Cancer stem cells (CSCs) are considered to be tumor‑initiating cells, responsible for
tumor invasive growth and dissemination to distant organ sites. Typically,
radiation treatment and
chemotherapy should target CSCs. However, current research investigating CSCs is impeded by the difficulty of isolating pure CSCs and maintaining them in vitro. In the present study, the synergistic inhibition of
glycogen synthase kinase 3 and mitogen‑activated
protein kinase kinase using small molecules,
CHIR99021 and
PD184352, efficiently generated CSCs from immortalized human mammary epithelial cells (HMLEs) and resulted in the acquisition of mesenchymal traits and the expression of epithelial‑mesenchymal transition markers. The cell proliferation, invasion and migration of HMLE cells were significantly promoted by
CHIR99021 and
PD184352 (P<0.05). Furthermore, the cell cycle was shifted from the G0/G1 phase to the G2/M phase, and the apoptotic rate was suppressed in HMLE cells following treatment with
CHIR99021 and
PD184352. Compared with control group, the stimulated cells exhibited an increased ability to form mammospheres and regenerate a
tumor. In addition to these properties, the induced cells also exhibited notable
chemotherapy resistance. In vivo, the treatment of cells with
CHIR99021 and
PD184352 promoted the growth of HMLE‑engrafted
tumor types. These results provide a practical strategy for the generation of CSCs using small molecules in vitro, which provides a cell resource that may be used for
drug screening. Additionally, the present results additionally highlighted the synergistic functions of Wnt and mitogen‑activated
protein kinase kinase signaling pathways in
tumorigenesis.