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.

PURPOSE: This study aimed to develop a decellularized three-dimensional testicular scaffold and evaluate the differentiation of human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs) into germ-like cells.

METHODS

Mouse testicular tissues were decellularized using three protocols: S1 : a solution containing 1% (W/V) sodium dodecyl sulfate (SDS) for 24 hours, followed by 1% Triton X-100 and 0.01% DNase/RNase for 4 h, S2: 1% Sodium Deoxycholate (SDC) for 24 hours, followed by 1% Triton X-100, and 0.01% DNase/RNase, for 4h. S3: 1% SDS for 24 hours, 1% SDC for 24 h and 1% Triton X-100 and 0.01% DNase/RNase for 4h". Decellularization efficiency and extracellular matrix (ECM) preservation were assessed by histology, DNA quantification, immunohistochemistry, Alcian Blue staining. hWJ-MSCs then seeded onto the selected scaffold and cultured for two weeks in differentiation media containing BMP4, retinoic acid, GDNF, and Sertoli cell-conditioned medium.

RESULTS

Among the protocols, S3 showed the highest DNA removal and best preservation of ECM architecture. Immunohistochemistry confirmed greater retention of collagen IV, fibronectin, and glycosaminoglycans in S3. A density of 30,000 cells/well isolated hWJ-MSCs provided optimal viability on the scaffold. After two weeks, the differentiation group supplemented with Sertoli cell-conditioned medium and growth factors exhibited the highest expression of germ cell-related genes (OCT4, Dmc1, and Tp2), increased Bcl2 / Bax expression. Electron microscopy study confirmed successful cell attachment on scaffold.

CONCLUSIONS

The SDS+SDC-derived scaffold provided a suitable microenvironment for hWJ-MSC attachment, proliferation, and differentiation into germ-like cells, highlighting its potential application in testicular tissue engineering and regenerative medicine.