HB-EGF preserves multilineage stem cell potential. MSCs were cultured for one week in the presence of CM without and with 50 ng/mL HB-EGF, which was added 2 hours before CM and left for the entire culture. Then both groups were cultured for 2 weeks with AM or OM, without HB-EGF and CM. MSCs treated with CM without HB-EGF underwent differentiation into chondrocytes, as suggested by morphology and Toluidine blue (A), while MSCs cultured with HB-EGF and CM did not (B). Adipogenic and osteogenic differentiation were not achieved with untreated MSCs (C,E), but only with HB-EGF–pretreated MSCs (D,F). The presence of HB-EGF in the preliminary culture with CM not only prevented MSC differentiation into chondrocytes, but also preserved their multilineage stem cell potential. The figure shows a representative case of 3 different experiments. Image visualization, acquisition, and processing were performed as for Figure 6.