Comparison of dissociation methods on pluripotency of stem cells

서지정보

ㆍ발행기관 : 한국예방수의학회(구 한국수의공중보건학회) ㆍ수록지정보 : 예방수의학회지 / 41권 / 4호
ㆍ저자명 : Na-Yeon Gu, Jienny Lee, Mi Jeong Park, Jeong Su Byeon, Da-Un Jeong, In-Soo Cho, Sang-Ho Cha

목차

서 론
재료 및 방법
세포 배양(cell culture)
계대 배양 방법(sub-culture)
알칼리성 인산가수분해효소 염색(alkaline phosphatase staining)
실시간 정량적 중합효소연쇄반응(quantitative real-time RT-PCR)
면역 형광 염색(immunofluorescent staining)
통계학적 분석
결 과
계대 배양 방법에 따른 돼지 유도만능줄기세포의 콜로니 모양과 알칼리성 인산가수분해효소 활성 비교
계대 배양 방법에 따른 돼지 유도만능줄기세포의 전사인자 발현 비교
고 찰
감사의 글
REFERENCES

영어 초록

Induced pluripotent stem cells (iPSCs) can be generated from adult cells. Somatic cells can be reprogrammed to form iPSCs by overexpressing transcription factors such as Oct4, Sox2, cMyc, and Klf4. To maintain undifferentiated state of iPSCs in vitro, cells have traditionally been maintained on mouse embryonic fibroblast feeders and passaged by enzymatic or mechanical dissociation methods. In this study, we compared the morphology and pluripotency of porcine iPSCs (piPSCs) after subsequent passaging using enzymatic and mechanical dissociation methods. Enzymatically and mechanically passaged piPSCs showed embryonic stem cell-like morphologies with compact cell adhesion and clear colony borders. In addition, alkaline phosphatase staining was positive for both enzymatically and mechanically passaged piPSCs. However, visual observation revealed that some colonies of enzymatically passaged piPSCs were spontaneously differentiated more than those of piPSCs mechanically passaged from 5 passage. Quantitative real-time RT-PCR demonstrated that enzymatically and mechanically passaged piPSCs expressed pluripotent genes such as Oct4, Sox2 and Nanog well at early passage. Immunofluorescent staining also confirmed that pluripotent markers such as Oct4, Sox2, and Nanog were positively expressed at early passage. However, expression levels of pluripotent genes in mechanically passaged piPSCs were also higher than those in enzymatically passaged piPSCs at early passage. Collectively, we found that mechanical passage method was better than enzymatic passage in terms of morphology and pluripotency of piPSCs at early passage. Further studies are needed to compare these dissociation methods with those obtained after more passages of piPSCs.

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