An in vitro model of the human ovary would greatly benefit the study of female sex development and reproduction. Current models do not recapitulate the human ovarian somatic cell environment and require extensive culture periods, necessitating innovation in engineering approaches. Here, we report overexpression of transcription factors (TFs) for rapid differentiation of human induced pluripotent stem cells (hiPSCs) to FOXL2+ granulosa-like cells and DDX4+ oogonia-like cells in vitro.
Through TF overexpression screening, we identified a core set of TFs capable of yielding FOXL2+granulosa-like cells from hiPSCs in five days. We systematically establish that overexpression ofNR5A1 and either RUNX1 or RUNX2 is sufficient to generate granulosa-like cells. Our granulosa cells have transcriptomes resembling human fetal ovarian cells and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with human primordial germ cell-like cells (hPGCLCs), our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the NANOS3+ premigratory to the DAZL+ gonadal stage in as little as four days.
In parallel, we assessed the effect of overexpression of putative oogenesis-regulating TFs on human germ cell specification. We show that overexpression of the TFs DLX5, HHEX, and FIGLA enhance hPGCLC specification from hiPSCs, and that DLX5 activity can substitute for BMP signaling requirements during hPGCLC differentiation. Additionally, we find that overexpression of the combination LHX8, SOHLH1, and ZNF281 can directly differentiate hiPSCs to DDX4-positive oogonia-like cells in a four-day monolayer culture system. We characterize these TF-based germ cells via gene expression analyses and demonstrate their broad similarity to in vivo and in vitro germ cells.
These results establish a first-in-human method for TF-directed differentiation of ovarian supportcells and post-migratory germ cells from hiPSCs. Together, these results increase our understandingof the genetic regulatory factors involved in human ovarian development and generate new tools forin vitro gametogenesis.