Osteoblast and adipocyte differentiation of Ad-MSCs

To test the multipotential properties of Ad-MSCs, we cultured them in different conditioned media.²⁴ In brief, fourth generation Ad-MSCs were cultured in two six-well plates with MSCM (ScienCell). The medium was changed to mesenchymal stem cell adipogenic differentiation medium (MADM, ScienCell) and mesenchymal stem cell osteogenic differentiation medium (MODM, ScienCell) when the cells reached 70–80% confluency. During the 3 weeks of culture, MADM and the MODM were exchanged every 3 days. Finally, all cells were fixed with methanol (−20°C, 2 min) and stained with Oil Red O and Von Kossa in accordance with the manufacturer’s protocols (Abcam).²⁵ Stained cells were observed by inverted microscopy (Olympus).