neural co-cultures enhance the microglial signature of pMGLs(a) top: schematic representation of transwell culture system exposing pMGLs to conditioned medium from differentiating neuro-glial cultures. Bottom: schematic representation of direct re-aggregation after GFP transduction, as free-floating spheroids or 3D stacks in transwells. (b) Principal component analysis comparing RNAseq profiles from differentiated neural cultures (full squares, NPC1 in red, NPC2 in blue, NPC3 in green) to those of primary fetal microglia (fMG1 and fMG2, open triangles. fMG1+ NCM, closed triangle), and pMGLs (circles, pMGL1 in red, pMGL2 in blue, and pMGL3 in green) in the absence (open circles) or presence (full circles) of neural progenitor conditioned medium (+NCM). N=1 for each sequencing data point. (c) phase contrast (left) and fluorescence (right) images depicting the relative tiling position of GFP-labeled pMGLs in 3d culture on transwell. Scale bar: 200 μm. (d) representative image of a GFP-positive pMGL (grey scale) after 4 weeks in 3D neural stacks (live observation). Scale bar: 10μm. (e): Optical section through a fixed 3D neuroglial culture (without embedded pMGLs), stained for DAPI (blue, top), neuronal MAP2 (green, middle) and astrocytic GFAP (magenta, bottom). Scale bar: 50μm. (f) Maximum projection of supplementary movie 5 (scale bar: 2μm) pointing to rapidly extending (yellow arrowhead) and retracting (red arrowhead) protrusions in the 3D neuroglial cultures. (g) 1/5 frames of supplementary movies S5 and S6, showing branch movements over 300s. (h) Example montage of time-lapse images depicting GFP-labeled pMGLs (grayscale) response to localized cellular damage in 3D culture. Yellow arrowhead indicates the site of two-photon laser ablation after 5 minutes of acquisition. Red arrowheads point to microglia-like cells further away from injury, not reacting to the damage. Bottom right panel represent the color-coded individual trajectories of pMGLs during acquisition, highlighting radial migration towards the injury. Scale bar = 100μm.
