In Vitro Generation of Sertoli-like and Haploid Spermatid-like Cells from Human Umbilical Cord Perivascular Cells

Background: First trimester (FTM) and term human umbilical cord-derived perivascular cells (HUCPVCs), which are rich sources of mesenchymal stem cells (MSCs), can give rise to Sertoli cell (SC)-like as well as haploid germ cell (GC)-like cells in vitro using culture conditions that recapitulate the testicular niche. Gamete-like cells have been produced ex vivo using pluripotent stem cells as well as MSCs. However, the production of functional gametes from human stem cells has yet to be achieved.

Methods: Three independent lines of FTM and term HUCPVCs were cultured using a novel 5-week step-wise in vitro differentiation protocol recapitulating key physiological signals involved in testicular development. SC- and GC-associated phenotypical properties were assessed by real-time polymerase chain reaction (RT-PCR), quantitative PCR immunocytochemistry, flow cytometry, and fluorescence in-situ hybridization (FISH). Functional spermatogonial stem cell-like properties were assessed using a xenotranplantation assay.

Results: Within 3 weeks of differentiation, two morphologically distinct cell types emerged including large adherent cells and semi-attached round cells. Both early GC-associated markers (VASA, DAZL, GPR125, GFR1α) and SC-associated markers (FSHR, SOX9, AMH) were upregulated, and 5.7 ± 1.2% of these cells engrafted near the inner basal membrane in a xenograft assay. After 5 weeks in culture, 10-30% of the cells were haploid, had adopted a spermatid-like morphology, and expressed PRM1, Acrosin, and ODF2. Undifferentiated HUCPVCs secreted key factors known to regulate spermatogenesis (LIF, GDNF, BMP4, bFGF) and 10-20% of HUCPVCs co-expressed SSEA4, CD9, CD90, and CD49f. We hypothesize that the paracrine properties and cellular heterogeneity of HUCPVCs may explain their dual capacity to differentiate to both SC- and GC-like cells.

Conclusions: HUCPVCs recapitulate elements of the testicular niche including their ability to differentiate into cells with Sertoli-like and haploid spermatid-like properties in vitro. Our study supports the importance of generating a niche-like environment under ex vivo conditions aiming at creating mature GC, and highlights the plasticity of HUCPVCs. This could have future applications for the treatment of some cases of male infertility.