Stempro osteogenesis differentiation media

Osteogenic differentiation: MSCs grown on 24 wells plate (TPP, Switzerland) for up to 80% of confluence. Then growth media was replaced with StemPro™ Osteogenesis Differentiation media (Gibco, Thermo Fisher Scientific, USA). MSCs were refeed every 3–4 days. After 21 days of osteogenic differentiation induction MSCs were stained with Alizarin Red S to stain calcium deposits, marker of mature osteoblasts [38 (link)].
Chondrogenic differentiation: MSCs grown on 24 wells plate (TPP, Switzerland) for up to 80% of confluence. Then growth media was replaced with MSCgo™ Chondrogenic Differentiation Medium (Sartorius, Germany). MSCs were refeed every 3–4 days. After 21 days of chondrogenic differentiation induction MSCs were stained with Alcian Blue to stain acidic polysaccharides [39 (link)].
Adipogenic differentiation: MSCs grown on 24 wells plate (TPP, Switzerland) for up to 80% of confluence. Then growth media was replaced with MesenCult™ Adipogenic Differentiation Kit (Human) (STEMCELL Technologies Inc., Canada). MSCs were refeed every 3–4 days. After 21 days of adipogenic differentiation induction MSCs were stained with Oil Red O to lipid drops [40 (link)].

Transfected human adipose-derived stem cells were cultured according to the manufacturer’s instructions in MesenPRO RS™ Medium in the absence, or presence, of ferric quinate at a final concentration of 34 mM. For assessment of specific lineage differentiation capacity mms6 transfected MSCs were cultured to confluence, after which the medium was replaced with specific differentiation media including StemPro^® Osteogenesis Differentiation media (Gibco), StemPro^® Chondrogenesis Differentiation medium (Gibco) and StemPro^® Adipogenesis Differentiation medium (Gibco) according to the manufacturer’s instructions. The MG63 osteosarcoma cell line was used in initial gene transfection and expression studies (see Supplementary Fig. 1).

The ADSCs were seeded into 6-well plates (5 × 10⁴ cells/ well). When cell confluency was approximately 80–90%, the culture media was replaced with Stempro osteogenesis differentiation media (Gibco, Grand Island, NY, USA) and cultured for 20 days. During osteogenic induction, the ADSCs were primed with 1 or 5 ng/mL of FGF2 (R&D systems, Minneapolis, MN, USA) or 10 or 50 ng/mL HGF (R&D systems, Minneapolis, MN, USA) or their combination for 1, 3, or 6 days after osteogenic induction. After completion of priming of FGF2 and/or HGF, the ADSCs were maintained in osteogenesis differentiation media for 20 days. On the 20th day after osteogenic induction, cells were fixed with 3.7% formaldehyde (Sigma-Aldrich, ST. Louis, MO, USA) and stained with 2% Alizarin red S solution (Sigma-Aldrich, ST. Louis, MO, USA) for 10 min to visualize calcium deposition. Alizarin red S was eluted using 10% cetylpyridinium chloride solution (Sigma-Aldrich, ST. Louis, MO, USA), and calcium deposition was quantified as the absorbance value at a wavelength of 560 nm (Molecular Devices, Sunnyvale, CA, USA).