exogenous addition of pMGLs. GFP-labeled pMGLs, which had adopted rounded to first-order ramified morphologies when cultured on plastic, integrated into the three-dimensional cultures, tiled the volume (Fig. 6c), and projected highly branched ramifications (Fig. 6d). The 3D culture matrix is a cortex-like mesh of MAP2 positive neurites and GFAP positive astrocytic branches (Fig. 6e). Live Imaging of the GFP positive pMGL branches in situ showed rapid extension and retraction of filopodial arbor termini (Fig. 6f, g and Supplementary Movies 6, 7). These results suggest that pMGLs can integrate into organotypic neural cultures, and mature into what is currently defined as resting yet dynamically motile microglia. We note that this magnitude of extension and retraction is not observed in 2D cultures on Primaria or glass coverslips, where non-amoeboid pMGLs only display terminal ruffle movement, on a much smaller scale.
