Osteogenic differentiation of sub-populations after > 8 pds. Osteogenic differentiation was induced using osteogenic induction medium, control cells were cultured in MSC growth medium (MSCGM). Three weeks after induction gene expression of the osteogenic markers, the transcription factor core-binding factor subunit alpha-1 (CBFA1) and alkaline phosphatase (ALP) were analyzed. Expression relative to GAPDH is presented for each individual culture (top). ALP activity was determined in the cell lysates at the end of the culture and was normalized to the corresponding DNA content in the well. In parallel, mineralization was visualized by Alizarin Red staining (bar = 200 μm). Representative pictures of four induced cultures are shown. None of the control cultures was positive. *: significant difference (p < 0.05) between all control cultures compared to all cultures under induction conditions. (For mean expression values see Additional file 1: Table S6).