The use of bioartificial livers (BALs) for the expansion of human adult liver stem cells and the production of growth factors could be a potential strategy for cell-based extracorporeal liver support. The present study aimed to assessing the differentiation of human adult liver stem cells in a rotary BAL. Liver stem cells were seeded into a polysulphone membrane filter at a density of 3 x 10(8) cells, and the filter was connected to a rotary bioreactor perfusion system (37 degrees C, 50 mL/min, 48 h). Viability, cell differentiation, and metabolic performances were evaluated at 24 and 48 h. Hepatocyte growth factor production from human adult liver stem cells, mature hepatocytes, and mesenchymal stem cells in adhesion and in the rotary BAL conditions was compared. Liver stem cells cultured in the rotary BAL produced the highest amounts of albumin (p = 0.002) and ammonia-induced urea (p = 0.0001), and had an increased cytochrome P450 expression in respect to liver stem cells in adhesion. Remarkably, liver stem cells in the rotary BAL produced very high amounts of hepatocyte growth factor (p = 0.005) in respect to hepatocytes and mesenchymal stem cells. Moreover, the cells lost their stem cell markers and acquired several markers of mature hepatocytes. In conclusion, the rotary BAL favored liver stem cell differentiation into mature hepatocyte-like cells.