Generation of Hematopoietic Progenitor Cells

Hematopoietic progenitor cells (HPCs) were generated as previously described [23 (link)]. Briefly, iPSCs were dissociated into single cells and quickly re-aggregated in Essential 8 Medium (Thermo Fisher Scientific) containing 80 ng/ml BMP4 (R&D SYSTEMS), 80 ng/ml VEGF (R&D SYSTEMS), 2 μM CHIR99021 and 30 μM Y-27632) (9000 cells/well) using a Nunclon Sphera Microplates 96U-Well Plate. Embryoid bodies were cultured at 37 °C, 5% CO₂ and 5% O₂ during differentiation. On day 2, the medium was removed, and Essential 6 Medium (Thermo Fisher Scientific) containing 80 ng/ml VEGF, 25 ng/ml bFGF (R&D SYSTEMS), 50 ng/ml SCF (R&D SYSTEMS) and 2 μM SB431542 (Tocris) was added. On day 4, the medium was replaced with Stemline® II Hematopoietic Stem Cell Expansion Medium (Sigma-Aldrich) containing 50 ng/ml SCF (R&D SYSTEMS), 20 ng/ml TPO (R&D SYSTEMS), 40 ng/ml VEGF and 50 ng/ml IL-3 (R&D SYSTEMS). HPCs were harvested by sorting with PE Mouse Anti-Human CD34 (BECKMAN COULTER, Brea, CA, USA #A07776) and FITC Mouse Anti-Human CD43 (Thermo Fisher Scientific #11-0439-42) at day 12 using BD FACSAria II (BD Biosciences).