Identification and enrichment of spermatogonial stem cells displaying side-population phenotype in immature mouse testis.
In mammals, spermatogenesis is maintained by spermatogonial stem cells (SSC). In their niche, SSC divide to self-maintain and to produce a transit-amplifying population that eventually enters the meiotic cycle to give rise to spermatozoa. The low number of SSC and the lack of specific markers hinder their isolation and enrichment. Stem cells in several adult tissues can be identified by using their verapamil-sensitive Hoechst dye-effluxing properties, which define the characteristic "side population" (SP). Here we show, by multicolor flow cytometric analysis, that immature mouse testis contains a "side-population" (T-SP), which is Sca-1pos, Ep-CAMpos, EE2 pos, alpha6-integrin pos, and alpha(v)-integrin neg. A 13-fold enrichment in SSC activity was observed when sorted T-SP cells from ROSA 26 mice were transplanted in busulfan-treated mouse testis. Whereas an incomplete range of spermatogenic stages was encountered two months after transplantation of unsorted testicular cells, the transplantation of T-SP cells generated all associations of mouse germ cells representing the full range of spermatogenic stages. These data suggest that Hoechst staining and cell sorting might provide a novel approach to SSC enrichment in mammals.
Falciatori
Ilaria
I
Department of Histology and Medical Embryology, University of Rome La Sapienza, Rome 00161, Italy.
Borsellino
Giovanna
G
Haliassos
Nikolaos
N
Boitani
Carla
C
Corallini
Serena
S
Battistini
Luca
L
Bernardi
Giorgio
G
Stefanini
Mario
M
Vicini
Elena
E
D000818:Animals; D000941:Antigens / Q000032:analysis; D002066:Busulfan / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D017690:Cell Transplantation; D005434:Flow Cytometry; D016130:Immunophenotyping; D008297:Male; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D010641:Phenotype; D013091:Spermatogenesis / Q000187:drug effects; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism* / Q000637:transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism*; D013737:Testis / Q000166:cytology* / Q000187:drug effects
D000941:Antigens; D008565:Membrane Proteins; D002066:Busulfan