Influence Of Wnt/β-catenin Signal Transduction Pathway On HUC-MSCs’ Hepatocyte-like Cells Differentiation

Objective:To explore the effect of Wnt/β-catenin signaling pathway on hUC-MSCs differentiation into hepatocyte-like cells and its potential molecular mechanism.Methods:1.hUC-MSCs were extracted by tissue adherent method from neonatal umbilical cord.After being cultured and purified,the 4th-passage of the adherent cells were prepared into a single cell suspension,of which molecule markers were detected and identified using flow cytometry,and the osteogenic differentiation and adipogenic differentiation were used to identify the multipotency differentiation of human umbilical cord mesenchymal stem cell,demonstrating that umbilical cord mesenchymal stem cell had been obtained successfully.2.Influence of Wnt/β-catenin signal transduction pathway on hUC-MSCs’hepatocyte-like differentiation by adding activator and inhibitor of Wnt in culture medium.The hUC-MSCs of passage 4 to 6 were divided into four groups as:Control group(DMEM culture group),the hepatocyte-like differentiation group,the activator Wnt3a group(adding activator Wnt3a in differentiation conditional medium),and the inhibitor Dkk-1 group(adding inhibitor Dkk-1 in differentiation conditional medium).Cells were collected respectively on day 7,14,21,28 and their mRNA expressions of AFP,ALB,HNF4α,CK-19,TAT,and key molecule of Wnt/β-catenin signaling pathway,like Wnt1,β-catenin,and GSK-3β were detected by real time quantitative PCR.Western blot was used to detect the protein expressions of AFP,HNF4α,CK-19,Wnt1,β-catenin,GSK-3β.Meanwhile,Periodic Acid-Schiff(PAS)staining,low density lipoprotein uptake experiments and indocyanine green absorption test experiment were applied to detect the function of hepatocyte-like cells on day 28.Results:1.hUC-MSCs were isolated using tissue adherent method.After one week culture,they were adherent and showed a fibroblast-like spindle-shape form.The result of flow cytometry analysis showed that they had a positive expression of CD105(99.3%±0.57%),CD90(99.5%±0.73%)and oocyte stem cell marker Oct4(47.160±1.32%)and they had a negative expression of hematopoietic stem cell markers,CD34 and CD45.This result was consistent with the properties of MSCs.After the cells were cultured in adipogenic conditional medium during 21 days,fat granules could be observed in cytoplasm,which could be stained into red by Oil Red O.Meanwhile,after 28 days culture in osteogenic conditional medium,calcium depositions could be found and stained as red compact nodules with Alizarin Red.These results demonstrated that we had isolated successfully MSCs from Wharton’s jelly of umbilical cord.2.Compared with control group,the mRNA of ALB and TAT,and the mRNA,as well as protein,expressions of AFP,HNF4a were significantly up-regulated(P<0.05)in all other three groups(the hepatocyte-like differentiation group,the activator Wnt3a group,and the inhibitor Dkk-1 group).Whereas,there was a down-regulation of CK-19 expression in mRNA and protein(P<0.01).Compared with the hepatocyte-like differentiation group,the mRNA and protein expressions of AFP,HNF4a,and the expression of ALB mRNA were down-regulated in activator Wnt3a group(P<0.05).This result was proved by the protein expression of AFP and HNF4a.The results also showed that the mRNA and protein expressions of Wntl andβ-catenin were up-regulated(P<0.05),which are higher than the hepatocyte-like differentiation and inhibitor Dkk-1 groups.Meanwhile,the mRNA and protein expression of GSK-3β were not significantly up regulated in activator Wnt3a group.Whereas,There was no significant difference of TAT mRNA expressions between the activator Wnt3a group and hepatocyte-like differentiation group.In comparison with the hepatocyte-like differentiation and activator Wnt3a groups,The inhibitor Dkk-1 group has the highest mRNA expressions of AFP,HNF4a,ALB and TAT(P<0.05)and the highest protein expressions of AFP and HNF4α(P<0.05).The mRNA and protein expressions of Wnt1 and β-catenin were down-regulated with the culture time(P<0.05),Their expressions were lower than the hepatocyte-like differentiation and activator Wnt3a groups at days 28.Only the expressions of GSK-3β mRNA and protein were up-regulated(P<0.05)in Dkk-1 inhibitor group than the hepatocyte-like differentiation and activator Wnt3a groups.It is shown by PAS staining,LDL uptake test and ICG absorption test that there were more positive induced cells in inhibitor Dkk-1 group than in differentiation group and the least positive cells were found in Wnt3a group.Conclusion:1.Tissue adherent method could isolated and purified hUC-MSCs successfully.2.Wnt/β-catenin signaling pathway has effect on hUC-MSCs’ differentiation into hepatocyte-like cells.Activation of Wnt/β-catenin signaling pathway promotes the differentiation of hUC-MSCs into Hepatocyte-like cells,conversely,it inhibits the differentiation of hUC-MSCs into Hepatocyte-like cells...