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BOR - Papers in Press, published online ahead of print January 22, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.014050
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Submitted December 9, 2002
Returned for revision January 6, 2003
Accepted January 15, 2003

Gamete Biology


Maintenance of Mouse Male Germ Line Stem Cells In Vitro

Makoto Nagano , Buom-Yong Ryu , Clayton J. Brinster , Mary R. Avarbock , Ralph L. Brinster *

* To whom correspondence should be addressed. E-mail: cpope{at}vet.upenn.edu.

Abstract

The proliferation and differentiation of a stem cell are regulated intrinsically by the stem cell and extrinsically by the stem cell niche. Elucidation of regulatory mechanisms of spermatogonial stem cells (SSCs), the stem cell of the postnatal male germ line, would be facilitated by in vitro studies that provide a defined microenvironment reconstituted ex vivo. In this study, we analyzed the effect of in vitro environment on the maintenance of adult and immature SSCs in a 7-day culture system. The results demonstrated that while the number of adult and immature SSCs remaining decreased in a time-dependent manner, nearly 1 in 4 stem cells (24%) could be maintained in vitro for 7 days. Stem cell maintenance was enhanced by coculture with OP9 bone marrow stroma or L fibroblast cell lines, addition of glial cell line-derived neurotrophic factor, or using specific culture medium. In contrast, coculture with TM4 or SF7 Sertoli cell lines as well as addition of activin A or bone morphogenetic protein 4 (BMP4) reduced stem cell maintenance in vitro. Only 4% of the stem cells remained in culture with TM4 cells or activin A, and 6% in culture with SF7 cells or BMP4. Together with the reported functions of Sertoli cells and the growth factors used, these results lead to the hypothesis that suppression of germ cell differentiation improves in vitro maintenance of SSCs by interrupting the unidirectional cascade of spermatogenesis and blocking stem cell differentiation.



Key words: Gamete Biology • Testis • Developmental biology • Gametogenesis • Spermatogenesis


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