Hematopoietic Differentiation of hPSCs

hPSCs were maintained and passaged on Matrigel in mTeSR1 media (WiCell). Hematopoietic differentiation was performed on collagen IV (ColIV)-coated plates in chemically defined serum-free medium as described.¹⁴ (link) The iSOX18 H1 line from hPSCs (H1 hESC line from WiCell) was maintained and passaged on Matrigel in mTeSR1 media (WiCell). The cell lines were differentiated on a collagen IV (ColIV)-coated plate.¹⁴ (link) To initiate differentiation, cells were plated at 5,000 cells/cm² onto 6 well plates with E8 media and 10 μM Rock inhibitor (Y-27632, Cayman Chemicals). This media was changed the following day to IF9S media with 50 ng/mL FGF2 (PeproTech), 50 ng/mL BMP4 (PeproTech), 15 ng/mL Activin A (PeproTech), and 2 mM LiCl (Sigma), and cells were cultured in hypoxia (5% CO₂, 5% O₂). On day 2, the media was changed to IF9S media with 50 ng/mL FGF2, 50 ng/mL VEGF (PeproTech), and 2.5 μM TGF-β inhibitor (SB-431542, Cayman), and cells were cultured in hypoxia (5% CO₂, 5% O₂). On days 4 and 6, the media was changed to IF9S media with 50 ng/mL FGF2, 50 ng/mL VEGF, 50 ng/mL TPO (PeproTech), 50 ng/mL IL-6 (PeproTech), 20 ng/mL SCF (PeproTech), and 10 ng/mL IL-3 (PeproTech), and cells were cultured in normoxia (20% CO₂, 5% O₂).