The synergic impact of decellularized testis scaffold and extracellular vesicles derived from human semen on spermatogonial stem cell survival and differentiation.

Abstract

Introduction: Decellularized scaffolds create a biomimetic niche to support spermatogonial stem cell (SSC) function and engraftment. Semen-derived extracellular vesicles (SEVs), containing proteins, lipids, and microRNAs with various functions, facilitate intercellular communication, enhance sperm maturation, and regulate the testicular microenvironment. This study explored the combined effects of rat decellularized testicular scaffolds and human SEVs on SSC survival and differentiation.

Materials and methods: The experimental approach involved decellularizing rat testis using detergents, followed by histological, immunohistochemical, DNA quantification, and scanning electron microscopy analyses to confirm extracellular matrix (ECM) preservation and cellular removal. SEVs were isolated from human seminal plasma via ultracentrifugation and characterized for size, morphology, and uptake by testicular cells. Whole testicular cells, including Dolichos Biflorus Agglutinin (DBA)-positive SSCs, were seeded onto scaffolds with or without SEVs, and the gene expression and cell viability were evaluated.

Results: DNA quantification and histochemical examinations revealed that the cell debris was removed, while the ECM constitution retained properly. Flow cytometery revealed 20% of the isolated cells from testis was SSCs. In vitro results demonstrated that SEV-enriched scaffolds significantly enhanced cell viability and upregulated DAZL and PIWI expression, indicating improved SSC survival and functionality, though meiosis (SCP1 expression) was not achieved.

Conclusions: The findings underscore the potential of integrating SEV-laden decellularized scaffolds to partially promote SSC differentiation for fertility restoration in spermatogenic failure.