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[ ABSTRACT] AIM:To investigate the effect of maxadilan, which specifically activates pituitary adenylate cycla-se-activating polypeptide type I receptor (PAC1 receptor), on the proliferation, apoptosis and differentiation potential of human adipose-derived stem cells ( ASCs) .METHODS:ASCs from human adipose tissue were isolated by enzymatic di-gestion and cultured.ASCs were confirmed by the analysis of the markers for cell phenotypes by flow cytometry ( FCM) and adipogenic/osteogenic induction.The effect of maxadilan on ASCs viability was analyzed by CCK-8 assay and FCM.ASCs were irradiated by ultraviolet C ( UVC) at 254 nm and the absorbance of apoptotic ASCs induced by various doses of UVC was measured by CCK-8 assay.ASCs were exposed to 702 J/m2 UVC for 24 h to induce apoptosis.The effect of maxadilan on ASC apoptosis was analyzed by FCM and the determination of caspase 3 and caspase 9 levels.RESULTS:Adipose-de-rived stem cells were confirmed by the detection of the positive expression of cell phenotypes including CD29, CD44, CD59 and CD105 by FCM.The data of CCK-8 assay revealed that ASCs treated with maxadilan (80 nmol/L) had the strongest ability of proliferation.The data of FCM also demonstrated that the addition of 80 nmol/L maxadilan to ASCs in experimen-tal group markedly improved the proliferation capacity of the cells compared with control group (P<0.05).The apoptosis of ASCs exposed to 702 J/m2UVC was dramatically inhibited by the treatment with maxadilan (80 nmol/L).Such process involved the caspase signaling pathway including caspase 3 and caspase 9.There was statistical significance (P<0.05) between experiment group ( ASCs irradiated by UVC and supplemented with maxadilan) and control group ( ASCs only irra-diated by UVC) .Meanwhile, adipogenic and osteogenic differentiation potentials were both positive in experiment group and control group.CONCLUSION:Maxadilan promotes proliferation and inhibits apoptosis of the ASCs.The differentia-tion potential of ASCs toward adipogenic and osteogenic lineages wouldn’ t be altered by maxadilan.Maxadilan would bene-fit to growth and expansion of ASCs in vitro.
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[ABSTRACT]AIM:ToinvestigatethepromotingroleofTranswellcontactco-culturesysteminthegrowthand differentiation of single-dissociated induced pluripotent stem cells (iPSCs).METHODS:Bovine corneal endothelial cells (CECs) at passage 1~2 (P1~2) were seeded on the underside of Transwell inserts placed into culture plates and were cultured in 37 ℃and 5%CO2 for 8 h.Accutase digestion and 40μm filter process disaggregated colony-aggregated iPSCs into single-dissociated iPSCs , and the cells were seeded on the inside of Transwell inserts with CECs in medium of mTeSR 1 for 3 d and then in low-glucose DMEM supplemented with 10% FBS for 2 weeks.The characteristics and differentiation markers were evaluated by real-time fluorescence quantitative polymerase chain reaction ( qPCR ) , immunofluorescence staining, live&dead cell staining and alkaline phosphatase (ALP) staining.The group of iPSCs cultured in conventional medium was used as control group 1.The group of single-dissociated iPSCs co-cultured with CECs was set as experimental group, while single-dissociated iPSCs without co-culture were as control group 2.RESULTS: The bovine CECs showed typical hexagonal cobblestone shape .iPSCs showed colony-like growth , while became single-dissociated cells after Tran-swell contact co-culture with bovine CECs for 3 d.The single-dissociated iPSCs positively expressed the undifferentiated markers, Nanog and Oct4.The mRNA expression levels of Nanog , Oct4 and Sox2 between experimental group and control group 1 were both positive and had no statistical significance difference (P>0.05).The dead cells in experimental group decreased significantly, and there was statistically significant difference compared to control group 2 (P<0.01).After 14 d of induced differentiation co-culture , the single-dissociated iPSCs showed rather uniform polygonal morphology , increased dimension and no obvious colony existence .Negative ALP staining, positive immunofluorescence staining for ZO-1, AQP1 and CD31, and negative for CD34 and CD133 were also observed.The results of qPCR showed that the mRNA expression of Oct4, Nanog and Sox2 significantly decreased , and had statistically significant difference compared with control group 1 (P<0.01).CONCLUSION: When co-cultured with bovine CECs, iPSCs morphologically changed to endothelial-like cells and expressed some markers of CECs .Transwell contact co-culture system not only enhances the growth of single-dis-sociated iPSCs , but also promotes their differentiation .