title: Alterations of proliferative and differentiation potentials of human embryonic stem cells during long-term culture.
abstract: Human embryonic stem cells (hESCs) are considered to be able to stably maintain their characteristics in vitro for prolonged periods, but we had previously encountered changes in proliferative ability and differentiation potential during extended culture of hESCs. Therefore, we investigated the proliferative ability and differentiation potential of hESCs during long-term culture. The hESCs, SNUhES3, were used to analyze population-doubling time, proliferation rate and differentiation potential. We classified hESCs into three groups according to culture period. Ten colonies of hESCs for each group were daily measured colony area and population-doubling time was assessed by the changes of colony area. Proliferation rate of hESCs was measured by 5-bromo-2'-deoxyuridine (BrdU) assay and telomerase activity. To evaluate differentiation potentials for hESCs, expression levels of undifferentiated and/or differentiated hESCs markers were examined by FACS, RT-PCR and immunostaining. Population-doubling time of early passage hESCs was longer than those of middle or late passage. Proliferative ability of hESCs was accelerated depending on culture periods. Cellular morphologies and the expression level of each three germ layer markers were obviously different from each passage of reattached embryoid bodies (EBs) after spontaneous differentiation. Differentiated cells of late passage expressed higher levels of undifferentiated markers such as Oct4 and SSEA4 than those of early and middle passage. But differentiated cells of early and middle passage expressed higher level of differentiated state markers, Nestin (ectoderm), Brachyury (mesoderm), HNF3beta (endoderm). From these results, it can be inferred that hESCs show higher proliferative abilities and reduced differentiation potentials as the passage number increased. Therefore, we conclude that early passage hESCs could be more suitable than middle and late passage hESCs in differentiation studies.
authors.affiliation: Central Research Institute, Sam Jin Pharm. Co. Ltd. Hwasung 445-746, Korea.
authors.forename: Yong Bin
authors.initials: YB
authors.lastname: Park
authors.forename: Yoon Young
authors.initials: YY
authors.lastname: Kim
authors.forename: Sun Kyung
authors.initials: SK
authors.lastname: Oh
authors.forename: Sun Gan
authors.initials: SG
authors.lastname: Chung
authors.forename: Seung Yup
authors.initials: SY
authors.lastname: Ku
authors.forename: Seok Hyun
authors.initials: SH
authors.lastname: Kim
authors.forename: Young Min
authors.initials: YM
authors.lastname: Choi
authors.forename: Shin Yong
authors.initials: SY
authors.lastname: Moon
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D001973:Bromodeoxyuridine / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D056741:Cyclin D; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000201:enzymology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007621:Karyotyping; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000378:metabolism; D013997:Time Factors
chemical_list: D015415:Biomarkers; D056741:Cyclin D; D016213:Cyclins; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D019098:Telomerase; D001973:Bromodeoxyuridine