Igf 1

Osteoblasts were cultured in α-MEM containing ribonucleosides and deoxyribonucleosides (GlutaMAX, Sigma) and 10% FBS (Autogen Bioclear). Primary osteoblasts were isolated from calvariae of neonatal mice (P1-P7) as previously described [38 (link)] and seeded at a density of 5.000 cells/cm². For differentiation, ascorbic acid (50 μg/ml) and β-glycerolphosphate (10 mM) were added to the culture medium. Bone nodules were stained at differentiation day 21 using Alizarin Red (Sigma). For BrdU stainings, osteoblasts were cultured until 70% confluency and incubated with 10 µM BrdU (Roche) for 4 h, before fixation with 70% ethanol and staining with an anti-BrdU antibody according to the manufacturer’s instructions (Becton Dickinson). Rapamycin (Wyeth), EGF (Roche) and IGF-1 (Promega) were used in concentrations indicated in the respective figure legends.

ES cell-derived HPCs were derived according to the protocol described previously by Chan et al. [3 ]. Briefly, HOXB4-transduced mouse ES cells (HM1 cell line) originally derived from the 129SvJ mouse strain were grown on feeder-seeded gelatinized flasks in ES cell culture medium with 15% fetal calf serum, 1% penicillin/streptomycin cocktail (GIBCO), 0.1 mM l-glutamine, and 1000 U/ml leukemia inhibitory factor for the maintenance of pluripotency. The ES cells were then subjected to embryoid body (EB) formation. The EBs were trypsinized and dissociated into a single cell dispersion before being replated onto ultralow-attachment Petri dishes in defined medium containing StemPro34 base media plus nutrient supplement (Life Technologies/BRL) and various hematopoietic cytokines: mIL-3 (2 ng/ml), mouse stem cell factor (100 ng/ml; R&D Systems), mIL-6 (5 ng/ml), IGF-1 (40 ng/ml; Promega), Flt3-L (10 ng/ml), and dexamethasone (1 μM; Sigma-Aldrich). The cell cultures were kept in a hypoxic incubator containing 9% CO₂.