Characterization of transcription factor function during human gametogenesis via directed differentiation

Abstract

It is estimated that one in ten people in the US struggle with infertility. Access to human germline cell types for study is limited, and little is known about the underlying genetic regulation of human gametogenesis. In vitro gametogenesis (IVG) holds promise for the complete modeling of human gametogenesis from stem cells and was recently successfully achieved in mice. However, challenges remain in translation of these methods to humans due to species-specific differences, extensive culture periods, low yielding differentiation, and lack of screening tools. In this dissertation, I aim to address these current challenges in human IVG to enable derivation of human germ cell types from human induced pluripotent stem cells (hiPSCs) via transcription factor (TF) overexpression. I develop an all-in-one integrated pipeline for mammalian genetic screening, developing in silico TF prediction tools, cDNA library cloning methods, and NGS-coupled screening readouts. I then leverage these tools to screen 47 TFs for their role in primordial germ cell (hPGCLCs) and oogonia-like formation. I identify three TFs, DLX5, HHEX, and FIGLA whose individual overexpression drive potent enhancement in hPGCLC formation in monolayer and aggregate co-culture. I demonstrate that HHEX and DLX5 are necessary to the germ cell development pathway and that DLX5 overexpression rescues loss of BMP4 during germ cell formation. I additionally identify LHX8, SOHLH1, and ZNF281, whose combinatorial overexpression generates DDX4+ induced oogonia-like cells (i-OLCs) in four days in monolayer culture. I further demonstrate higher order TF combinations to drive increased yield i-OLC formation and develop methods for feeder-free i-OLC expansion. I elucidate the TF binding and regulatory activity of all six TFs and show these TFs regulate the core gametogenesis gene regulatory network (GRN). I likewise uncover mechanisms of how these TFs drive specification of the germ line program via trajectory analysis during directed differentiation. Together, these results establish a foundational platform for targeted derivation and genetic screening of human germ cell types from stem cells via TF-directed differentiation. Using these tools, we uncover TFs that play central roles in human gametogenesis and expand the basic knowledge of how diverse TFs regulate and direct human germ cell development.