Emerging evidence indicates that human mesenchymal stem cells (hMSCs) can be 
recruited to tumor sites, and affect the growth of human malignancies. However, 
little is known about the underlying molecular mechanisms. Here, we observed the 
effects of hMSCs on the human cholangiocarcinoma cell line, HCCC-9810, using an 
animal transplantation model, and conditioned media from human umbilical 
cord-derived mesenchymal stem cells (hUC-MSCs). Animal studies showed that 
hUC-MSCs can inhibit the growth of cholangiocarcinoma xenograft tumors. In cell 
culture, conditioned media from hUC-MSCs inhibited proliferation and induced 
apoptosis of tumor cells in a dose- and time-dependent manner. The proliferation 
inhibition rate increased from 6.21% to 49.86%, whereas the apoptosis rate 
increased from 9.3% to 48.1% when HCCC-9810 cells were cultured with 50% hUC-MSC 
conditioned media for 24 h. Immunoblot analysis showed that the expression of 
phosphor-PDK1 (Ser241), phosphor-Akt (Ser 437 and Thr308), phosphorylated 
glycogen synthase kinase 3β (phospho-GSK-3β(Ser9)), β-catenin, cyclin-D1, and 
c-myc were down-regulated. We further demonstrated that CHIR99021, a GSK-3β 
inhibitor reversed the suppressive effects of hUC-MSCs on HCCC-9810 cells and 
increased the expression of β-catenin. The GSK-3β activator, sodium 
nitroprusside dehydrate (SNP), augmented the anti-tumor effects of hUC-MSCs and 
decreased the expression of β-catenin. IGF-1 acted as an Akt activator, and also 
reversed the suppressive effects of hUC-MSCs on HCCC-9810 cells. All these 
results suggest that hUC-MSCs could inhibit the malignant phenotype of HCCC-9810 
human cholangiocarcinoma cell line. The cross-talk role of Wnt/β-catenin and 
PI3K/Akt signaling pathway, with GSK-3β as the key enzyme bridging these 
pathways, may contribute to the inhibition of cholangiocarcinoma cells by 
hUC-MSCs.