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Sertoli Cell–Conditioned Medium Induces Differentiation of Bone Marrow–Derived Mesenchymal Stem Cells to Male Germ-Like Cells in Busulfan-Induced Azoospermic Mouse Model

  • Male Reproduction: Original Article
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Abstract

Non-obstructive azoospermia is a severe form of male infertility, with limited effective treatments. Bone marrow mesenchymal stem cells (BMSCs) can differentiate to different cell lines; therefore, transplantation of these cells is used for treatment of several diseases. Since these cells require induction factors to differentiate into germ cells, we co-transplanted bone marrow stem cells (BMSCs) with Sertoli cell–conditioned medium (SCCM) into the testis of azoospermic mice. This study was carried out in two sections, in vitro and in vivo. For in vitro study, differentiating factors (c-kit and ID4) were examined after 15 days of co-culture of bone marrow cells with Sertoli cell–conditioned medium, while for in vivo study, the azoospermia model was first created by intraperitoneal administration of a single-dose busulfan (40 mg/kg) followed by single-dose CdCl2 (2 mg/kg) after 4 weeks. Mice were divided into 4 groups including control (azoospermia), BMSC, SCCM, and BMSC + SCCM. Eight weeks after transplantation, samples were assessed for proliferation and differentiation via the expression level of MVH, ID4, SCP3, Tp1, Tp2, and Prm1 differentiation markers. The results showed that BMSC co-cultured with SCCM in vitro differentiated BMSC to germ-like cells. Similarly, in vivo studies revealed a higher level of BMSC differentiation into germ-like cells with significant higher expression of differentiation markers in transplanted groups compared to the control. This study confirmed the role of SCCM as an inductive factor for BMSC differentiation to germ cells both in vivo and in vitro conditions.

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Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Code Availability

In this study, we used ImageJ (ImageJ, RRID:SCR_003070) and GraphPad Prism (GraphPad Prism, RRID:SCR_002798) software. We also used anti-MVH (Abcam Cat# ab13840, RRID: AB_443012), anti-ID4 polyclonal (Geno Tex Cat# GTX89728, RRID: AB_ 10,726,681), anti-SCP3 (Abcam Cat# ab15093, RRID: AB_301639), anti-c-kit (Fitzgerald Industries International Cat# 10R-3024, RRID: AB_11193325), Goat anti-Rabbit IgG-H&L polyclonal, HRP conjugated (Abcam Cat# ab721, RRID: AB_955447), goat anti-Mouse IgG (H&L)-HRP conjugate (Bio-Rad Cat# 170–6516, RRID: AB_11125547), goat anti-Rabbit IgG-H&L Polyclonal (FITC conjugated) (Abcam Cat# ab6717, RRID: AB_955238), and hamster anti-rat CD29 (β1-integrin) (FITC) (BD Biosciences Cat# 555,005, RRID: AB_395639) antibodies.

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Acknowledgements

The authors thank the Tehran University of Medical Sciences and Health Services, Tehran, Iran.

Funding

This study was supported in part by a grant received from the Tehran University of Medical Sciences and Health Services, Tehran, Iran (grant number:48758).

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Authors and Affiliations

  1. Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Nasrin Khanmohammadi, Fatemeh Malek, Nasrin Takzaree & Tayebeh Rastegar

  2. Department of Anatomy, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

    Mehrnoush Malekzadeh & Maryam Khanehzad

  3. Department of Anatomy, Olabisi Onabanjo University, Ago-Iwoye, Sagamu, Ogun State, Nigeria

    Omotosho Dhulqarnian Akanji

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  1. Nasrin Khanmohammadi
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Contributions

All authors participated in the design, interpretation of the studies, analysis of the data, and review of the manuscript. Nasrin Khanmohammadi and Tayebeh Rastegar designed the study, Fatemeh Malek supervised the histological studies, Nasrin Takzaree, Mehrnoush Malekzadeh and Maryam Khanehzad supervised the data collection and analyzed the data, Omotosho Dhulqarnain Akanji prepared the manuscript for publication. All authors have read and approved the manuscript.

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Correspondence to Tayebeh Rastegar.

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This article does not contain any studies with human participants performed by any of the authors. All experiments involving the use of animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the Tehran University of Medical Science. All applicable international, national, and institutional guidelines for the care and use of animals were followed.

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Khanmohammadi, N., Malek, F., Takzaree, N. et al. Sertoli Cell–Conditioned Medium Induces Differentiation of Bone Marrow–Derived Mesenchymal Stem Cells to Male Germ-Like Cells in Busulfan-Induced Azoospermic Mouse Model. Reprod. Sci. 31, 375–392 (2024). https://doi.org/10.1007/s43032-023-01332-7

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