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Effective Isolation of Endodermal Lineage Cells Derived from Human Embryonic Stem Cells Post Activin-A Treatment  

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)
Publication Information
Reproductive and Developmental Biology / v.34, no.3, 2010 , pp. 135-141 More about this Journal
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
Human embryonic stem cells; EB formation; Endoderm differentiation; Activin-A; Micro-dissection;
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