Creation and Evaluation of iPSCs from Children with ASD with Megalencephaly

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PROJECT SUMMARY – PROJECT 3 Autism spectrum disorder (ASD) is a complex condition characterized by important changes to the brain and behavior. 15% of boys with ASD have disproportionate megalencephaly (ASD-DM), or enlarged brain relative to body size. An increase in brain size often precedes the first clinical signs of the disorder, suggesting that understanding the mechanisms leading to brain overgrowth could provide a window of opportunity to intervene and possibly prevent disease onset. Here, the research team will use human induced pluripotent stem cell (hiPSC) technology to model ASD-DM and investigate the underlying cellular and molecular mechanisms involved. They will obtain skin fibroblasts from 40 individuals in Project 2 and derive human iPSCs from: A) 10 ASD subjects with megalencephaly, ASD-DM; B) 10 ASD subjects with normal sized brains, ASD-N C) 10 Typically developing (TD) subjects with megalencephaly, TD-DM, and D) 10 TD subjects with normal sized brains, TD-N. Following iPSC generation, they will differentiate each of the iPSC lines into neural progenitor cells (NPCs), oligodendrocyte progenitor cells (OPCs), and microglia (the primary immune cells in the brain that maintain homeostasis). The overarching goals of their project are two-fold: 1) to investigate whether ASD-DM is due to an increase in cell proliferation, increase in cell survival, improper elimination of damaged cells, and/or a combination of all; and 2) to identify therapeutic targets by understanding the underlying cellular and signaling mechanisms involved. In Specific Aim 1, they will identify the cellular mechanisms underlying ASD-DM by investigating changes in the cell cycle, cell proliferation, and apoptosis of iPSC-derived NPCs, OPCs, and microglial cells. In Specific Aim 2, they will investigate the functional activity of microglia in ASD-DM by directly differentiating each of the iPSC lines into microglia and assessing their phagocytic capacity by co-culturing them with mixed neuroglial cultures derived from the same lines. This will test their hypothesis that microglia are compromised in ASD-DM, failing to eliminate damaged cells and synapses and contributing to brain overgrowth. In Specific Aim 3, they will identify the underlying regulatory signaling mechanisms that lead to the changes at the cellular level. They will differentiate the iPSCs into NPCs, OPCs, and microglia, sort them by flow cytometry using antibodies specific for each cell type, and perform RNA-sequencing to identify gene networks and signaling mechanisms that are significantly regulated in each condition. Using these mechanistic insights, they will identify therapeutic targets to directly test in the in vitro models. Their overall goal across the projects is to collect imaging, behavioral, and mechanistic data on the same cohort of subjects. In Specific Aim 4, they will correlate the cellular and mechanistic data obtained in Project 3 with the imaging and behavioral data from Project 2 to identify broader trends and characteristics specific to ASD-DM. This comprehensive body of data will be a valuable resource for the broader research and medical communities in identifying predictive biomarkers of ASD and/or ASD-DM and potentially more tailored therapies.