Comprehensive Techniques to Study Activity-dependent and Neurogenic Mechanisms in Stem Cell-derived Neuron Models

Accurately modeling human-related neuronal phenomena remains at the forefront of neuroscience. This thesis utilizes already-established in vitro models of mouse embryonic stem cells, designing an efficient method to optogenetically stimulate neurons derived from mouse stem cells and expounds upon their scope with novel protocols to generate hippocampal neurons from human induced pluripotent stem cells. First, a novel platform for optogenetic stimulation was built and tested on mouse embryonic stem cells to demonstrate functionality of optogenetic channels in mouse embryonic stem cell-derived neurons. The device was built from 3D printed materials and validated with oscilloscopy and spectrophotometry while neurons were cultured for over 30 days in vitro and assayed first for electrical activity by electrophysiology, calcium signaling, and small molecule activation of glutamatergic receptors. When verified that both device and neurons were functional, cells were transduced with a ChannelRhodopsin variant, ChR2-eYFP-NpHR, and were stimulated over several light cycle parameters and assayed for CFOS expression. Having shown that neurons responded in an activity-dependent manner to the device, I established preliminary studies into human hippocampal embryonic neurogenesis. I derived a novel protocol to differentiate hiPSCs to hippocampal neural progenitors using small molecules and specific laminar substrates unique to the subgranular zone. Hippocampal progenitors were assayed for literature-established genetic markers including WNT7b, WNT8a, PROX1, FOXG1, and ZBTB20, and then allowed to spontaneously differentiate into neurons expressing canonical neural, synaptic, glutamatergic, and constitutive hippocampal markers. These cells were expanded over 200 days in vitro. When allowed to spontaneously differentiate or forced to differentiate under NOTCH inhibition, neuronal cultures sustained ZBTB20 and FOXG1 coexpression over the terminal differentiation path though cultures at ~200 days old did not differentiate at the same rate as cultures from ~30 days. When transplanted in vivo, human hippocampal progenitors differentiated fully after 4 months, projected toward the CA3 from the dentate gyrus, and established synaptic connections with host neurons identified by staining synaptic markers. In conclusion, several novel findings are demonstrated throughout this thesis, though the most pertinent include: 1.) mESC-derived neurons may be optogenetically stimulated by ergonomic device fabrication. 2.) Sustained or adult neurogenesis is dependent on the laminin isoform expressed in the subgranular zone. 3.) Hippocampal progenitors from human induced pluripotent stem cells behave like neurons and can be optogenetically targeted and are transplantable in vivo hippocampus in which they integrate into pre-existing hippocampal networks. Future investigations include merging activity-dependent Tau phosphorylation in mESC- and hiPSC-derived human hippocampal neurons and transplantation of human hippocampal