2.5. Differentiation of iPSCs towards monocytes and macrophages
iPSC-derived monocytes (iMCs) were harvested from embryoid bodies (EBs) according to van Wilgenburg et al (2013). For this, iPSCs were made single cell using Accutase and resuspended in mTeSR-1 medium supplemented with 50 ng ml-1 BMP4, 20 ng ml-1 SCF (Peprotech, 300–07), 50 ng ml-1 VEGF and
RevitaCell (1/100) (spin EB medium) at a concentration of 150 000 cells ml-1. Cells were seeded in 10 µl
per well in a 96 ultra-low attachment U-bottom plate (Thermo Fisher Scientific, 174929) and 100 µl of spin
EB medium was added to each well. The plate was centrifuged for 3 min at 100 g. From day 1–3, medium was changed every day with spin EB medium. At day 4 all EBs were resuspended in 20 ml of differentiation medium (X-VIVO 15 medium (Lonza, BE02-060 F), 100 ng ml-1 M-CSF (PeproTech, 300-25), 25 ng ml-1 IL-3 (PeproTech, 200-03), 2 mM L-Glutamine (Lonza, BE17-605E/U1), 100 U ml-1 PenStrep (Gibco, 15140122), 0.055 mM ß-mercaptoethanol (Gibco, 31350–010)) and cultured in 5 wells of a 6-
well plate with weekly medium changes. iMCs can be harvested from the supernatant of the EBs by filtering medium containing EBs through an invertiblestrainer (40 µM, Corning, 352340). Medium containing iMCs was centrifuged at 300 g for 5 min and resuspended in Microglia Medium (ScienCell, 1901). iMCs were seeded in 12-well plates with a density of 120 000 cells/well to transdifferentiate towards iMF. Medium was changed on day 3 and 5 after seeding in 2D and iMF were harvested on day 7.