Characterization, recellularization, and transplantation of rat decellularized testis scaffold with bone marrow-derived mesenchymal stem cells

Background: Regenerative medicine potentially offers the opportunity for curing male infertility. Native extracellular matrix (ECM) creates a reconstruction platform to replace the organs. In this study, we aimed to evaluate the efficiency of the testis decellularized scaffold as a proper niche for stem cell differentiation toward testis-specific cell lineages. Methods: Rats' testes were decellularized by freeze-thaw cycle followed by immersion in deionized distilled water for 2h, perfused with 1% Triton X-100 through ductus deferens for 4h, 1% SDS for 48h and 1% DNase for 2h. The decellularized samples were prepared for further in vitro and in vivo analyses. Result: Histochemical and immunohistochemistry studies revealed that ECM components such as Glycosaminoglycans (GAGs), neutral carbohydrate, elastic fibers, collagen I & IV, laminin, and fibronectin were well preserved, and the cells were completely removed after decellularization. Scanning electron microscopy (SEM) showed that 3D ultrastructure of the testis remained intact. In vivo and in vitro studies point out that decellularized scaffold was non-toxic and performed a good platform for cell division. In vivo implant of the scaffolds with or without mesenchymal stem cells (MSCs) showed that appropriate positions for transplantation were the mesentery and liver and the scaffolds could induce donor-loaded MSCs or host migrating cells to differentiate to the cells with phenotype of the sertoli- and leydig-like cells. The scaffolds also provide a good niche for migrating DAZL-positive cells; however, they could not differentiate into post meiotic-cell lineages. Conclusion: The decellularized testis can be considered as a promising vehicle to support cell transplantation and may provide an appropriate niche for testicular cell differentiation.