Human Germ Cell Specification Via Combinatorial Expression of Prioritized Transcription Factors

Abstract The generation of germline cell types from human induced pluripotent stem cells (hiPSCs) represents a key milestone toward in vitro gametogenesis, which has the potential to transform reproductive medicine. Methods to recapitulate germline cell specification in vitro have relied on extensive, long term culture methods, the most notable of which is a four-month culture protocol employing xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. Recently, transcription factor (TF)-based methods have demonstrated the feasibility of exogenous factor expression to directly differentiate hiPSCs into cell types of interest, including various ovarian cell types. The protocols leveraged in these studies, however, utilize more local methods of factor selection, such as basic differential gene expression analysis, and lower-throughput screening strategies via iterative testing of a small set of TFs. In this work, we integrate our recently-described, more globally-representative graph theory and highly-parallelized screening protocols to globally identify and screen 46 oogenesis-regulating TFs for their role in human germline formation. We identify DLX5, HHEX, and FIGLA whose individual overexpression enhances hPGCLC formation from hiPSCs, as well as an additional set of three TFs, ZNF281, LHX8, and SOHLH1, whose combinatorial overexpression drives DDX4+ germ cell formation from hiPSCs. In contrast to previous methods, our protocol employs a simple four-day, feeder-free monolayer culture condition. We characterize these TF-based germ cells via gene expression analyses, and demonstrate their broad similarity to in vivo and in vitro-derived germ cells. Together, these results identify new regulatory factors that enhance in vitro human germ cell specification and further establish unique computational and experimental tools for human in vitro oogenesis research.