A fundamental characteristic of microglia is the surveillance of the CNS environment with their highly ramified processes. To investigate how iMGLs might interact within a human brain environment, iMGLs were cultured with hiPSC 3D brain-organoids (BORGs). BORGs include neurons, astrocytes, and oligodendrocytes that self-organize into a cortical-like network, but lack microglia (Figure 6). To test if iMGLs invade BORGs similarly to how microglia enter the developing neural tube (Chan et al., 2007; Rezaie and Male, 1999), iMGLs were added to BORG cultures. By day three, iMGLs had embedded into the BORGS and were not detected in the media, suggesting rapid iMGL chemotaxis toward CNS cues (Figure 6A–C). By day 7, iMGLs (green) also tiled and extended varying degrees of ramified processes within the 3D organoid environment (Figure 6D–F). To determine whether iMGLs respond to neuronal injury, BORGS were pierced with a 25-gauge needle. After injury, iMGLs clustered near the injury site and adopted a more amoeboid morphology, resembling “activated” microglia found in injured or diseased brains (Kettenmann et al., 2011) (Figure 6G–I). Collectively, these data demonstrate that iMGLs can integrate within an in vitro 3D CNS environment, mature, ramify, and respond to injury similar to brain microglia.
