Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Establishment of a Simple and Effective Method for Isolating Male Germline Stem Cells (GSCs) from Testicular Cells of Neonatal and Adult Mice

  • Kim Kye-Seong (Department of Anatomy and Cell Biology, Hanyang University College of Medicine) ;
  • Lim Jung-Jin (Department of Anatomy and Cell Biology, Hanyang University College of Medicine) ;
  • Yang Yun-Hee (Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University) ;
  • Kim Soo-Kyoung (Department of Anatomy and Cell Biology, Hanyang University College of Medicine) ;
  • Yoon Tae-Ki (Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University) ;
  • Cha Kwang-Yul (Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University) ;
  • Lee Dong-Ryul (Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University)
  • Published : 2006.09.01
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Abstract

The aims of this study were to establish a simple and effective method for isolating male germline stem cells (GSCs), and to test the possibility of using these cells as a new approach for male infertility treatment. Testes obtained from neonatal and adult mice were manually decapsulated. GSCs were collected from seminiferous tubules by a two-step enzyme digestion method and plated on gelatin-coated dishes. Over 5-7 days of culture, GSCs obtained from neonates and adults gave rise to large multicellular colonies that were subsequently grown for 10 passages. During in vitro proliferation, oct-4 and two immunological markers (Integrin ${\beta}1,\;{\alpha}6$) for GSCs were highly expressed in the cell colonies. During another culture period of 6 weeks to differentiate to later stage germ cells, the expression of oct-4 mRNA decreased in GSCs and Sertoli cells encapsulated with calcium alginate, but the expression of c-kit and testis-specific histone protein 2B(TH2B) mRNA as well as the localization of c-kit protein was increased. Expression of transition protein (TP-l) and localization of peanut agglutinin were not seen until 3 weeks after culturing, and appeared by 6 weeks of culture. The putative spermatids derived from GSCs supported embryonic development up to the blastocyst stage with normal chromosomal ploidy after chemical activation. Thus, GSCs isolated from neonatal and adult mouse testes were able to be maintained and proliferated in our simple culture conditions. These GSCs have the potential to differentiate into haploid germ cells during another long-term culture.

Keywords

References

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