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BOR - Papers in Press, published online ahead of print April 28, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.029207
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BIOLOGY OF REPRODUCTION 71, 722–731 (2004)
DOI: 10.1095/biolreprod.104.029207
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Culture Conditions and Single Growth Factors Affect Fate Determination of Mouse Spermatogonial Stem Cells1

Hiroshi Kubota, Mary R. Avarbock, and Ralph L. Brinster2

Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Cell fate determination between self-renewal or differentiation of spermatogonial stem cells (SSCs) in the testis is precisely regulated to maintain normal spermatogenesis. However, the mechanisms underlying the process remain elusive. To address the problem, we developed a model SSC culture system, first, by establishing techniques to obtain enriched populations of stem cells, and second, by establishing a serum-free culture medium. Flow cytometric cell sorting and the SSC transplantation assay demonstrated that Thy-1 is a unique surface marker of SSCs in neonatal, pup, and adult testes of the mouse. Although the surface phenotype of SSCs is major histocompatibility complex class I– Thy-1+ {alpha}6-integrin+ {alpha}v-integrin–/dim throughout postnatal life, the most enriched population of SSCs was obtained from cryptorchid adult testes by cell-sorting techniques based on Thy-1 expression. This enriched population of SSCs was used to develop a culture system that consisted of serum-free defined medium and STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders, which routinely maintained stem cell activity for 1 wk. Combining the culture system and the transplantation assay provided a mechanism to study the effect of single growth factors. A negative effect was demonstrated for several concentrations of basic fibroblast growth factor and leukemia inhibitory factor, whereas glial cell line-derived neurotrophic factor and stem cell factor appeared to have a positive effect on stem cell maintenance. The stem cell enrichment strategies and the culture methods described provide a reproducible and powerful assay system to establish the effect of various environmental factors on SSC survival and replication in vitro.

1 Support for the research was from the National Institute of Child Health and Human Development (NICHD 044445); The Commonwealth and General Assembly of Pennsylvania; and the Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation.

2 Correspondence: R.L. Brinster, School of Veterinary Medicine, University of Pennsylvania, 3850 Baltimore Ave., Philadelphia, PA 19104. FAX: 215 898 0667; cpope{at}vet.upenn.edu






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Copyright © 2004 by the Society for the Study of Reproduction.