。我们通过培养来优化植入前的培养条件 生物反应器系统中的有机化合物。我们还将比较生物反应器中的长期培养，在那里培养 移植的有机体的条件是最优的。人们的期望是血管共培养和 通过生物反应器培养，在小鼠体内移植将产生更多成熟和有活力的神经组织。最后，我们 将通过3D免疫染色来表征IPSC来源的3D神经组织、单个器质块和单个 细胞RNAseq. 这些组合实验的影响将是对简单和中高吞吐量的定义 长期神经元分化分析的方法学，旨在产生不同亚群的成熟神经元 种群和更高程度的结构分化模仿人类的端脑发育，具有 神经发育障碍的潜在影响，如自闭症，以及潜在的障碍，如 精神分裂症、阿尔茨海默病和帕金森病及其相关药物的发现。

Neuropsychiatric disorders have been primarily studied from post-mortem brain samples, given the inability to isolate neurons from living affected individuals. Unfortunately post-mortem samples offer a limited view of the disorders, for the disorder onset is typically several decades earlier than the brain sample. In addition, cellular physiology is difficult to analyze in postmortem tissue, limiting the experimental design. As a result, progress toward understanding the cellular and molecular mechanisms behind neuropsychiatric disorders has been poor. We will take advantage of recent discoveries, which have demonstrated that iPSC derived 3D cerebral organoid culture allows large variety of neuronal specification mimicking human brain development. These efforts have been greatly limited by necrosis near the center of the 3D spheroids due to poor metabolite exchange with the inner most cells, and overall limited cell culture survival. We propose to transplant the organoids in mouse as a way to achieve long-term culture conditions, via active blood perfusion through the host vascular system. We expect the host vascular system to vascularize the transplanted organoids. We propose to reduce the time to vascularization by developing a vasculature in the organoids using human endothelial cells and mesenchymal stem cells. This has the added benefit of limiting the number of mouse cells in the human organoid. We optimize culture condition pre-implantation by culturing the organoids in a bioreactor system. We will also compare long-term culture in the bioreactor, where culture conditions are optimal, with the transplanted organoids. The expectation is that vascular co-culture and transplantation in mouse will generate more mature and viable neural tissue the bioreactor culture. Finally, we will characterize the iPSC-derived 3D neural tissue by 3D immunostaining, individual organoid bulk and single cell RNAseq. The impact of these combined experiments will be the definition of a simple and medium to high throughput methodology for long-term neuronal differentiation assays, aimed at producing mature neurons of diverse sub- populations and a higher degree of structural differentiation mimicking human telencephalic development, with potential implications for neurodevelopmental disorders, like Autism, and potentially also disorders like Schizophrenia, Alzheimer and Parkinson disease and related drug discovery.