Influence of Wnt/beta-catenin signal transduction pathway on the differentiation of umbilical cord mesenchymal stem cells into hepatocyte-like cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Umbilical cord mesenchymal stem cells can be induced to differentiate into hepatocyte-like cells in vitro and in vivo.However,the exact mechanism is still unknown. Existing studies have shown that the Wnt/β-catenin signaling pathway is closely related to this process. OBJECTIVE: To explore the effect of Wnt/β-catenin signaling pathway on the differentiation of umbilical cord mesenchymal stem cells into hepatocyte-like cells and its potential molecular mechanism. METHODS: Human umbilical cord mesenchymal stem cells were extracted from the neonatal umbilical cord by tissue adherent method. After being cultured and purified, the umbilical cord mesenchymal stem cells at passages 4-6 were divided into four groups control group (DMEM culture group), hepatocyte-like differentiation group, activator Wnt3a group (adding 20 μg/L Wnt3a, an activator of Wnt/β-catenin signaling pathway, under the differentiation condition), and inhibitor Dkk-1 group (adding 20 μg/L Dkk-1, an inhibitor of Wnt/β-catenin signaling pathway, under the differentiation condition). Induced cells were collected respectively on days 7, 14, 21, 28. Their mRNA and protein expressions of α-fetoprotein (AFP), albumin (ALB), hepatocyte nuclear factor 4α (HNF4α) and Cytokeratin-19 (CK-19) in the cells were detected by real-time quantitative PCR and western blot respectively. Meanwhile, Periodic Acid-Schiff staining, low-density lipoprotein uptake test and indocyanine green absorption test were applied to detect the function of hepatocyte-like cells. RESULTS AND CONCLUSION: Compared with the control group, expressions of AFP and HNF4α mRNA and protein as well as ALB mRNA were significantly up-regulated in the hepatocyte-like differentiation group, activator Wnt3a group and inhibitor Dkk-1 group (P < 0.05). Whereas, there was a decrease in the CK-19 expression at mRNA and protein levels (P < 0.01) in these three groups. Compared with the hepatocyte-like differentiation group, the mRNA and protein expressions of AFP and HNF4α, and the mRNA expression of ALB were significantly down-regulated in the activator Wnt3a group (P < 0.05). Compared with hepatocyte-like differentiation group and activator Wnt3a group, the inhibitor Dkk-1 group had higher expression of AFP, HNF4α mRNA and their proteins as well as the mRNA expression of ALB (P <0.05). Findings from the Periodic Acid-Schiff staining, low-density lipoprotein uptake test and indocyanine green absorption test showed more positive cells in the inhibitor Dkk-1 group than in the hepatocyte-like differentiation group and least positive cells in the activator Wnt3a group. Overall, these findings suggest that the inhibition of Wnt/β-catenin signaling pathway promotes the differentiation of umbilical cord mesenchymal stem cells into hepatocyte-like cells;conversely,the cell differentiation can be inhibited via the Wnt/β-catenin pathway.