Differentiation of iPSCs into Microglia

Human iPSCs (Thermo Fisher #A18945) were maintained in mTeSR1 medium (StemCell, #85857) and routinely passaged as clumps onto Geltrex (Thermo Fisher, #A1413301)-coated plates. The differentiation protocol used was established previously^{24 (link)}. Briefly, iPSCs were first differentiated into hematopoietic progenitor cells (HPCs) using the STEMdiff Hematopoietic Kit (StemCell, #05310). HPCs that were positive for identity markers CD34, CD43, and CD45 were transferred to plates containing primary human astrocytes and co-cultured in Media C adapted from Pandya et al.^{53 (link)} for 14–21 days, during which time HPCs were progressively removed. The media included IMDM (Thermo Fisher), FBS, PenStrep, 20 ng/mL each of IL3, GM-CSF, M-CSF (Peprotech) . Once non-adherent cells in co-culture were predominantly mature microglia (> 80%), the microglia were collected and plated for subsequent analysis.