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Amniotic membrane mesenchymal stem cells can differentiate into germ cells in vitro

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Abstract

This is the first report on differentiation of mouse amniotic membrane mesenchymal stem cells (AM-MSCs) into male germ cells (GCs). AM-MSCs have the multipotent differentiation capacity and can be differentiated into various cell types. In the present study, AM-MSCs were induced for differentiation into GCs. AM-MSCs were isolated from mouse embryonic membrane by enzymatic digestion. AM-MSCs were characterized with osteogenic and adipogenic differentiation test and flow cytometric analysis of some CD-markers. AM-MSCs were induced to differentiate into GCs using a creative two-step method. Passage-3 AM-MSCs were firstly treated with 25 ng/ml bone morphogenetic protein 4 (BMP4) for 5 d and in continuing with 1 μM retinoic acid (RA) for 12 d (total treatment time was 17 d). At the end of the treatment period, real-time reverse transcription (RT)-PCR was performed to evaluate the expression of GC-specific markers—Itgb1, Dazl, Stra8, Piwil2, Mvh, Oct4, and c-Kit- in the cells. Moreover, flow cytometry and immunofluorescence staining were performed to evaluate the expression of Mvh and Dazl at protein level. Real-time RT-PCR showed that most of the tested markers were upregulated in the treated AM-MSCs. Furthermore, flow cytometric and immunofluorescence analyses both revealed that a considerable part of the treated cells expressed GC-specific markers. The percentage of positive cells for Mvh and Dazl was about 23 and 46%, respectively. Our results indicated that a number of AM-MSCs successfully differentiated into the GCs. Finally, it seems that AM-MSCs would be a potential source of adult pluripotent stem cells for in vitro generation of GCs and cell-based therapies for treatment of infertility.

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Acknowledgments

The authors would like to thank the manager and staff of Tashkhis Baft Aragene Company (TBA) for their helpful technical support.

This study carried out at Pharmaceutical Sciences Research Center (PSRC) of Tehran University of Medical Sciences in collaboration with Infertility and Reproductive Health Research Center of the Health Research Institute of Babol University of Medical Sciences.

All experimental protocols were approved by the ethical committee of Tehran Medical University on Care and Use of Animals that were in accordance with the guidelines of National Institute of Health, USA.

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    Authors and Affiliations

    1. Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

      Zohreh Afsartala

    2. Department of Pharmacology and Toxicology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center (PSRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran

      Mohammad Amin Rezvanfar & Mohammad Abdollahi

    3. Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

      Mahshid Hodjat & Shima Tanha

    4. Cellular and Molecular Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran

      Vahideh Assadollahi

    5. Tashkhis Baft Aragene Company Ltd (TBA), Tehran, Iran

      Khosro Bijangi

    6. Infertility and Reproductive Health Research Center, Health Research Institute, Babol University of Medical Sciences, P.O. Box: 47318-38711, Amirkola, Babol, Iran

      Mohammad Ghasemzadeh-Hasankolaei

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    Zohreh Afsartala and Mohammad Amin Rezvanfar contributed equally to this work.

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    Afsartala, Z., Rezvanfar, M.A., Hodjat, M. et al. Amniotic membrane mesenchymal stem cells can differentiate into germ cells in vitro. In Vitro Cell.Dev.Biol.-Animal 52, 1060–1071 (2016). https://doi.org/10.1007/s11626-016-0073-6

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