Granulocyte colony stimulating factor

All cell lines were grown at 37°C under 5% CO₂. RPE1 and derived cell lines were grown in DMEM/F12 (Sigma-Aldrich) supplemented with 10% FBS (Biochrom), 2 mM l-glutamine (Thermo Fisher Scientific), and 0.348% sodium bicarbonate (Sigma-Aldrich). HEK293T and GP2-293 cells were cultured in DMEM with high glucose supplemented with 10% FBS. MCF10A, MCF10A, and MCF10AT1 cell lines were maintained in DMEM/F12 supplemented with 0.1 µg/ml cholera toxin, 10 µg/ml insulin, 0.5 µg/ml hydrocortisone, 0.02 µg/ml EGF, and 5% horse serum. Primary CD34⁺ cells (HSPCs) were provided by the Department of Internal Medicine, University Hospital Heidelberg (Heidelberg, Germany). Primary HSPCs were cultured in Stemline II Hematopoietic Cell Expansion Medium (Sigma-Aldrich), supplemented with l-glutamine (2 mmol/liter; Thermo Fisher Scientific), thrombopoietin (100 ng/ml; R&D Systems), stem cell factor (100 ng/ml; R&D Systems), granulocyte–colony-stimulating factor (100 µg/ml; R&D Systems), and Flt-3 Ligand (500 ng/ml; R&D Systems). HSPCs were expanded for 4 d after isolation on retronectin-coated dishes (T100B Recombinant Human Fibronectin Fragment; Takara Bio). The coating was performed by incubating the dish for 2 h at 37°C with retronectin at a final concentration of 20 µg/ml in PBS, followed by washing in PBS.

Hematopoietic differentiation was performed as previously described (Hong et al., 2011 (link)). Briefly, undifferentiated hPSC colonies were prepared with low density (approximately 5–7 colonies in a 35 mm culture dish). Cells were grown to 1 mm size, media were changed to Stemline II serum-free medium (Sigma) supplemented with Insulin-Transferrin-Selenium and BMP4 (20 ng/ml) for 4 days, followed by treatment with SCF (50 ng/ml) and VEGF (40 ng/ml) for 2 days. On day 6, the cultures were given fresh hematopoietic induction medium supplemented with hGFs cocktail (50 ng/ml SCF, 10 ng/ml Thrombopoietin, 50 ng/ml Interleukin-3, 50 ng/ml FMS-like tyrosine kinase 3 ligand and 50 ng/ml Granulocyte colony-stimulating factor, R&D Systems) with and without 10 μM Im and cultured for 11 days. The hematopoietic differentiation was assessed by the frequencies of CD34⁺CD45⁺ and CD34⁻CD45⁺ populations on day 17.