title: Development of quantitative microscopy-based assays for evaluating dynamics of living cultures of mouse spermatogonial stem/progenitor cells.
abstract: Spermatogonial stem cell (SSC) self-renewal and differentiation are required for continuous production of spermatozoa and long-term fertility. Studying SSCs in vivo remains challenging because SSCs are rare cells and definitive molecular markers for their identification are lacking. The development of a method for propagating SSCs in vitro greatly facilitated analysis of SSCs. The cultured cells grow as clusters of a dynamic mixture of "true" stem cells and differentiating progenitor cells. Cells in the stem/progenitor culture system share many properties with spermatogonia in vivo; however, to fully exploit it as a model for spermatogonial development, new assays are needed that account for the dynamic heterogeneity inherent in the culture system. Here, assays were developed for quantifying dynamics of cultures of stem/progenitor cells that expressed histone-green fluorescent protein (GFP). First, we built on published results showing that cluster formation in vitro reliably predicts the relative number of SSCs. The GFP-based in vitro cluster assay allows quantification of SSCs with significantly fewer resources than a transplantation assay. Second, we compared the dynamics of differentiation in two experimental paradigms by imaging over a 17-day time frame. Finally, we performed short-term live imaging and observed cell migration, coordinated cell proliferation, and cell death resembling that of spermatogonia in the testes. The methods that we present provide a foundation for the use of fluorescent reporters in future microscopy-based high-throughput screens by using living spermatogonial stem/progenitor cultures applicable to toxicology, contraceptive discovery, and identification of regulators of self-renewal and differentiation.
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, USA.
authors.forename: Crystal N
authors.initials: CN
authors.lastname: Heim
authors.forename: Danielle A
authors.initials: DA
authors.lastname: Fanslow
authors.forename: Christina Tenenhaus
authors.initials: CT
authors.lastname: Dann
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D018929:Cell Culture Techniques; D016923:Cell Death; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008811:Mice, Inbred DBA; D008856:Microscopy, Fluorescence / Q000379:methods; D013093:Spermatogonia / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D013234:Stem Cells / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D014162:Transfection
chemical_list: D049452:Green Fluorescent Proteins