Activin-A 처리에 의해 분화 촉진된 인간 배아 줄기세포 유래 내배엽성 세포의 효과적인 정제

서지정보

ㆍ발행기관 : 한국동물번식학회 ㆍ수록지정보 : Reproductive & developmental biology / 34권 / 3호
ㆍ저자명 : 김문규, 문성환, 박순경, 이경일, 신정민, 장재우, 정형민

목차

ABSTRACT 서론 재료 및 방법 배상체 형성 및 내배엽성 세포로의 분화 내배엽성 세포의 정제 및 배양 RNA 추출 및 실시간 중합효소 연쇄 반응 분석 면역 염색 분석 통계처리 및 분석 결과 Actvin-A가 배상체 형성에 미치는 형태학적 분석 Activin-A에 의한 배상체 내에서의 내배엽성 세포의 발현 패턴 및 분포도 분석 부착된 배상체에서의 AFP발현 패턴을 이용한 고순도 내배엽성 세포의 정제 고찰 인용문헌

영어 초록

Embryoid bodies (EBs) generated from human embryonic stem cells (hESCs) include spontaneously induced endodermal lineage cells (ELCs). Activin-A plays important roles in the endoderm differentiation of hESCs. Despite studies on the generation of ELCs from hESCs with treatment of Actvin-A, it was unclear for localization and pattern of ELCs by Activin-A during differentiation of hESCs. Accordingly in this study, we knew that Actvin-A increased the cystic EBs formation, including the highly enriched AFP (endoderm lineage specific marker)-expressing cells in the surface of cystic EBs. To induce the EBs formation from undifferentiated hESCs, cells were transferred onto petri-dish and cultured in suspension condition without bFGF removed hESC media (EB media) for 3 days. Next to investigate the effect of Activin-A, EBs were subsequently cultured in EB media supplement with 100 ng/ml Activin-A for 3 days. After 5～7 days of Activin-A treatment, cystic EBs began to appear which increased in numbers reaching ～60% of initially formed EBs over 5 days. Endoderm lineage marker, AFP were highly expressed and specifically localized at the surface region of cystic EBs comparison with normal EBs. We next attached the cystic EBs onto gelatin-coated plates and cultured for 5 days. In the results of real-time PCR and immunocytochemistry analysis, AFP-expressing cells migrated and localized at the outgrowth region of attached cystic EBs. To obtain the AFP-expressing cells of the outgrowth region, we manually isolated by using micro- dissection and cultured them. These cells strongly express AFP over 70% of isolated cells post re-plating. Here, we first showed an expression pattern of specifically localized ELCs by Activin-A during differentiation of hESCs. From this observation, we could highly purified ELCs from undifferentiated hESCs. Taken together, our system will provide a novel and efficient option to generate ELCs from hESCs.

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