Dickkopf Wnt signaling pathway inhibitor 1 regulates the differentiation of mouse embryonic stem cells in vitro and in vivo
Embryonic stem cells (ESCs) are pluripotent stem cells derived from early stage embryos. It remains unclear whether inhibiting the Wnt/β-catenin signaling pathway using dickkopf Wnt signaling pathway inhibitor 1 (DKK1) impacts on the differentiation potential of mouse ESCs in vitro and in vivo. In the present study, immunohistochemical staining was used to measure the expression of markers of the three germ layers in ESCs and teratomas derived from ESCs. The expression of markers for the Wnt/β-catenin signaling pathway were detected by reverse transcription-polymerase chain reaction (RT-qPCR). Immunohistochemistry and western blotting indicated that the expression levels of octamer-binding transcription factor 4 in the DKK1-treated ESC group were significantly greater compared with the control ESCs. Reduced expression levels of NeuroD and bone morphogenetic protein 4 were observed in the DKK1-treated ESCs and teratomas derived from DKK1-treated ESCs compared with the control group. Increased expression levels of SOX17 were observed in the DKK1-treated ESCs compared with the control group. RT-qPCR indicated that β-catenin expression was significantly reduced in DKK1-treated ESCs and teratomas derived from DKK1-treated ESCs compared with the control groups. Western blotting indicated no alterations in the expression of GSK-3β, however, the levels of phosphorylated-GSK-3β were significantly greater in the DKK1 treatment groups, while cyclin D1 and c-Myc expression levels were significantly reduced in the DKK1 treatment groups compared with the control groups. These results suggest that inhibiting Wnt signaling in ESCs using DKK1 may promote mouse ESCs to differentiate into endoderm in vitro and in vivo, and suppress the tumorigenicity of ESCs.Five C57BL/6 mice used to establish ESC lines were provided by the Experimental Animal Center of Chongqing Medical University (Chongqing, China). The Balb/c mice (10 male, 10 female; HFK Bioscience Co., Ltd., Beijing, China) were kept under standard conditions and sacrificed at 6–8 weeks old and 18–25 g. This study was approved by the ethics committee of Chongqing Medical University (Chongqing, China).Isolated murine ESCs in the control group exhibited morphological features characteristic of ESCs: Active proliferation, closely connected cells, “nest” growth, clear colony edges with smooth surfaces, dense structure and clear boundaries between the layers of cells (Fig. 1A). The colony of ESCs in the DKK1-treated group appeared to be clearer and more rounded compared with those in the control group (Fig. 1A).The current study was supported by a project grant from the National Basic Research Program of China (973 program; grant no. 2011CB707900).