Efficient Human Germ Cell Specification from Stem Cells via Combinatorial Expression of Transcription Factors Christian Kramme, 1,2,* Merrick Pierson Smela, 1,2,* Bennett Wolf, 1,2 Patrick R. Fortuna, 1,2 Garyk Brixi, 1,2,3 Kalyan Palepu, 1,2,3 Edward Dong, 1,2 Jessica Adams, 1,2 Suhaas Bhat, 1,2,3 Sabrina Koseki, 4,5 Emma Tysinger, 4,5 Teodora Stan, 4,5 Richie E. Kohman, 1,2 Songlei Liu, 1,2 Mutsumi Kobayashi, 6 Toshi Shioda, 6 George M. Church, 1,2 Pranam Chatterjee 1,2,3,4,5,† 1. Wyss Institute, Harvard Medical School 2. Department of Genetics, Harvard Medical School 3. Department of Biomedical Engineering, Duke University 4. Center for Bits and Atoms, Massachusetts Institute of Technology 5. Media Lab, Massachusetts Institute of Technology 6. Massachusetts General Hospital Center for Cancer Research, Harvard Medical School *These authors contributed equally † Corresponding author: pranam.chatterjee@duke.edu Abstract Germ cells are the vehicle of human reproduction, arising early in embryonic development and developing throughout adult life until menopause onset in women. Primordial germ cells are the common precursors of germline cells in both sexes, undergoing sexual specification into oogonia or gonocytes which further develop into oocytes or spermatocytes during development. Methods for recapitulation of primordial germ cell and oogonia formation have been developed extensively in recent decades, but fundamental technical limitations in their methodologies, throughput, and yield limit their utilization. Recently, transcription factor (TF)-based methods for human primordial germ cell-like cell (hPGCLC) formation, mouse meiotic entry, and mouse oocyte maturation have demonstrated the feasibility of gene overexpression screening in identifying potent regulators of germ cell development. Here we screened 47 folliculogenesis-regulating TFs for their role in hPGCLC and oogonia formation, identifying DLX5, HHEX, and FIGLA whose individual overexpression enhances hPGCLC formation from hiPSCs. Additionally, we identify a set of three TFs, ZNF281, LHX8, and SOHLH1, whose combinatorial overexpression drives direct oogonia-like formation from hiPSCs in a four-day, feeder-free monolayer culture condition with additional feeder-free culture capabilities post-isolation. We characterize these TF-based germ cells via gene and protein expression analyses, and demonstrate their broad similarity to in vivo germ cells. Together, these results identify novel regulators of human germ cell development and establish new TF-based tools for human in vitro oogenesis research. . CC-BY-NC 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.11.499564 doi: bioRxiv preprint