Colonies were treated with collagenase IV (1.5mg/mL) and mildly triturated to form a suspension of uniform clumps, transferred directly to 5mL MGdM (NGD + 10ng/mL IL-34 + 10ng/mL CSF1), in ultra-low attachment 6-well plates (corning). 6 confluent wells (~3×106 cells) were pooled into one suspension well. Embryoid bodies were monitored for appearance of two main identifiable types. The first group was composed of compact phase-bright neuralized spheroids, the second group were large, expanding cystic bodies, YS-EBs. Every 5 days, EBs were gently triturated to shear off loose cells of interest, settled, and the supernatant placed in a single well of a Primaria 6-well plate. Unattached cells and small EBs were washed with fresh MGdM. Attached cells were monitored for morphological characteristics of microglia/microglial precursors (compact nucleus, vacuoles, membrane ruffles, motility), and wells from 6 consecutive productions (one month) were pooled to constitute one synchronized population. Further maintenance was performed in MGM (NGD + 100ng/mL IL-34 + 5ng/mL CSF1). Cells are sensitive to passaging, but can be lifted with Accutase, or preferentially with ice-cold PBS with 5ng/mL CSF1. Assays and imaging were performed 4–5 days after passaging or feeding. pMGL staining displays cells derived from iPS-wt5.
