Abstract
Differentiation of Leydig cells plays a key role in male reproductive function. Bone marrow mesenchymal stem cells (BMSCs) have emerged as a potential cell source for generating Leydig-like cells due to their multipotent differentiation capacity and accessibility. This study aimed to investigate the morphological and genetic expression changes of BMSCs during differentiation into Leydig-like cells. Testicular extract liquid, which simulates the microenvironment in vivo, induced the third passage BMSCs differentiated into Leydig-like cells. Changes in cell morphology were observed by microscopy, the formation of lipid droplets of androgen precursor was identified by Oil Red Staining, and the expression of testicular specific genes 3β-HSD and SF-1 in testicular stromal cells was detected by RT-qPCR. BMSCs isolated from the bone marrow of Sprague-Dawley (SD) rats were cultured for 3 generations and identified as qualified BMSCs in terms of morphology and cell surface markers. After 14 days of induction with testicular tissue lysate, lipid droplets appeared in the cytoplasm of P3 BMSCs by Oil Red O staining. RT-qPCR detection was performed on BMSCs on the 3rd, 7th, 14th, and 21st day after induction. Relative expression levels of 3β-HSD mRNA significantly increased after 14 days of induction, while the relative expression of SF-1 mRNA increased after 14 days of induction but was not significant. BMSCs can differentiate into testicular interstitial cells with reserve androgen precursor lipid droplets after induction by testicular tissue lysate. The differentiation ability of BMSCs provides the potential to reconstruct the testicular microenvironment and is expected to fundamentally improve testicular function and provide new treatment options for abnormal spermatogenesis diseases.
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All processed data used in this study can be obtained from the corresponding author upon reasonable request.
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Funding
Self-Financing Project of Reproductive Hospital of Guangxi Zhuang Autonomous Region (No. 2019GXZJK01), the Chinese Medicine Bureau Self-Financing Project of the Medical and Health Commission of Guangxi Zhuang Autonomous Region (No. GXZYZ20210107), Self-Financing Project of Guangxi Medical and Health Committee (No. Z20190633), Guangxi Natural Science Foundation Project (No. 2019GXNSFAA185016) and the National Natural Science Foundation of China (No. 82060286).
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R.H. performed duty as experiment chief executor. F.L. raised the laboratory animal. F.G., H.H., and Y.X. performed the isolation, culture, and identification of bone marrow mesenchymal stem cells and induced differentiation. M.H., Y.F., and Y.W. performed the Oil Red O staining identification and RT-qPCR analysis. W.Z. performed the formal analysis. A.M. professionally edited and proofread this manuscript. Y.M. and Z.L. supervised the study and contributed to funding acquisition.
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The experiments were performed at The Reproductive Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, and were approved by the Experimental Ethics Committee of The Reproductive Hospital of Guangxi Zhuang Autonomous Region (experimental animal use license number: SYXK (Gui) 2021–0007). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The methods used for animal experiments are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). This article does not contain any studies with human participants performed by any of the authors.
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Hua, R., Liang, FF., Gong, FQ. et al. Differentiation of bone marrow mesenchymal stem cells into Leydig-like cells with testicular extract liquid in vitro. In Vitro Cell.Dev.Biol.-Animal (2024). https://doi.org/10.1007/s11626-024-00902-6
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DOI: https://doi.org/10.1007/s11626-024-00902-6