OBJECTIVE: To test the efficiency of direct spherification in supporting differentiation of mouse embryonic stem cells (mESCs) into meiotic male germ cells.DESIGN: Biological 3D scaffolding was utilized to culture and differentiate mESCs. Two main methods were investigated: 1) mESCs were injected into pre-formed spheres (IN), and 2) a suspension of mESCs was directly encapsulated (DE) into spheres. To coax differentiation, spheres were bathed in a growth factor cocktail. After mechanical breaching of the spheres, serial isolation at different time intervals was carried out. These isolated cells were analyzed with germ cell stage-specific markers.MATERIALS AND METHODS: MESCs were initially cultured on a 6well dish, trypsinized, and resuspended in stem cell medium. For the IN method, the suspension was injected by using a 1 mL syringe into pre-formed spheres. For the DE method, spheres were formed by suspending mESCs in a base spherification solution and encapsulated by exposure to sodium alginate. Four spheres were bathed in EpiLC differentiating medium containing Activin A, bFGF, and KSR for 3 days, and then submerged in spermatogonial stem cell (SSC) medium composed of DMEM, GDNF, FGF2, 2-mercaptoethanol, L-glutamine, B27 supplement, and 1 mM retinoic acid (RA) for 7 days. Each sphere measured at a diameter of 8 to 10 mm, containing approximately 6.3x10⁵ cells per sphere. Cells were isolated and analyzed after 3 days for OCT4 and Nanog, and DAZL and VASA at day 10.RESULTS: Both IN and DE proved successful in supporting the growth of mESCs. However, optic visualization through the membrane of the IN sphere showed a lower concentration compared to the DE approach. Embryoid body formation was first observed in the DE group at day 4, indicating greater efficiency with this approach. Confident with the sustainability and efficiency of DE, we attempted differentiation solely by this method, by bathing the spheres in EpiLC medium. After 3 days of culture, two spheres were breached and their cellular content was analyzed, showing OCT4 expression in about 85% of cells and a more conspicuous decrease in Nanog expression (<40%), indicating successful progression to EpiLCs. The remaining two spheres cultured in SSC medium with RA from day 4 to 10 showed an increase in embryoid body size that even extruded through the sphere wall. At day 10, the spheres were mechanically breached, and isolated cells showed positivity for VASA ($5%) and DAZL ($5%), confirming meiotic differentiation into germ cells.CONCLUSIONS: Successful sustenance and propagation of mESCs using direct spherification indicates the feasibility of this 3D culture system to promote differentiation towards the male germ line. The ability to support differentiation to post-meiotic stages confirmed by biological proof would render this culture method sustainable for in vitro neogametogenesis.
