Femur bones were obtained from 4-to 6-week old germ-free pigs. BMCs were isolated by previously described methods [54, 55, 56] . Briefly, the tip of each bone was removed and the marrow was harvested by inserting a syringe needle into one end of the bone and flushing with Dulbecco's Modified Eagle's Medium (DMEM; Gibco). The bone marrow cells were filtered through a 70-mm nylon mesh filter (BD, Falcon, USA) and mononuclear cells were obtained by density gradient centrifugation over Ficoll-Hypaque (gradient density 1.077). Cells (1-5610 5 /cm 2 ) were plated in 25 cm 2 cell culture flasks in DMEM containing 10% fetal bovine serum, 2 mm L-glutamine, 1% antibiotic solution (Gibco, USA). Cultures were incubated at 37uC in a humidified atmosphere containing 95% air and 5% CO 2 . The non-adherent cells were removed after 72 h of culture. Individual epithelial colonies surrounded by mesenchymal cells were plucked and were expanded in vitro by culturing in DMEM media.