Study On In Vitro Culture Of Mouse Spermatogonial Stem Cells

Objective and significance:To establish a long-term proliferation culture system for mouse spermatogonial stem cells and examine the role of leukemia inhibitory factor(LIF)in this culture system.These improvements not only provide a good platform for the study of spermatogenesis in vitro and self-renewal of stem cells,but also have important implications for the development of cell replacement therapy, transgenic technology,germ cell genetic manipulations,et al.Material and methods:Section One:Testis tissues were obtained from newborn male ICR mice on postnatal day 2 to 6.Testis cell suspension was collected by two-step enzymatic digestion prior to culture.The dissociated cells were aliquoted into tissue culture plates and cultivated with a modified system composed of serum-free defined medium included GDNF,LIF,EGF and bFGF on mouse embryonic fibroblasts(MEF)feeders.BrdU incorporation test was used to determine its proliferation at passage 2, passage 5 and passage 11.Alkaline phosphatase(AP)activity, immunofluorescence staining and RT-PCR assay were carried for the identification of the cultured cells at passage 11.Section Two:For the further study of the role of LIF in this culture system,we used two kinds of defined medium with different cytokine combination in the presence(groupⅠ,GDNF+bFGF+EGF+LIF)or absence(groupⅡ,GDNF+bFGF+EGF)of LIF to determine their effect. Then the number counting of germ cell colonies,BrdU labeling and TUNEL assay was carried out to detect the differences between these two groups at passage 2 and passage 5.Section Three:We obtained enriched populations of SSCs from adult male ICR mice at 9 to 12 weeks of age by two-step enzymatic digestion and concentrated by Ficoll fractionation.The germ cells were cultured by the method described above.BrdU incorporation test was used to determine its proliferation on day 5,day 10 and day 15.AP activity and immunofluorescence staining assay were used for the identification of the cultured cells.Result:The cultures were maintained for 15 passages in vitro in a steady state and continued to generate germ cell colonies.The undifferentiated state was confirmed by the strong positive for AP activity, immunofluorescence staining of GFRα-1~+/Oct-4~+/VASA~+/SCP3~- and GFRα-1~+/Oct-4~+/SCP3~- at the gene expression levels.The average number of germ cell colonies in individual wells of the culture plate in the presence of LIF(groupⅠ)or absence of LIF(groupⅡ) were 70.40±8.06 and 37.60±7.15 at passage 2,75.20±7.83 and 44.60±6.35 at passage 5.There was significant difference between the two groups.The index of BrdU positive cells on day 3 and day 5 reached 46.07±7.95%,45.09±7.80%(groupⅠ)and 31.47±3.34%,31.73±3.28%(groupⅡ)at passage 2,day 3 and day 5,separately.While the number were 66.22±7.51%,62.40±6.38%(groupⅠ)and 49.68±7.32 %,46.30±6.15(groupⅡ)on the third and fifth days of passage 5.These results demonstrated significant differences between the two groups with or without LIF(P＜0.05).While the percentage of TUNEL positive cells in groupⅠshowed no significant difference in contrast to groupⅡ.As a result,LIF can promote the in vitro proliferation of SSCs,but it has no effect on the anti-apoptosis.Interestingly,SSCs could also expand effectively without LIF in groupⅡindicating that LIF was not always indispensable in this culture system.Adult mouse spermatogonial stem cells grew slowly and colonies could be formed in about 10 to 15 days.The index of BrdU positive cells on day 5,10 and 15 were 14.57±2.01%,26.17±3.36%,16.73±2.46%. The germ cell colonies were positive for AP,GFRα-1 and VASA,while only parts of single cells were SCP3 posive.The cultured cells committed to apoptosis gradually after subculture.Conclusion:1.Spermatogonial stem cells from neonatal mouse could be expanded in our defined serum-free culture system and maintained for at least 15 passages steadily in vitro.The harvested cells remained in an undifferentiated state,which provides a good platform for the further study of spermatogenesis in vitro.2.The addition of LIF to this culture system enhanced the formation of germ cell colonies,however,it was not always indispensable.3.Spermatogonial stem cells from adult mouse testis could be amplified short-termly in vitro.