Spermatogenesis is initiated from spermatogonial stem cells (SSCs) that has an ability of self-renewal and unipotency to generate differentiating germ cells. The objective of this study is to develop the simple method for derivation of SSCs using non-sorting of both spermatogonia and feeder cells. Simply uncapsulated mouse testes were treated with enzymes followed by surgical mincing, and single cells were cultured in stempro-34TM cell culture media at 37℃. After 5 days of culture, aciniform of SSC colony was observed, and showed a strong alkaline phosphatase activity. Molecular characterization of mouse SSCs showed that most of the mouse SSC markers such as integrin α6 and β1, CD9 and Stra8. In addition, pluripotency embryonic stem cell (ESC) marker Oct4 were expressed, however Sox2 expression was lowered. Interestingly, expression of SSC markers such as Vasa, Dazl and PLZF were stronger than mouse ESC (mESC). This data suggest that generated mouse SSCs (mSSCs) in this study has at least similar biomarkers expression to mESC and mSSCs derived from other study. Immunocytochemistry using whole mSSC colony also confirmed that mSSCs generated from this study expressed SSC specific biomarkers such as c-kit, Thy1, Vasa and Dazl. In conclusion, mSSCs from 5 days old mouse testes were successfully established without sorting of spermatogonia, and this cells expressed both mESC and SSC specific biomarkers. This simple derivation method for mSSCs may facilitate the study of spermatogenesis.

ABSTRACT   
서론   
재료 및 방법    
생쥐 정원 줄기세포 분리 및 배양 방법    
Alkaline Phosphate (AP) 염색    
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)    
Western Blot    
Immunocytochemistry   
결과 및 고찰   
인용문헌

• 이원영(건국대학교 의료생명대학 식품생명과학부) | Won Young Lee
• 김희찬(건국대학교 의료생명대학 식품생명과학부) | Hee Chan Kim
• 김동훈(농촌진흥청 국립축산과학원) | Dong Hoon Kim
• 정학재(농촌진흥청 국립축산과학원) | Hak Jae Chung
• 박진기(농촌진흥청 국립축산과학원) | Jin Ki Park
• 송혁(건국대학교 의료생명대학 식품생명과학부) | Hyuk Song Corresponding author