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.
This application investigates methodologies for long-term differentiation culture of human induced pluripotent stem cells (iPSCs)-derived neuronal progenitors, for which no viable and robust protocol has yet been identified. Our studies will establish whether co-culturing neuronal progenitors with endothelial cells in the presence of mesenchymal stem cells and transplanting these cultured cells in mouse will generate more mature neurons with improved efficiency. We will compare the transplanted cells with cells cultured in a bioreactor, which provides optimal culture conditions.
