Cd200

iHPCs and iPS-microglia were differentiated according to the protocol published by McQuade et al. (McQuade et al., 2018 (link)). To begin iHPC differentiation, iPSCs were passaged in mTeSR-E8 to achieve a density of 80 colonies of 100 cells each per 35mm well. On day 0, cells were transferred to Medium A from the STEMdiff™ Hematopoietic Kit (Stem Cell Technologies). On day 3, flattened endothelial cell colonies were exposed to Medium B and cells remained in medium B for 7 additional days while iHPCs began to lift off the colonies. On day 10, non-adherent CD43+ iHPCs were collected by removing medium and cells with a serological pipette. At this point, d10-d11 iHPCs can be frozen in Bambanker (Wako). Cells used for early-postnatal iHPC transplantation were thawed in iPS-Microglia medium (DMEM/F12, 2X insulin-transferrin-selenite, 2X B27, 0.5X N2, 1X glutamax, 1X non-essential amino acids, 400 μM monothioglycerol, and 5 μg/mL human insulin freshly supplemented with 100ng/mL IL-34, 50ng/mL TGFβ1, and 25 ng/mL M-CSF (Peprotech)) and allowed to recover for 24 hours, then resuspended at 62,500 cells/uL in 1X DPBS (low Ca²⁺, low Mg²⁺). Cells utilized for in vitro experiments continued microglial differentiation for 28 days. During the last 3 days in culture, 100ng/mL CD200 (Novoprotein) and 100 ng/mL CX3CL1 (Peprotech) were added to mature microglia in a brain-like environment.

iPSC-microglia were generated as described (McQuade et al., 2018 (link)). Briefly, iPSCs were directed down a hematopoietic lineage using the STEMdiff Hematopoesis kit (StemCell Technologies). After 10–12 days in culture, CD43⁺ hematopoteic progenitor cells are transferred into a microglia differentiation medium containing DMEM/F12, 2× insulin-transferrin-selenite, 2× B27, 0.5× N2, 1× glutamax, 1× non-essential amino acids, 400 µM monothioglycerol, and 5 µg/mL human insulin. Media was added to cultures every other day and supplemented with 100 ng/mL IL-34, 50 ng/mL TGF-β1, and 25 ng/mL M-CSF (Peprotech) for 28 days. In the final 3 days of differentiation 100 ng/mL CD200 (Novoprotein) and 100 ng/mL CX3CL1 (Peprotech) were added to culture.

iMGLs were differentiated according to a previously published protocol (55 (link)). Briefly, iPSCs were differentiated to hematopoietic progenitor cells using the STEMdiff Hematopoietic kit for 10 to 15 days before passage into microglia differentiation medium including DMEM/F12, 2× insulin transferrin-selenite, 2× B27, 0.5× N2, 1× glutamax, 1× nonessential amino acids, 400 μM monothioglycerol, and human insulin (5 μg/ml). Cultures were maintained in this basal medium supplemented with interleukin-34 (100 ng/ml), transforming growth factor–β1 (TGF-β1; 50 ng/ml), and macrophage colony-stimulating factor (25 ng/ml) (PeproTech) for 28 days. For the last 3 days in culture, two additional cytokines were added [CD200 (100 ng/ml, Novoprotein) and CX3CL1 (100 ng/ml, PeproTech)] to mature the microglia in a homeostatic brain-like environment.