P–803 Novel culture conditions for the improvement of the in vitro expansion of human male fetal germ cells

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Abstract Study question Do different ECMs/substrates and growth media culture conditions improve in vitro male human primordial germ cell (hPGC) expansion? Summary answer We achieved in vitro expansion improvement of male hPGCs with specific growth factors such as LIF, EGF, FGF2 and GDNF on gelatin- and vitronectin-coating cultures. What is known already PGCs are the precursors of male and female gametes, which are specified during early mammalian post-implantation embryonic development. PGCs undergo sequential cell divisions to differentiate into pro-spermatogonia (pSPG). In vitro propagation of pSPG could be important to understand the transition to spermatogonial stem cells (SSCs), important for fertility preservation in patients with infertility. Here, we aimed at performing a comparative analysis on in vitro feeder-free culture systems, based on different extracellular matrix (ECM) and growth media culture conditions, to support the expansion of the male germline stem cell populations using second trimester human male gonads as primary material. Study design, size, duration We collected human 2nd trimester male fetal gonads from elective abortions. Male gonads were dissected in saline solution (0.9% NaCl) and were either fixed overnight in 4% paraformaldehyde (PFA) for immunohistochemistry or disaggregated by enzymatic digestion for in vitro culture. Participants/materials, setting, methods After differential plating, fetal cells were cultured for 6 days. Disaggregated gonads were cultured with two different growing media (Medium 1 supplemented with LIF, EGF, FGF–2 and GDNF and Medium 2 supplemented with RA, BMP 4 and Activin A) on gelatin, laminin, vitronectin or matrigel coated plates. Cultured cells were immunostained, quantified for the expression of DDX4 and POU5F1 after 3 days (D3) and 6 days (D6) of culture. Main results and the role of chance We pursued to evaluate whether germ cells dissociated from a pool of male fetal gonads could propagate in vitro when cultured for D6 in different conditions. We observed that expansion of POU5F1-positive early PGCs and DDX4-positive late PGCs was only observed when cells were plated on gelatin or vitronectin and cultured with Medium 1, containing the growth factors LIF, EGF, FGF2 and GDNF. However, a reduced percentage of PGCs was observed in all four different coatings when grown with Medium 2, containing RA, BMP4 and Activin A. We analyzed the relative expression of the PGC markers POU5F1, DDX4 and MAGEA4 in histological sections of gonads from embryos at 18.5 weeks of gestation. Two populations of hPGCs were observed: ∼10–30% of the gonadal cells expressed solely DDX4 (late PGCs), whereas less than 10% of gonadal cells expressed POU5F1 (early PGCs). SOX9 and STARD1 expression was evaluated, confirming the presence of Sertoli cells and Leydig cells, respectively. Limitations, reasons for caution Due to the limited and difficulty to obtain human fetal tissue, a limited number of samples were used. Wider implications of the findings: We expanded human male fetal germ cells in vitro for D6 on gelatin and vitronectin coated plates with Medium 1, containing growth factors LIF, EGF, FGF2 and GDNF. Our findings provide a 2D culture system to expand hPGCs that could be useful to study propagation to pSPGs and eventually SSCs. Trial registration number Not applicable

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