Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold

ABSTRACT

Electrospun nanofiber matrices sufficiently mimic the structural morphology of natural extracellular matrix. In this study, we aimed to examine the effects of agar/polyvinyl alcohol nanofiber (PVA) scaffold on the proliferation efficiency and differentiation potential of neonate mouse spermatogonial stem cells (SCCs). Testicular cells were isolated from testes of 40 mouse pups and were seeded in: 1) 2D cell culture plates in the absence (2D/−GF) or presence (2D/+GF) of growth factors and 2) onto agar/PVA scaffold in the absence (3D/−GF) or presence (3D/+GF) of growth factors. The cells were subsequently cultured for 4 weeks. First 2 weeks were dedicated to proliferative phase, whereas the next 2 weeks emphasized the differentiation phase. The identity of the SCCs was investigated at different time-points by flow cytometry and quantitative reverse transcription PCR (qRT-PCR) analyses against the germ cell markers, including PLZF, Id-4, Gfrα-1, Tekt-1, and Sycp-3. After 2 weeks of culture, the 3D/+GF group showed the highest percentage of PLZF-positive cells among culture systems (P < 0.05). The expression levels of pre-meiotic markers (Id-4 and Gfrα-1) decreased significantly in all groups, particularly in 3D/+GF group after 28 days of culture. Additionally, the cells in the 3D/+GF group displayed the highest expression of meiotic (Sycp-3) and post-meiotic markers (Tekt-1) 14 days after differentiation induction. Seemingly, the combination of the agar/PVA scaffold and growth factor-supplemented medium synergistically increased the differentiation rate of mouse SSCs into meiotic and post-meiotic cells. Thus, agar/PVA nanofiber scaffolds may have the potential for applications in the restoration of infertility, especially in azoospermic males.

Abbreviations

2D: two dimentional; 3D: three dimentional; bFGF: basic fibroblast growth factor; BMP-4: bone morphogenetic protein 4; DMEM: Dulbecco’s modified Eagle’s medium; ECM: extracellular matrix; FCS: fetal calf serum; FTIR: Fourier-transform infrared spectroscopy; GDNF: glial cell line-derived neurotrophic factor; GF: growth factors; Gfrα-1, GDNF family co-receptor α1; Id-4, Inhibitor of DNA Binding 4; MTT: methylthiazoltetrazolium; PLZF: promyelocytic leukemia zinc finger; PVA: polyvinyl alcohol; qRT-PCR: quantitative reverse transcription PCR; RA: retinoic acid; SACS: soft agar culture system; SD: standard deviation; SEM: scanning electron microscope; SSCs: spermatogonial stem cells; Sycp-3, Synaptonemal complex protein 3; Tekt-1, Tektin 1.

KEYWORDS:

• Agar/PVA nanofibers
• spermatogonial stem cells
• self-renewal
• differentiation
• three dimensional culture

Acknowledgments

This study was financially supported by the Deputy of Research of Iran University of Medical Sciences (IUMS), Tehran, Iran (grant number 30476).

Disclosure statement

No potential conflict of interest was reported by the authors.

Authors’ contributions

Conception and design of the study, analyzed data, obtained funding, and supervised the project: FM, MK; Conducted SEM analysis and participated in the design and preparation of nanofibers: ZB; Took specimens, performed qRT-PCR and flow cytometry analyses, contributed in data interpretation, and wrote the first draft: MZK; Interpreted flow cytometry and qRT-PCR data and helped in manuscript evaluation: HRA, MN. All authors read and revised the manuscript. Additionally, all authors confirmed the final version of the manuscript to be submitted.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the Iran University of Medical Sciences [under Grant number 30476].