α mem medium

h-iPSCs and h-MSCs (used as control) were seeded at 25.000 cells/cm² in 24-well cell-culture plasticware and were cultured in α-MEM medium (PAN Biotech; USA) containing 5 g/L glucose without any added serum in a humidified, 37 °C, 5% CO₂, 95% N₂ and 0.1% oxygen environment for 3 days. All experiments were performed in triplicate at three separate occasions.

For osteoblast cultures mesenchymal cells were isolated from the calvariae of 3–6 days old neonatal hTNF-α tg mice. In brief, calvariae were digested in α-MEM medium (PAN Biotech, Aidenbach, Germany) with 0.1% collagenase type IA (Sigma-Aldrich) and 0.2% dispase II (Roche, Basle, Switzerland) at 37°C on a shaker for 5 × 10 min. Fractions 2–5 were collected and cells were cultured and expanded until P2 to P3. At subconfluency state, cells were plated at 104/cells/cm². Mineralization assays were carried out in 12-well plate by changing the medium to osteoblast mineralization medium (PromoCell, Heidelberg, Germany) at 100% confluency and cells were irradiated 24 h after the first medium change. Mineralization media was used according to the manufacturer’s recommendation and formation of bone nodules was evaluated at d21 using Alizarin red stain (Millipore, Darmstadt, Germany). Total wells were scanned and images were then analyzed using ImageJ software (Version 1.46r).

For osteoblast cultures mesenchymal cells were isolated from the calvariae of 3–6 days old neonatal mice. In brief, calvariae were digested in α-MEM medium (PAN Biotech, Aidenbach, Germany) supplemented with 0.1% collagenase type IA (Sigma-Aldrich) and 0.2% dispase II (Roche, Basel, Switzerland) at 37 °C on a shaker for 5 × 10 min. Fractions 2–5 were collected and cells were cultured and expanded until P2 to P3. At subconfluency state, cells were plated at 1 × 10⁴ cells/cm². Mineralization assays were carried out in 12-well plates by changing the medium to osteoblast mineralization medium (PromoCell, Heidelberg, Germany) at 100% confluency and cells were irradiated 24 h after the first medium change. Mineralization media was used according to the manufacturer’s recommendation and formation of bone nodules was evaluated at day 21 using Alizarin red stain (Millipore, Darmstadt, Germany). For analyses, total wells were scanned and images then were analyzed using ImageJ software (Version 1.46r).