[Immunomagnetic beads sorting and functional identification of human spermatogonial stem cells].
OBJECTIVE
The very nature of spermatogonial stem cells (SSC) is still poorly understood. The objective of this study is to explore the specific markers of human SSC and search for the suitable method for their isolation and functional identification.


METHODS
Adults testicular cell suspensions were sorted by immunomagnetic beads method using alpha6,beta1 integrin and Thy-1 markers. The light-scattering properties and DNA Ploidy of the resultant subpopulations were analyzed by flow cytometry. The efficacies of the sorting on human SSC were evaluated by germ cell transplantation.


RESULTS
(1) The alpha6+ Thy-1+ c-kit- and beta1+ Thy-1+ c-kit- cells were relatively uniform subpopulations in size and morphology, which represented about 2%-3% and 0.5%-1% of the unsorted testis cells, respectively. The analysis of light-scattering properties showed that both of the subpopulations had low side light-scattering properties. The DNA Ploidy analysis showed significant changes of these two cell subpopulations in DNA Ploidy. The percentage of diploid cells in alpha6+ Thy-1+ c-kit- cell subpopulation significantly increased to 51.2% and synthesis phase and tetraploid cells disappeared. (2) The functional evaluation showed that the SSC in the alpha6+ Thy-1+ c-kit- cells were enriched 40 times and the SSC in the beta1+ Thy-1+ c-kit- cells 20 times that of the unsorted cells.


CONCLUSIONS
The alpha6,beta1 integrin and Thy-1 may be used for the SSC isolation as positive markers. The immunomagnetic beads sorting using alpha6,beta1 integrin and Thy-1 markers can result in significant enrichment of human SSC. It will open up a wide prospect for the researches on the biology of human SSC and the treatment of male sterility.
Li
Yanfeng
Y
Department of Urology, Daping Hospital, Institute of Field Surgery, the Third Military Medical University, Chongqing 400042, China. lyf1000@yahoo.com.cn
Guo
Yinglu
Y
Li
Xiaohong
X
Jin
Fengshuo
F
Jiang
Jun
J
Bi
Gang
G
D000328:Adult; D000818:Animals; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D039503:Integrin alpha6 / Q000032:analysis; D019012:Integrin beta1 / Q000032:analysis; D008297:Male; D051379:Mice; D013093:Spermatogonia / Q000166:cytology*; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology*; D013737:Testis / Q000166:cytology*; D018800:Thy-1 Antigens / Q000032:analysis
D039503:Integrin alpha6; D019012:Integrin beta1; D018800:Thy-1 Antigens