A Comparison between the Colony Formation of Adult Mouse Spermatogonial Stem Cells in Co cultures with Sertoli and STO (Mouse Embryonic Fibroblast Cell Line)

作者：Mohamamadi Syede Momeneh; Movahedin Mansoureh^*; Koruji Seyed Morteza

来源：Yakhteh, 2010, 12(2): 231-240.

• Adult Spermatogonial Stem Cells
• Colony Formation
• Co Culture
• Mouse

Objective: The aim of this study was to compare the colony formation of spermatogonial stem cells (SSCs) on sertoli and STO (Mouse embryonic fibroblast cell line) feeder cell layers during a two-week period.
Materials and Methods: Initially, sertoli cells and SSCs were isolated from adult mouse testes using a two-step enzymatic digestion and lectin immobilization. Characteristics of the isolated cells were immunocytochemically confirmed by examining for the presence of Oct-4, CDH1, promyelocytic leukaemia zinc finger factor (PLZF), SSC C-kit, and the distribution of Sertoli cell vimentin. SSCs were then cultured above the Sertoli, STO and the control (without co-culture) separately for two weeks. In all three groups, the number and diameter of colonies were evaluated using an invert microscope on the 3(rd), 7(th), ath and 14(th) day. beta(1) and alpha(6)-integrin m-RNA expressions were assessed using a reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. Furthermore, Oct-4 m RNA expression was assessed using real time PCR. Statistical analysis was performed using ANOVA; and the paired two-sample t test and Tukey's test were used as post-hoc tests for the data analysis of the three sertoli, STO and control cocultures.
Results: At the four specified time points, our results showed significant differences (p<0.05) in colony numbers and diameters among the sertoli, STO and control groups. The number and diameter of colonies increased more rapidly in the sertoli coculture than in the other two Our results at all four time points also showed significant differences (p<0.05) in the mean colony numbers and diameters between the three groups, with the Sertoli coculture having the highest mean values for colony numbers and diameters. The RT-PCR results, after two-weeks of culturing, showed that beta(1)-integrin was expressed in all three groups cocultures, but alpha(6)-integrin was not expressed. Additionally, based on real time PCR results, the three genes (beta(1)-integrin, alpha(6)-integrin, Oct-4) mentioned were also expressed in all three co cultures groups.
Conclusion: Based on the optimal effects of sertoli feeder cells on spermatogonial stem cells in a co culture system, as also confirmed by several other studies, their use is suggested to achieve better colonization of SSCs.

• 出版日期2010