Perfusable brain organoids: a long-term culture approach

可灌注脑类器官：长期培养方法

• 批准号: 9973237
• 负责人: Gianfilippo Coppola
• 金额: $ 20.94万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-05 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9973237
• 关键词: 3-Dimensional; Affect; Alzheimer' s Disease; Anastomosis - action; Animals; Autopsy; Biological Assay; Bioreactors; Blood - brain barrier anatomy; Blood Vessels; Blood capillaries; Brain; Cardiovascular system; Cell Culture System; Cell Culture Techniques; Cell Line; Cell Transplantation; Cell physiology; Cells; Cerebrum; Characteristics; Coculture Techniques; Cultured Cells; Data; Dermal; Development; Diffusion; Disease; Endothelial Cells; Environment; Ethics; Experimental Designs; Generations; Genetic Engineering; Goals; Growth; Human; Hypoxia; In Vitro; Individual; Invaded; Literature; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Methodology; Molecular; Mus; Names; Necrosis; Neurodevelopmental Disorder; Neuronal Differentiation; Neurons; Onset of illness; Organ; Organoids; Parkinson Disease; Pathology; Perfusion; Procedures; Protocols documentation; Publishing; Regenerative Medicine; Research; Sampling; Schizophrenia; Site; Speed; Stem cell transplant; Structure; System; Time; Tissue Engineering; Tissues; Transplantation; Vascular System; Vascularization; Work; Zika Virus; angiogenesis; autism spectrum disorder; blood perfusion; body system; cell type; drug discovery; expectation; experimental study; improved; in vivo; induced pluripotent stem cell; insight; minimally invasive; nerve stem cell; neuropsychiatric disorder; novel; preimplantation; relating to nervous system; single-cell RNA sequencing; stem; three dimensional cell culture; vascular bed

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.

神经精神疾病主要通过死后大脑样本进行研究，因为无法 从活着的受影响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。 疾病，因为疾病的发生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，限制了实验设计。结果，进步了 对神经精神疾病背后的细胞和分子机制的理解一直很差。 我们将利用最近的发现，这些发现证明 iPSC 衍生的 3D 大脑 类器官培养允许模仿人类大脑发育的多种神经元规格。这些 由于代谢物较差，3D 球体中心附近的坏死极大地限制了努力 与最内部细胞的交换，总体上限制了细胞培养物的存活。 我们建议将类器官移植到小鼠体内，作为实现长期培养条件的一种方式，通过主动 通过宿主血管系统的血液灌注。我们期望宿主血管系统使 移植的类器官。我们建议通过在血管内发育脉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类器官。这还有一个额外的好处：限制 人类类器官中小鼠细胞的数量。我们通过培养优化植入前的培养条件 生物反应器系统中的类器官。我们还将比较生物反应器中的长期培养，其中培养 移植类器官的条件是最佳的。期望血管共培养和 移植到小鼠体内将在生物反应器培养物中产生更成熟和有活力的神经组织。最后，我们 将通过 3D 免疫染色、单个类器官体和单个器官来表征 iPSC 衍生的 3D 神经组织 细胞RNA测序。 这些组合实验的影响将是简单和中高通量的定义 长期神经元分化测定的方法，旨在产生不同亚型的成熟神经元 群体和模仿人类端脑发育的更高程度的结构分化， 对神经发育障碍（如自闭症）的潜在影响，以及对诸如自闭症等疾病的潜在影响 精神分裂症、阿尔茨海默病和帕金森病以及相关药物的发现。