Spermatogenic differentiation of spermatogonial stem cells on three-dimensional silk nanofiber scaffold

Abstract

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.

In vivo and in vitro sperm production: an overview of the challenges and advances in male fertility restoration

“…In recent years, a wide range of synthetic polymers (synthetic carbon [79,80], polycaprolactone [81], poly-L-lactic acid [82,83], polyvinyl alcohol [84], polyamide [85], and glycolic acid [83]) and natural polymers (alginate [86,87], gelatin [88], methyl cellulose [89], collagen [90,91], fibroin [92,93], chitosan [94], Matrigel [95][96][97], and agarose [75,98]) have been used to fabricate scaffolds for the purpose of improving spermatogenesis. Most synthetic scaffolds were found unsuitable for SSC differentiation, whereas natural biomaterials demonstrated superior performance.…”

Advances of three-dimensional (3D) culture systems for in vitro spermatogenesis

“…Number of spermatogonial stem-like cell colonies was significantly higher than control group after transplantation into the seminiferous tubules of busulfan-treated adult mice. Narimanpouret al [ 153 ] Mouse testicular cells Silk scaffold 1 week Expression of VASA, DAZL, and Piwil2 markers was significantly higher in silk scaffold group. Baert et al [ 177 ] Mouse testicular cells Alginat 3D bioprinted scaffold 40 days The elongated spermatids were detected in 66% of TC/CFS, round and elongated spermatids were found in all and 33% of CD49f + /CLS constructs.…”

“…After 1 week of culture, SSCs colonies were slowly formed. The results showed that the expression of VASA, DAZL, and Piwil2 markers was significantly higher in silk scaffold than the control group [ 153 ] (Table 1 ).…”