Osteoblast differentiation

To induce osteoblast differentiation, iPSCs were plated (in colony pieces) at a density of 7×10⁴ cells per well of a 24-well OsteoAssay surface coated plate (Corning, Wiesbaden, Germany) in iPSC media. The following day, the media was replaced with osteogenic media [DMEM/F12, supplemented with: 15% FCS, 2 mM L-glutamine, 1% non-essential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol (all Invitrogen), 10 mM β-glycerophosphate, 10 nM dexamethasone and 28 µM ascorbic acid (all Sigma-Aldrich)]. Cells were cultured for 21 days with the media replaced every 2–3 days prior to analysis.

To determine matrix mineralisation, von Kossa staining (Abcam, Cambridge, UK) was carried out according to the manufacturer's instructions. Alizarin Red S staining for calcium deposition was also performed by incubating differentiated iPSCs with 2% Alizarin Red S pH 4.2 for 5 min. Hydroxyapatite deposition was detected using the OsteoImage bone mineralisation assay (Lonza, Slough, UK) according to the manufacturer's instructions. Alkaline phosphatase activity was measured using a quantitative colorimetric test on cell culture supernatant (Abcam) according to the manufacturer's instructions.