Isolating highly pure rat spermatogonial stem cells in culture

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

Research output: Chapter in Book/Report/Conference proceedingChapter

21 Scopus citations

Abstract

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press
Pages163-179
Number of pages17
Volume450
ISBN (Print)9781603272131
DOIs
StatePublished - 2008

Publication series

NameMethods in Molecular Biology
Volume450
ISSN (Print)10643745

Keywords

  • Germ cell
  • Germline
  • Multipotent
  • Pluripotent
  • Spermatogenesis
  • Spermatogonia
  • Spermatogonial
  • Stem cell
  • Testis

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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