Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
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Effective Isolation of Endodermal Lineage Cells Derived from Human Embryonic Stem Cells Post Activin-A Treatment

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

  • Kim, Mun-Kyu (Graduate School of Life Science, CHA Stem Cell Institute, CHA University, College of Medicine) ;
  • Moon, Sung-Hwan (CHA Biotech & Diostech Co., Ltd.) ;
  • Park, Soon-Jung (Graduate School of Life Science, CHA Stem Cell Institute, CHA University, College of Medicine) ;
  • Lee, Kyung-Il (CHA Biotech & Diostech Co., Ltd.) ;
  • Shin, Jeong-Min (CHA Biotech & Diostech Co., Ltd.) ;
  • Jang, Jae-Woo (Graduate School of Life Science, CHA Stem Cell Institute, CHA University, College of Medicine) ;
  • Chung, Hyung-Min (Graduate School of Life Science, CHA Stem Cell Institute, CHA University, College of Medicine)
  • 김문규 (차의과학대학 의생명과학대학원 줄기세포 연구실) ;
  • 문성환 (차바이오앤디오스텍) ;
  • 박순정 (차의과학대학 의생명과학대학원 줄기세포 연구실) ;
  • 이경일 (차바이오앤디오스텍) ;
  • 신정민 (차바이오앤디오스텍) ;
  • 장재우 (차의과학대학 의생명과학대학원 줄기세포 연구실) ;
  • 정형민 (차의과학대학 의생명과학대학원 줄기세포 연구실)
  • Received : 2010.06.10
  • Accepted : 2010.08.30
  • Published : 2010.09.30
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Abstract

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.

Keywords

References

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