Macrophage Differentiation from Stem Cells

The PSC were differentiated into macrophages according to protocol as described previously by our group (Han et al., 2019 (link)). hESC H9 (WiCell) was maintained on a Matrigel-coated 35-mm dish using mTeSR 1 (STEMCELL), and the medium was replenished every day. ThehiPSC line CMC-hiPSC-003 was obtained from Korea National Stem Cell Bank (Kim et al., 2021 (link)) and maintained on a vitronectin-coated 35 mm dish using E8 media (STEMCELL). When colonies of both H9 or CMC-hiPSC-003 grew to approximately 500 μm in diameter, cellular differentiation was induced using mesoderm differentiation medium (APEL 2 supplemented with 1X insulin-transferrin-selenium-X [Invitrogen] and 100 ng/ml BMP4). After 2 days, 100 ng/ml BMP4 was replaced with 20 ng/ml BMP4 and incubated for 2 days. On day 4, BMP4 was replaced with 40 ng/ml VEGF and 50 ng/ml SCF. Two days later, the medium was replaced with hematopoietic differentiation medium (APEL 2 supplemented with 1X insulin-transferrin-selenium-X [Invitrogen], 50 ng/ml SCF, 10 ng/ml TPO, 50 ng/ml IL-3, 50 ng/ml IL-6, and 50 ng/ml Flt-3L). On day 15, floating cells were harvested and incubated in macrophage differentiation medium (RPMI 1640 supplemented with 100 ng/ml M-SCF).