Germline Stem Cells pp 163-179

Part of the Methods in Molecular Biology™ book series (MIMB, volume 450) | Cite as

Isolating Highly Pure Rat Spermatogonial Stem Cells in Culture

  • F. Kent Hamra
  • Karen M. Chapman
  • Zhuoru Wu
  • David L. Garbers

Summary

Methods are detailed for isolating highly pure populations of spermatogonial stem cells from primary cultures of testis cells prepared from 22- to 24-day-old rats. The procedure is based on the principle that testicular somatic cells bind tightly to plastic and collagen matrices when cultured in serum-containing medium, whereas spermatogonia and spermatocytes do not bind to plastic or collagen when cultured in serum-containing medium. The collagen-non-binding testis cells obtained using these procedures are thus approx. 97% pure spermatogenic cells. Stem spermatogonia are then easily isolated from the purified spermatogenic population during a short incubation step in culture on laminin matrix. The spermatogenic cells that bind to laminin are more than 90% undifferentiated, type A spermatogonia and are greatly enriched in genetically modifiable stem cells that can develop into functional spermatozoa. This method does not require flow cytometry and can also be applied to obtain enriched cultures of mouse spermatogonial stem cells. The isolated spermatogonia provide a highly potent and effective source of stem cells that have been used to initiate in vitro and in vivo culture studies on spermatogenesis.

Keywords

Germ cell germline multipotent pluripotent spermatogonia spermatogonial spermatogenesis stem cell testis 

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Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • F. Kent Hamra
    • 1
  • Karen M. Chapman
    • 2
  • Zhuoru Wu
    • 3
  • David L. Garbers
    • 4
  1. 1.Department of Pharmacology and Cecil H. and Ida Green Center for Reproductive Biology SciencesUniversity of Texas Southwestern Medical CenterDallasTX
  2. 2.Southwestern Medical CenterHoward Hughes Medical Institute, University of TexasDallasTX
  3. 3.Department of PharmacologyUniversity of Texas Southwestern Medical CenterDallasTX
  4. 4.Department of Pharmacology and Cecil H. and Ida Green Center for Reproductive Biology SciencesHoward Hughes Medical Institute, University of Texas Southwestern Medical CenterDallasTX

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