AIM: To isolate, culture and identify the human fetal pancreatic ductal stem cells in vitro, and to observe the potency of these multipotential cells differentiation into insulin-producing cells.METHODS: The human fetal pancreas was digested by 1 g/L collagease type Ⅳ and then 2.5 g/L trypsin was used to isolate the pancreatic ducta stem cells, followed by culture in serum-free, glucose-free DMEM media with some additional chemical substrates in vitro (according to the different Stage). The cells were induced by glucose-free (control),5 mmol/L, 17.8 mmol/L and 25 mmol/L glucose, respectively.The cell types of differentiated cells were identified using immunocytochemical staining.RESULTS: The shape of human fetal pancreatic ductal stem cells culturedin vitro was firstly fusiform in the first 2 wk,and became monolayer and cobblestone patt after another 3 to 4 wk. After induced and differentiated by the glucose of different concentrations for another 1 to 2 wk,the cells formed the pancreatic islet-like structures. The identification and potency of these cells were then identified by using the pancreatic ductal stem cell marker, cytokeratin-19 (CK-19), pancreatic β cell marker, insulin and pancreatic α cell marker, glucagons with immunocytochemical staining.At the end of the second week, 95.2% of the cells were positive for CK-19 immunoreactivity. Up to 22.7% of the cells induced by glucose were positive for insulin immunoreactivity, and less than 3.8% of the cells were positive for glucagon immunoreactivity in pancreatic isletlike structures. The positive ratio of immunoreactive staining was dependent on the concentration of glucose, and it was observed that the 17.8 mmol/L glucose stimulated effectively to produce insulin- and glucagons-producing cells.CONCLUSION: The human fetal pancreatic ductal stem cells are capable of proliferation in vitro. These cells have multidifferentiation potential and can be induced by glucose and differentiated into insulin-producing cells in vitro.