Mouse stem cell factor

CD105⁺ Sca-1⁺ enriched bone marrow culture was initiated with 1 × 10⁶ cells per well of a 6 well plate. When cells reached confluence they were cultured in a 10 cm plate until harvest at day 14. Cells were maintained in StemSpan^TM media (StemSpan serum-free expansion medium, Stemcell Technologies, Vancouver, BC, Canada) supplemented with recombinant human fibroblast growth factor 1 (10 ng ml⁻¹), mouse thrombopoietin (50 ng ml⁻¹), mouse insulin growth factor II (20 ng ml⁻¹) and mouse stem cell factor (20 ng ml⁻¹); (R&D Systems, Minneapolis, MN). Cells were incubated at 37°C. Fourteen days later, cells were collected for flow cytometric analysis of EGFP and genomic DNA isolation for shuttle vector rescue.

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.