A simple method to generate germ cells from male embryonic stem cells

Abstract

Many complex methods are available for creating and maintaining differentiated germ cells. We evaluated the hanging drop (HD) system to expeditely induce germ cell (GC) appearance in an embryonic stem cell culture. Embryoid bodies were induced and maintained in medium supplemented with differentiating factors in either HD or in culture dishes (CD). PGC appearance and differentiation was assessed for germ cell markers by microscopy staining, immunofluorescence, and molecular epigenetic assay by next-generation sequencing (NGS). Male mESCs were maintained on mouse embryonic fibroblast (MEF) cells until confluency was achieved, after which colonies were passaged by trypsinization. Male mESCs were separated from MEF cells by sedimentation. Approximately 1000 feeder-free mESCS were placed in 25 uL HD. Two days later, EBs were either transferred to HD containing retinoic acid (RA) or CD also supplemented with RA. On day 8, EBs were treated with collagenase IV and stained for GCAP/OCT4/DAZL or processed for gene expression analysis by NGS. Of a total of 260 EBs, 100 EBs were re-plated on day 2 to corresponding HD containing RA, while the remaining 160 EBs were transferred to CD supplemented with RA. On day 8, HD-cultured EBs reached approximately 150um in diameter, while EBs in CD reached 280-300um. EBs cultured in HD yielded an average of 77% GCAP positive cells, while those in CD only presented 22% GCAP positivity. EBs were also assessed for OCT4 activity to detect loss of stemness. In HD-cultured EBs, 40% of isolated cells were strongly positive for OCT4, compared to 60% of cells isolated from EBs in CD, indicating a higher level of cell differentiation in the former. To undoubtedly detect male germ cells, EBs were stained for DAZL. EBs cultured in HD and CD yielded 5% and 1% DAZL-positive cells, respectively. RNA sequencing revealed significant upregulation on Gdf9, Sox17, Kit, and Prm2 genes in HD-cultured EBs. These genes are critical for gametogenesis, confirming the presence of pre-meiotic and post-meiotic germ cells. Our attempt to induce the appearance and eventual maturation of male germ cells by the HD approach, by minimizing environment cell-surface interference, appears the most promising. Once the ability of these germ cells to progress through meiosis is confirmed, EB formation through HD may provide an inexpensive and reproducible model to experiment with in vitro gametogenesis.