Optimization of the Decellularization Process and Evaluation of the Differentiation Potential of Wharton's Jelly Mesenchymal Stem Cells on a Decellularized 3D Scaffold of Testicular Tissue

This study aimed to develop a three-dimensional biological model of testicular tissue and assess the differentiation of human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs) into germ-like cells. In the first phase, three decellularization protocols were compared. Quantitative DNA assays and histological analysis revealed that the combination of SDS (sodium dodecyl sulfate) and SDC (sodium deoxycholate) (S3) achieved the greatest DNA reduction and the best preservation of the extracellular matrix (ECM) structure. Immunohistochemistry and Alisian Blue staining confirmed superior retention of type IV collagen, fibronectin, and glycosaminoglycans in this group. The MTT assay indicated no significant cytotoxicity from residual detergents. In the second phase, MSCs were isolated from Wharton's Jelly and cultured, maintaining their morphology and expressing CD105 and CD44 while lacking CD34. Viability and cell-counting tests showed that an initial density of 30 000 cells per well provided optimal survival on the selected scaffold. Finally, hWJ-MSCs were seeded on the Group 3 scaffold and cultured in different differentiation systems. After 2 weeks, qRT-PCR analysis showed that the G3 differentiation group (supplemented with Sertoli cell-conditioned medium and growth factors) had the highest expression of pluripotency and germ cell-related genes (OCT4, Dmc1, Tp2) as well as the anti-apoptotic gene Bcl2, while Bax expression was significantly reduced. Electron microscopy and BrdU assays confirmed cell presence and proliferation on the ECM surface within 2 weeks. Overall, the SDS + SDC-derived scaffold, with its preserved ECM architecture and favorable biocompatibility, provided a suitable microenvironment for the attachment, proliferation, and differentiation of hWJ-MSCs into germ-like cells, offering a promising model for testicular regeneration.