Human urine-derived stem cells (hUSCs) show multipotential differentiation ability and can differentiate into mesodermal cell lineages. Interstitial cells of Cajal-like cells (ICC-LCs) are crucial for the pace-making function of spontaneous contraction in the bladder. However, the mechanisms by which hUSCs generate ICC-LCs have not been elucidated. In this study, we developed a strategy for directional differentiation of hUSCs into ICC-LCs. hUSCs were transfected with lentiviral vectors encoding c-Kit, stem cell factor (SCF), hyperpolarization activated cyclic nucleotide gated potassium channel 4 (HCN4), and 5-azacytidine induced 2 (AZI2) genes, and the cells were cultured for an additional 7 days in specific medium. The expression of the surface marker c-Kit on ICC-LCs was determined at 7 days after transfection. hUSCs were successfully expanded and transfected with the four lentiviral vectors. hUSCs transfected with lentiviral-c-Kit, lentiviral-HCN4, and lentiviral-AZI2 showed higher expression of c-Kit 7 days after transfection, but only the lentiviral-HCN4-transfected cells showed morphological alterations in ICC-LCs. These cells also displayed visible HCN current amplitude and density. This approach may provide a new strategy for the treatment of underactive bladder. (C) 2019 Published by Elsevier Inc.