Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold
Creators: name: Kashani, Marzieh Ziloochi
name: Bagher, Zohreh
name: Asgari, Hamid Reza
name: Najafi, Mohammad
name: Koruji, Morteza
name: Mehraein, Fereshteh
Publisher: name: Taylor & Francis
Year: 2020
Subjects: subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Computational Biology

description: 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, <i>Id-4, Gfrα-1, Tekt-1</i>, and <i>Sycp-3</i>. After 2 weeks of culture, the 3D/+GF group showed the highest percentage of PLZF-positive cells among culture systems (P Id-4 and <i>Gfrα-1</i>) 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 (<i>Sycp-3</i>) and post-meiotic markers (<i>Tekt-1</i>) 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. 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.