The Effects of Conditioned Medium from Adipose Tissue-derived Mesenchymal Stem Cells on Differentiation of Spermatogonial Stem Cells
The Effects of Conditioned Medium from Adipose Tissue-derived Mesenchymal Stem Cells on Differentiation of Spermatogonial Stem Cells
Hoda Fazaeli,1Mohsen Sheykhhasan,2Naser Kalhor,3Faezeh Davoodi,4Azar Sheikholeslami,5,*
1. Department of Mesenchymal Stem Cells, Academic Center for Education, Culture, and Research (ACECR), Qom Branch, Qom, Iran 2. Department of Mesenchymal Stem Cells, Academic Center for Education, Culture, and Research (ACECR), Qom Branch, Qom, Iran 3. Department of Mesenchymal Stem Cells, Academic Center for Education, Culture, and Research (ACECR), Qom Branch, Qom, Iran 4. Department of Mesenchymal Stem Cells, Academic Center for Education, Culture, and Research (ACECR), Qom Branch, Qom, Iran 5. Department of Mesenchymal Stem Cells, Academic Center for Education, Culture, and Research (ACECR), Qom Branch, Qom, Iran
Introduction: Spermatogenesis is a biological process essential for the male germline's continuity by which haploid spermatozoa are produced. Throughout the male's life, spermatogenesis is a continuous and coordinated process of cell proliferation and differentiation that results in the development of unrestricted numbers of spermatozoa. Spermatogonial stem cells (SSCs), which have the remarkable ability to self-renew and produce differentiated daughter cells that will eventually form spermatozoa, are at the heart of this scheme. In seminiferous tubules, SSCs are highly rare stem cells found in niches surrounded by Sertoli cells and differentiating spermatogonia. In vitro differentiation of stem cells into male or female germ cells has recently been proposed as a new strategy to the treatment of infertility by researchers. The use of mesenchymal stem cells (MSCs) is gaining popularity since they face to no ethical problems of embryonic stem cells and can be collected from a variety of sources such as adipose tissue, bone marrow, and menstrual blood which all have a strong ability to develop into various tissues. The adipose tissue-derived mesenchymal stem cells (AD-MSCs), as almost easy access and well characterized source of MSCs, have been widely employed for therapeutic purposes. In vitro effects of conditioned medium (CM) from MSCs on germ cell regeneration has been shown. The aim of this study was to compare and evaluate the proliferation and differentiation of spermatogonia stem cells using their co-culture with Sertoli cells and supernatant from fat-derived mesenchymal stem cells.
Methods: This experimental study was conducted on Wistar Rats from Medical Sciences Animal Lab (Iran, Qom), under standard conditions with free access to Food and water. The testicular tissues were separated from 2-7 days old neonate Wistar Rats and transferred into the laboratory. After mechanical and 2-step enzymatic digestion with collagenase I and 0.25% trypsin enzymes, the SSCs and Sertoli cells were isolated and cultured in DMEM with 10% FBS, 1X antibiotic, bFGF, and GDNF and incubated in 95% humidity, 5% CO2 and 34°C. The isolation of SSCs and Sertoli cells was confirmed with immunocytochemistry (ICC) for CD49f and Vimentin, respectively. The cells were treated with the conditioned medium from adipose tissue-derived MSCs for 12 days and then the genes related to differentiation were measured with Real-Time PCR. Also, the expression level of two major spermatogenic markers of DAZL and DDX4 was calculated with ICC and Western Blotting (WB).
Results: CD49f and Vimentin were positive for SSCs and Sertoli cells, respectively (Fig. 1). The expression level of Scp3, Dazl, and Prm1 genes was significantly increased after treatment compared to the control group. However, no significant difference was observed in Stra8 expression between the group treated with conditioned medium and the control group (Fig. 2). The ICC results showed that DAZL and DDX4 were positive in the experimental group compared with the control (Fig. 3). Also, WB revealed that both DAZL and DDX4 were higher in the treated group than the control group, however, no significant difference was observed (Figure 4).
Conclusion: Given that SSCs are essential sources for male germline continuity, maintaining and restoring their differentiation potential sounds critical. In this study, we concluded that the conditioned medium obtained from adipose tissue-derived MSCs could be considered as a suitable biological material to induce the differentiation in spermatogonial stem cells.