Neurodifferentiation/Stem Cell Unit

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Specific aim 1: To develop in vitro 3D brain organoid models derived from human adult peripheral CD34+ cells to study neural development and degeneration and infectious diseases involving human brain. We expended our capability to derive 3D neural organoids to mimic human brain. Using improved clearance techniques and advanced confocal imaging system, we confirmed that pericytes can be produced along endothelial cells and neural cells in the 3D organoid spontaneously with sequential induction media, making the model perfect for virus infection studies, as pericytes have been reported as a main target by viruses including SARS-CoV2. In another 3D model with incorporating CD34 cells, we treated them with cytokines to promote microglial differentiation and used scRNA-Seq analysis to study the resulting microglial cells. We found that although the treatment of cytokines is not necessary for producing microglia, it did increase the numbers of microglia compared to organoids without cytokine stimulation. When the organoids were treated with LPS, which induced inflammatory reactions by increasing transcriptions of certain microglial genes in the organoids, indicating the differentiated microglia in the organoids are functional. A manuscript is prepared for publication based on the results. Specific aim 2: To study the roles of HERV-K on brain development and tumorigenicity. We supported Dr. Ashish Shah on studying the mechanism of HERV-K on the pathogenesis of glioma. We transfected astroglia with plasmid containing HERV-K and detected higher level of gene transcription responsible for stemness. This result indicates that marker for stem cells is activated by HERV-K and maybe the mechanism underlying its effect on tumorigenesis. We are further studying HERV-K effect in cell proliferation and tumorigenesis using 3D models. Specific aim 3: To study the association of HERV-K and ALS. We differentiated motor neurons from four sets of C9orf72 and control iPSCs and did RNA-Seq analysis. The results showed top differential pathways were tumorigenesis related. As we have found that in C9orf72 motor neurons, there was increased DNA repairing element followed by HERV-K activation, this result imply that DNA damaging events in motor neurons leads to HERV-K Env activation, which may reverse the neurons to a status more similar to stem or tumor cells, causing motor neuron malfunction. Specific aim 4: To facilitate research and therapeutic developments for neurological disorders using our models and methods. We are continuing to collaborate with other investigators by providing material support and technique trainings of the iNSC/iPSC generation and 3D modeling. We are working with Dr. In-Hong Yang in UNC to develop protocol to culture motor neurons in a microfluidic chamber. We collaborated with Dr. Kousa from National Childrens Hospital to study the pathogenesis of ZIKA virus on human brain development. We derived neural stem cells from targeting and control iPSC cells and found ZIKA virus infection was enhanced significantly by a target malfunction. We also helped Dr. Farinaz Safavi to generate 3D brain organoids from iPSCs with an mutative gene. Our result showed the mutation decreased the growth of the derived 3D organoids significantly compared to corrected iPSCs.