Differentiating mouse-induced pluripotent stem cells into male germ cells through embryoid bodies

Abstract

We examined the ability of the hanging drop (HD) method to differentiate induced pluripotent stem cells into male germ cells in the presence of specific growth factors. Over an eight-month period, 130 embryoid bodies (EBs) were created from mouse-induced pluripotent stem cells (miPSCs) using the hanging drop (HD) method. EBs were cultured in HDs after formation and differentiated in a 2-step culture system, each with a specific medium composition. Immunofluorescence staining was used to estimate germ cell appearance. miPSCs were maintained on mouse embryonic fibroblast (MEF) cells until confluent. MEF cells provided a cellular matrix for the miPSCs to grow on. Once confluent, the colonies were isolated by trypsinization. HDs were formed from 25-uL droplets containing miPSCs at a concentration of 1x105 cells/ml. Two male 14-week-old B6D2F1 mice were biopsied for testicular cells. Growth factors were extracted from adult mouse testes and added to the step-one medium containing Activin A, bFGF, and KSR. After 3 days, EBs were transferred to HDs containing step-two medium composed of LIF, BMP4, BMP8b, SCF, EGF, and testicular growth factors. On day 8, EBs were treated with collagenase and stained for OCT4, VASA, and DAZL. OCT4, VASA, and DAZL expression was compared between EBs cultured in the two-stage growth factor-conditioned medium system and control EBs maintained in standard culture medium to determine the stage of spermatogenesis reached. Of the isolated cells cultured from the two-step medium system, 86% were strongly positive for OCT4. In comparison, only 54% of cells isolated from control EBs cultured without differentiating factors were positive for OCT4. In addition, EBs cultured with differentiating factors yielded 31% VASA-positive cells, whereas control EBs only yielded 1% VASA-positive cells. Furthermore, EBs cultured with differentiating factors yielded 4% DAZL-positive cells, while control EBs only yielded 1% DAZL-positive cells. The retained expression of OCT4, along with the presence of VASA-positive and DAZL-positive cells, strongly indicates that the cells reached the spermatogonia/spermatocyte cell stage. Cells that were positive for VASA and DAZL without OCT4 expression also suggest that a fraction of cells were able to progress to the advanced meiotic stages of in vitro spermatogenesis. Our attempt to use the HD method together with the two-step culture system to induce the appearance and eventual maturation of male germ cells has shed light on the mechanisms of spermatogenesis in vitro. Using pluripotent stem cells to generate germ cells may benefit men afflicted by spermatogenic failure.