earticle

영어

Spermatogonial stem cells (SSCs) can produce sperms transferring genetic information into the next generation in seminiferous tubule of testes. Accordingly, an efficient isolation technique of SSCs with extremely low numbers from testes is required for successful downstream researches related to maintenance, differentiation and cryopreservation of SSCs. To date, a variety of isolation techniques in a variety of species have been used for retrieving SSCs from testicular tissue. However, comparison of their efficiency has been not revealed clearly. Accordingly, among isolation methods described previously, we tried to elucidate a technique showing the best isolation efficiency in the retrieval of SSCs from testes derived from mice. For these, SSCs were isolated from mouse testis as follows: differential plating (DP), EpCAM, Thy1 or GFRα1 antibody-based magnetic-activating cell sorting (MACS) post-DP, EpCAM, Thy1 or GFR α1 antibody-based MACS, EpCAM, Thy1 or GFRα1 antibody-based MACS post-MACS based on GFRα1 antibody (double MACS for positive selection), and CD34 or α-SMA antibody-based MACS post-MACS based on GFRα1 antibody (double MACS for negative selection). Subsequently, SSCs isolated from each method were stained by alkaline phosphatase (AP) staining and percentage of AP positive SSCs was compared to find an optimal method among isolation method candidates. As the results, SSCs isolated by DP for 8 h showed numerically the highest percentage of AP positive SSCs. In case of MACS post-DP, SSCs isolated by MACS based on Thy1 antibody post-DP (MACSThy1 post-DP) revealed numerically higher percentage of AP positive cells than those on EpCAM and GFRα1 antibodies post-DP. Moreover, numerically the highest percentage of AP positive SSCs was detected when SSCs were isolated from mouse testis by MACS based on GFRα1 (MACSGFRα1) compared to EpCAM and Thy1 antibodies. In case of double MACS for positive selection, the usage of EpCAM antibody in the second MACS post-GFRα1 antibody-based MACS (double MACSGFRα1/EpCAM) showed numerically the highest percentage of AP positive SSCs compared to Thy1 and GFRα1 antibodies. On the other hands, after GFRα1 antibody-based MACS sorting, the usage of α-SMA antibody in the second MACS (double MACSGFRα1/α-SMA) for negative selection showed numerically higher percentage of AP positive SSCs than CD34 antibody. The subsequent comparison of isolation efficiency derived from each method demonstrated that MACSGFRα1 and double MACSGFRα1/EpCAM resulted in significantly the best isolation efficiency. Accordingly, we could elucidate that MACSGFRα1 and double MACSGFRα1/EpCAM were techniques sorting effectively SSCs from mouse testes.