Differentiation of embryonic stem cells into male germ line cells in a bioreactor using decellularized seminiferous tubule matrix immersed in a conditioned medium

Abstract

To propose a method to sustain mouse embryonic stem cells (mESCs) utilizing a decellularized seminiferous tubule matrix (DSTMs) as a biomechanical scaffold, and induce differentiation into male germ line cells in conditioned medium. We tested the potential of a bioreactor concept–based culture system supported by a biological scaffold to allow de novo generation of meiotic male germ line cells from mESCs. Male mESCs were cultured in epiblast cell–like cell (EpiLC) medium containing activin A, bFGF, and KSR for 3 days to allow differentiation into EpiLCs. Subsequent exposure to primordial germ cell–like cell (PGCLC) medium subsidized with BMP4, BMP8b, SCF, LIF, and EGF in hanging droplet (HD) allowed the formation of embryoid bodies (EBs) rich in PGCLCs. Isolated cells from 80 EBs were utilized in the bioreactor, in directed contact with DSTMs, and loaded with DMEM in a gelatin-treated culture well equipped with a 0.4-μm pore size mesh inlet. 14-week-old adult male B6D2F1 mice were sacrificed for DSTMs and conditioned medium. DSTMs were prepared by immersion in 1% sodium dodecyl sulfate for 24 hours. Eighty 3-mm sections of DSTM, longitudinally sliced open and flattened, were placed below the mesh; interstitial cells were isolated from the respective contralateral testes by differential plating and loaded above the mesh. Cell characteristics were analyzed by germ cell stage–specific markers on an H&E-stained background. After culturing mESCs in EpiLC medium, the continuing expression of OCT4 (>90%) and the decreased positivity of Nanog (45%) indicated progression to EpiLCs. EBs rich in PGCLCs expressed positive surface SSEA-1 after six days of culture in HD with PGCLC medium. Isolated cells of PGCLCs were derived from the digestion of EBs and layered on the DSTMs. The earliest attachment of PGCLCs onto DSTM occurred on day 3, and complete recellularization was observed at approximately day 10. Following complete recellularization, about half of all isolated cells obtained from the enzymatic digestion of recellularized tubule displayed decreased expression of OCT4, while 5% displayed nuclear DAZL positivity at day 10. In 1% of the cells, perinuclear DAZL confirmed spermatocyte differentiation at day 21. At around day 16, cytoplasmic VASA positivity in 5% of the cells suggested meiotic/post-meiotic germ line differentiation. The timeline of our bioreactor system was comparable with in vivo spermatogenesis in the mouse, occurring in the course of 21 days. Once the ability of a 3D biocompatible scaffold to induce late-stage gametogenesis is confirmed, it will be possible to study spermatogenesis in vitro. Neogametogenesis from genotyped stem cells performed in a scaled-down microfluidic device may help to treat men afflicted by Sertoli cell–only syndrome.