To test whether physical embedding and maturation of pMGLs in a 3D organotypic neuroglial environment would accentuate their branching patterns and allow us to observe their surveying behavior, we transduced immature pMGLs with a GFP lentivirus and re-aggregated the GFP-positive pMGLs with pre-differentiated neural cultures, at a stage where neurons have already become post-mitotic and gliogenesis is ongoing (>4weeks). The re-aggregates were either kept in spinning suspension cultures to maximize viability, or as 200μm-thick cultures in transwells, so as to avoid triggering pMGL activation from hypoxia of the surrounding tissue and allow live observation. In this context, an entire spheroid or cellular stack is self-assembling, laying down its own extracellular matrix without need for additional scaffold (Fig. 6a, lower panel). The neural progenitors progressively differentiated into neurons and macro-glia (astrocytes, oligodendrocytes), and would be devoid of any microglia without the 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
