Stepwise Differentiation of hPSCs into Hematopoietic Cells

Stepwise direct HSC differentiation was performed as previously described^{9 (link),11 (link)}. Undifferentiated colonies of hPSCs were prepared at a density of <5 colonies per well. When the colonies grew to ~500 µm in diameter, they were cultured in hematopoietic differentiation medium (HDM) containing Stemline II serum-free medium (Sigma), Insulin-Transferrin-Selenium (Gibco) and BMP4 (20 and 100 ng/mL) for the first 2 days. The colonies were incubated with HDM containing 20 ng/mL BMP4 for 2 days, followed by treatment with 40 ng/mL vascular endothelial growth factor and 50 ng/mL stem cell factor (SCF) for 2 days. On day 6, the cultures were given fresh HDM supplemented with hematopoietic cocktail (50 ng/mL SCF, 10 ng/mL thrombopoietin, 50 ng/mL interleukin-3 (IL-3), 50 ng/mL granulocyte colony-stimulating factor and 50 ng/mL Feline McDonough Sarcoma (FMS)-like tyrosine kinase 3 ligand (all from R&D Systems)) and cultured for 13 days. The medium was changed every 3 days. The induction efficiency of hPSCs into the hematopoietic lineage was assessed by measuring the frequencies of hemogenic precursors (CD45-CD31+), hematopoietic progenitors (CD34+CD45+ or CD34+CD43+) and mature blood cells (CD34-CD45+ and CD34-CD43+) by flow cytometry.