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Isolation of Human Male Germ-Line Stem Cells Using Enzymatic Digestion and Magnetic-Activated Cell Sorting

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Germline Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 825))

Abstract

Mammalian spermatogenesis is a process whereby male germ-line stem cells (spermatogonial stem cells) divide and differentiate into sperm. Although a great deal of progress has been made in the isolation and characterization of spermatogonial stem cells (SSCs) in rodents, little is known about human SSCs. We have recently isolated human G protein-coupled receptor 125 (GPR125)-positive spermatogonia and GDNF family receptor alpha 1 (GFRA1)-positive spermatogonia using a 2-step enzymatic digestion and magnetic-activated cell sorting (MACS) from adult human testes. Cell purities of isolated human GPR125- and GFRA1-positive spermatogonia after MACS are greater than 95%, and cell viability is over 96%. The isolated GPR125- and GFRA1-positive spermatogonia coexpress GPR125, integrin, alpha 6 (ITGA6), THY1 (also known as CD90), GFRA1, and ubiquitin carboxyl-terminal esterase L1 (UCHL1), markers for rodent or pig SSCs/progenitors, suggesting that GPR125- and GFRA1-positive spermatogonia are phenotypically the SSCs in human testis. Human GPR125-positive spermatogonia can be cultured for 2 weeks with a remarkable increase in cell number. Immunocytochemistry further reveals that GPR125-positive spermatogonia can be maintained in an undifferentiated state in vitro. Collectively, the methods using enzymatic digestion and MACS can efficiently isolate and purify SSCs from adult human testis with consistent and high quality. The ability of isolating and characterizing human SSCs could provide a population of stem cells with high purity for mechanistic studies on human SSC self-renewal and differentiation as well as potential applications of human SSCs in regenerative medicine.

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Author information

Authors and Affiliations

  1. Clinical Stem Cell Research Center, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Zuping He

  2. Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC, USA

    Maria Kokkinaki, Jiji Jiang & Martin Dym

  3. Northwest Agricultural and Forest University, Shanxi, China

    Wenxian Zeng

  4. Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada

    Ina Dobrinski

Authors
  1. Zuping He
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  2. Maria Kokkinaki
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  3. Jiji Jiang
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  4. Wenxian Zeng
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  5. Ina Dobrinski
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  6. Martin Dym
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Corresponding author

Correspondence to Zuping He .

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Editors and Affiliations

  1. , School of Biomedical Sciences, The Chinese University of Hong Kong, n/a, Shatin, New Territories, n/a, Hong Kong/PR China

    Wai-Yee Chan

  2. , Animal Biosciences, ARS-USDA, Baltimore Avenue 10300, Beltsville, 20705, Maryland, USA

    Le Ann Blomberg

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He, Z., Kokkinaki, M., Jiang, J., Zeng, W., Dobrinski, I., Dym, M. (2012). Isolation of Human Male Germ-Line Stem Cells Using Enzymatic Digestion and Magnetic-Activated Cell Sorting. In: Chan, WY., Blomberg, L. (eds) Germline Development. Methods in Molecular Biology, vol 825. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-436-0_4

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  • DOI: https://doi.org/10.1007/978-1-61779-436-0_4

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  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-61779-435-3

  • Online ISBN: 978-1-61779-436-0

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