In vitro Complete Differentiation of Human Spermatogonial Stem Cells to Morphologic Spermatozoa Using a Hybrid Hydrogel of Agarose and laminin

Abstract

Background Spermatogenesis refers to the differentiation of spermatogonial stem cells (SSCs) located in the basal part of seminiferous tubules into haploid spermatozoa. Prerequisites for in vitro spermatogenesis include an extracellular matrix, a culture medium containing factors influencing the survival, proliferation and differentiation of spermatogenic cells, and testicular somatic cells that play a supporting role for SSCs. Accordingly, in this paper, the potential of co-culture of Sertoli cells and SSCs embedded in a hybrid hydrogel of agarose and laminin as one of the main components of the extracellular matrix of the basal compartment of seminiferous tubules in the presence of culture medium containing growth factors was investigated. Methods After three–week conventional culture of the cellular suspension of enzymatic digestion of human testicular tissue, the cells were cultured in agarose hydrogel alone or hybrid hydrogel (agarose/laminin) for 74 days. Then, the presence of spermatogonial cells and other cells of the different stages of spermatogenesis were analysed by immunocytochemistry, real-time PCR, electron microscopy and morphological staining. Results The results showed that colonies with positive spermatogenesis markers were observed in both culture systems. The presence of different levels of spermatogenesis in two groups media was confirmed, but morphological spermatozoa were observed only in hybrid hydrogel group. Conclusions Based on the results of this paper, it can be concluded that an agarose and extracellular matrix-based scaffold which maintains the mechanical strength of the scaffold during culture and also increases the biological activity of the scaffold, can support from all physiological activities of SSCs such as survival, proliferation and differentiation.