Differentiating iPSCs into Microglia

IPSCs were differentiated into microglia following an established protocol (McQuade et al., 2018 (link)). In brief, iPSCs were maintained in mTeSR Plus Kit (Stemcell Technologies # 05825) in 10 cm dishes. At DIV−1 of HPCs differentiation, 5- to 7-day-old iPSC cultures were then used to generate a cluster of differentiating 43 positive (CD43⁺) hematopoietic progenitor cells (HPCs) following a 12-day commercially available kit (STEMdiff Hematopoietic Kit; Stemcell Technologies # 05310). IPSCs were cleaned and 1/3 dish was gently dissociated with dispase (Stemcell Technologies # 07923) for 12–15 min at 37°C. IPSCs were then collected and spun down at 500 rpm for 1–2 min and resuspended in 2 mL of mTeSR Plus media with 20 μM ROCK inhibitor Y-27632 (Stemcell Technologies # 72304). Matrigel, hESC-Qualified Matrix (Corning # 354277), coated six-well plates were used to start the HPC differentiation. Using a 5-mL serological pipette, one drop or two drops of iPSCs were seeded per well into matrigel. Then, the next day, on DIV 0 of HPCs differentiation, wells with 80 small colonies per well were selected to start HPCs differentiation. IPSCs were fed following the manufacturer's instructions. On DIV 12 of HPCs differentiation, only the non-adherent HPCs were transferred to a new six-well plate to start microglia differentiation (DIV 12/0).