Accelerating the Fate Specification of Human Pluripotent Stem Cell-Derived Forebrain Progenitors into Cortical Neurons

The human neocortex consists of excitatory projection neurons and inhibitory interneurons, which undergo an intricate cascade of cellular events during development. These two populations both arise from a similar type of neuronal precursor cell, and diverge into two distinct neuronal lineages through a series of molecular events causing cell fate specification. Using human Induced Pluripotent Stem cells (hIPS) harboring stable transgenic green fluorescent protein (GFP) reporters, we are able to detect and accelerate the generation of early forebrain neural progenitor cells that are fate-specified towards interneurons or projection neurons. By screening different combinations of 20 small molecule inhibitors of multiple cell-signaling pathways, we report a novel method to enhance the generation of fate-specified neurons within 2-3 weeks. Current protocols take between 8-12 weeks to generate neurons from pluripotency, with limited efficiency of patterning. By employing this small molecule combination, we can enhance the specification of forebrain-patterned neural progenitor cells into post-mitotic neurons in three weeks with better maturation and efficiency. We characterize these neuronal subtypes by using immunocytochemistry, electrophysiology, and fluorescence activated cell sorting (FACS). Our results indicate that our hIPS-derived neurons express the appropriate neurochemical, morphological, and electrophysiological properties associated with functional cortical neurons, which can be used to better understand cell vulnerability to neurodegenerative disease progression.