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Enrichment and In Vitro Culture of Spermatogonial Stem Cells from Pre-Pubertal Monkey Testes

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Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Spermatogonial stem cells (SSCs) are essential for spermatogenesis throughout the lifespan of the male. However, the rarity of SSCs has raised the need for an efficient selection method, but little is known about culture conditions that stimulate monkey SSC proliferation in vitro. In this study, we report the development of effective enrichment techniques and in vitro culturing of germ cells from pre-pubertal monkey testes. Testis cells were analyzed by fluorescence-activated cell sorting techniques and were transplanted into the testes of nude mice to characterize SSCs. Thy-1-positive cells showed a higher number of colonies than the unselected control after xenotransplantation. Extensive colonization of monkey cells in the mouse testes indicated the presence of highly enriched populations of SSCs in the Thy-1-positive sorted cells. Furthermore, monkey testis cells were enriched by differential plating using extracellular matrix, laminin, and gelatin, and then cultured under various conditions. Isolation of monkey testicular germ cells by differential plating increased germ cell purity by 2.7-fold, following the combinational isolation method using gelatin and laminin. These enriched germ cells actively proliferated under culture conditions involving StemPro medium supplemented with bFGF, GDNF, LIF, and EGF at 37 °C. These results suggest that the enrichment and in vitro culture method proposed in the present study for harvesting a large number of functionally active monkey SSCs can be applied as the basis for efficient in vitro expansion of human SSCs.

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Acknowledgements

This work has been supported by the Korean Health Technology R&D Project (HI12C0055), Ministry of Health and Welfare, Republic of Korea. We also appreciate the Chung-Ang University Research Scholarship Grant in 2017, Republic of Korea.

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Author notes

  1. Bang-Jin Kim

    Present address: Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

Authors and Affiliations

  1. Department of Animal Science and Technology, College of Biotechnology and Natural Resource, Chung-Ang University, Anseong, Gyeonggi-Do, 17546, Republic of Korea

    Yong-Hee Kim, Hyun-Gu Kang, Bang-Jin Kim, Sang-Eun Jung, Polash C. Karmakar, Seok-Man Kim & Buom-Yong Ryu

  2. Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju, Jeollabuk-do, 55365, Republic of Korea

    Seongsoo Hwang

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  1. Yong-Hee Kim
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Correspondence to Buom-Yong Ryu.

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Ethical statement

All animal procedures were approved by the Animal Care and Use Committee of Chung-Ang University (IACUC no. 2015-00016) in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and the Guide for Care and Use of Laboratory Animals.

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Kim, YH., Kang, HG., Kim, BJ. et al. Enrichment and In Vitro Culture of Spermatogonial Stem Cells from Pre-Pubertal Monkey Testes. Tissue Eng Regen Med 14, 557–566 (2017). https://doi.org/10.1007/s13770-017-0058-x

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  • DOI: https://doi.org/10.1007/s13770-017-0058-x

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