Anat Cell Biol.  2023 Dec;56(4):508-517. 10.5115/acb.22.246.

Evaluating the effect of conditioned medium from mesenchymal stem cells on differentiation of rat spermatogonial stem cells

• Fazaeli H
• Sheykhhasan M
• Kalhor N
• Asl FD
• Kashani MH
• Sheikholeslami A

• KMID: 2548908
• DOI: http://doi.org/10.5115/acb.22.246

Abstract

In cancer patients, chemo/radio therapy may cause infertility by damaging the spermatogenesis affecting the selfrenewal and differentiation of spermatogonial stem cells (SSCs). In vitro differentiation of stem cells especially mesenchymal stem cells (MSCs) into germ cells has recently been proposed as a new strategy for infertility treatment. The aim of this study was to evaluate the proliferation and differentiation of SSCs using their co-culture with Sertoli cells and conditioned medium (CM) from adipose tissue-derived MSCs (AD-MSCs). Testicular tissues were separated from 2–7 days old neonate Wistar Rats and after mechanical and enzymatic digestion, the SSCs and Sertoli cells were isolated and cultured in Dulbecco’s modified eagle medium with 10% fetal bovine serum, 1X antibiotic, basic fibroblast growth factor, and glial cell linederived neurotrophic factor. The cells were treated with the CM from AD-MSCs for 12 days and then the expression level of differentiation-related genes were measured. Also, the expression level of two major spermatogenic markers of DAZL and DDX4 was calculated. Scp3, Dazl, and Prm1 were significantly increased after treatment compared to the control group, whereas no significant difference was observed in Stra8 expression. The immunocytochemistry images showed that DAZL and DDX4 were positive in experimental group comparing with control. Also, western blotting revealed that both DAZL and DDX4 had higher expression in the treated group than the control group, however, no significant difference was observed. In this study, we concluded that the CM obtained from AD-MSCs can be considered as a suitable biological material to induce the differentiation in SSCs.

Keyword

Spermatogonial stem cells; Mesenchymal stem cells; Conditioned medium; Differentiation

Figure

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  Fig. 1 Colonies of SSCs and Sertoli cells 72 hours after isolation. SSCs (arrowheads), Sertoli cells (arrows) (scale bar: 100 µm).

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  Fig. 2 The expression level of some germ cell genes. (A) Integrin-α6 (ITGA6), (B) c-Kit, (C) integrin-β1 (ITGB1), and (D) Nanog were clearly expressed in the co-culture of SSCs and Sertoli cells.

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  Fig. 3 Immunocytochemical staining to confirm spermatogonial stem cells (SSCs) and Sertoli cells. (A) CD49f (spermatogonial cell marker) was expressed in SSCs. (B) Isolated cells were also positive for vimentin (a marker of Sertoli cells). Nuclei were stained with DAPI (left figures).

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  Fig. 4 The mRNA expression of (A) osteogenic, (B) adipogenic, and (C) chondrogenic markers was assessed after 14 days under specific media by real-time polymerase chain reaction in adipose tissue-derived mesenchymal stem cells (AD-MSCs) to confirm their capacity to differentiate into bone, fat and cartilage. (D) Inverted microscope image of living MSCs isolated from human adipose tissue showing spindle cells in the third passage. ALPL, alkaline phosphatase; COL2A1, type II collagen. **P≤0.01.

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  Fig. 5 Expression level of (A) Stra8, (B) Dazl, (C) Scp3, and (D) Prm1 genes using real-time polymerase chain reaction in spermatogonial stem cells treated with CM after 12 days. CM, conditioned medium; AD-MSCs, adipose tissue-derived mesenchymal stem cells. *P≤0.05, **P≤0.001, ***P≤0.001.

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  Fig. 6 Immunostaining of germ cell markers in the control and treat groups after 12 days. (A) Groups stained with anti-DAZL. (B) Groups stained with anti-DDX4. Right-hand images show nuclei stained with 4’,6-diamidino-2-phenylindole.

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  Fig. 7 Western blot for DAZL and DDX4 expression. (A) The expression pattern of DAZL, DDX4, and GAPDH on membrane. (B) Relative protein expression level of DAZL and DDX4.

Reference

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