Silk Nanofibrous Electrospun Scaffold Amplifies Proliferation and Stemness Profile of Mouse Spermatogonial Stem Cells

Abstract: Electrospun nanofibrous scaffolds provide a structural microenvironment similar to the native extracellular matrix, which leads to the maintenance and propagation of the cultured cells under in vitro conditions. Furthermore, we used the sertoli cells to improve SSC proliferation due to its secretory role in growth factors. In this study, a silk electrospun nanofibrous scaffold has been used as a microenvironment for guiding the proliferation of spermatogonial stem cells (SSCs). SSCs were seeded on the prepared scaffolds and then cultured in vitro for 2 weeks. The attachment of SSCs was examined by scanning electron microscopy (SEM). Cell attachment and viability were evaluated via 4′, 6-diamidino-2-phenylindole staining and 3-(4, 5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide assessment, respectively. The expression of the specific SSC genes including stimulated by retinoic acid gene 8 (Stra8), deleted in azoospermia-like (DAZL) and piwil-like RNA-mediated gene silencing 2 (Piwil2), was quantified using real-time-PCR and confirmed by immunofluorescent staining. The data indicated that the prepared scaffold guided more effective SSC attachment and proliferation in comparison to the monolayer control. However, a significant upregulation of DAZL, Stra8, and piwill2 genes was observed in the cells grown on the silk scaffold. Also, the expression of Srta8 and Oct4 in the cultured cells was confirmed by immunofluorescence staining 2 weeks post-culture. Silk scaffold increases the in vitro viability of SSCs and amplifies their proliferation during the culture period. It is concluded that silk scaffold can serve as an appropriate substrate with broad implications for SSC proliferation under in vitro culture medium. Lay Summary: We used a silk scaffold for guiding the proliferation of spermatogonial stem cell (SSC) population. Spermatogonia stem cells were seeded and cultured on silk scaffolds for 2 weeks. The attachment of SSCs was examined via SEM and viability was evaluated by DAPI staining and MTT assessments. The expression of specific spermatogonial stem cell markers including Stra8 gene, DAZL, and Piwill2 was quantified via Q-PCR and immunofluorescent staining. Scaffold guides firm attachment of SSCs and proliferates more than a monolayer layer. The significant upregulation of DAZL, Stra8, piwill2, and Oct4 in the experiment was confirmed by the real-time PCR and immunofluorescence staining 2 weeks after culture. Silk scaffold increases the in vitro viability of SSCs and amplifies their proliferation during the culture period. Silk scaffold can serve as an appropriate substrate with wide implications for SSC proliferation under in vitro condition.