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.
Biological Markers/metabolism ; Bromodeoxyuridine/metabolism ; *Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Cyclins/metabolism ; DNA-Binding Proteins/genetics/metabolism ; Embryonic Stem Cells/*cytology/enzymology ; Flow Cytometry ; Gene Expression Regulation ; Homeodomain Proteins/genetics/metabolism ; Humans ; Karyotyping ; Octamer Transcription Factor-3/genetics/metabolism ; Telomerase/metabolism ; Time Factors

Background::Psoriatic arthritis (PsA) is an inflammatory arthropathy characterized by psoriasis and bone erosion on radiology. Dickkopf-1 (Dkk-1) is considered to be the main inhibitor of the Wnt signaling pathway and results in reduced osteoblast proliferation. The aim of this study was to investigate the serum level of Dkk-1 and its association with bone erosion in PsA patients.Methods::Serum Dkk-1 levels were measured by enzyme-linked immunosorbent assay (ELISA) in 69 patients with PsA and 60 controls, including 39 rheumatoid arthritis (RA) patients, and 21 healthy controls (HCs). Rheumatoid factor and anti-cyclic citrullinated peptide levels were also determined by ELISA. The association of Dkk-1 level with clinical and laboratory features of PsA was analyzed. Logistic regression analysis was used to analyze the risk factors for bone erosion in PsA.Results::Dkk-1 was elevated in 68.1% (47/69) of the patients with PsA, 46.2% (18/39) of RA patients, and 9.5% (2/21) of HCs. Serum Dkk-1 concentration was significantly higher in PsA patients compared with that in HCs. The level of serum Dkk-1 was correlated with a swollen joint count, and levels of complement components 3 and 4. Elevated Dkk-1 level (odds ratio = 4.440, 95% confidence interval: 1.246-15.817, P = 0.021) was identified as the risk factor for bone erosion in PsA. Conclusions::The serum level of Dkk-1 is abnormally elevated in PsA patients. The elevation of Dkk-1 might be involved in the mechanism of bone erosion in patients with PsA.