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Background Nano-fibrous scaffolds provide a three-dimensional matrix that guides sufficient orientation of seeded cells similar to a natural niche. In this research, we designed a silk scaffold to improve the differention of mouse spermatogonial stem cells to spermatogenic cell lines. Spermatogonial stem cells were collected from neonatal mouse (2–6 days) testes (n=60) using a two steps mechanical and enzymatic method. Cells were seeded on a silk scaffold and were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 15 % fetal bovine serum and 1000 units/ml leukemia inhibitory factor, and incubated at 32°C in a humidified atmosphere of 5% CO2 in air. SEM technique was done for confirmation of seeding cells. In this study two major groups (i.e., 2D and 3D culture groups) of 30 mice each. Isolated testicular cells from each group were cultured in the absence of silk scaffold or the presence of silk scaffold. For induction of differentiation, seeded cells on a scaffold were exposed to 1 μM and 50 ng/ml BMP-4. The specific spermatogenic genes, e.g.; VASA, DAZL, PLZF, and Piwil2, were assessed via real-time PCR and immunocytochemistry techniques. P values less than 0.05 were assumed significant. All experiments were performed at least three times. Results SEM analysis confirmed the homogeneity of fabricated silk scaffold and average diameter of 450 nm for nanofibers fibers. Silk scaffold induces attachment of SSCs in comparison to the monolayer group. Spermatogonia stem cell colonies were observed gradually after 1 week of culture. Electrospun scaffold supports the differentiation of SSCs to spermatogenic lines. Dates of real-time PCR showed that the expression of meiotic markers, VASA, DAZL, and Piwil2 as related to specific spermatogenic genes, had a significant upregulation in cell-seeded silk scaffold compared to the control group (P < 0.05). Immunocytochemistry founding approved the expression of specific spermatogenic markers; DAZL and PLZF were higher in the experiment group compared to the control (P < 0.05). Conclusion It is concluded silk scaffold induces spermatogenic differentiation of mouse spermatogonial stem cells in vitro.

Introduction: Proliferation of spermatogonial stem cells (SSCs) can be a treatment for infertile men. Here, we design an efficient method based on culturing in the presence of Sertoli cells to improve the expression level of some specific spermatogonia stem cell genes during two weeks post culture. Materials and Methods: Cells were derived from neonatal (2-6 days old) mice testes and were cultured in DMEM medium with FBS. The colonization of cultured SSCs in days 4, 7, and 14 of culture was counted via phase-contrast microscope and Image J software. Methyl thiazolyl tetrazolium (MTT) test was performed to evaluate the viability of cultured SSCs in days 3, 7, and 14 of culture. The expression level and the alteration pattern of specific spermatogonial markers, i.e., Stra8, DAZL, and Piwill2 was examined via real-time polymerase chain reaction (PCR) during two weeks post culture. Results: The number and the diameters of colonies showed a significant increase in cultured cells. MTT results proved the higher viability of testicular cells during the culture period. The results of ALP staining detected a positive reaction in spermatogonia colonies. Real-time PCR data showed that culturing SSCs in the presence of interstitial cells of the testis, amplified the level and alteration pattern of specific spermatogonia stem cells genes beneficial in the enrichment of SSCs propagation. Conclusion: Providing a similar culture environment to testicular niche increases viability, forms SSCs colonies, and regulates the level and alteration pattern of spermatogonia stem cell genes.