Human ES or iPS cells were grown in hES medium on feeder layers of murine embryonic fibroblasts. Following enzymatic passaging, uniform cell clusters, free of single cells, were used for initial embryoid body seeding. These clusters were re-suspended directly in NGD (without stepwise adaptation) containing 10ng/mL CSF1 and IL-34 (microglial differentiation medium, MGdM) in Corning ultra-low adherence plates. After one week, we observed the formation of two types of structures: dense neuralized spheroids appearing alongside embryoid bodies forming cystic structures, bound by a single cell layer (Fig. 1a). When plated at these early stages on poly-D-lysine, the neuralized EBs flattened into typical rosette-forming neuroepithelium, while the small cystic EBs flattened into cell lawns reminiscent of endothelial cells 18 (Supplementary Fig. 1b). Island whorls could be seen giving rise to three-dimensional clusters of round cells at their borders, organizing themselves in ropes (Fig. 1b). Proliferation and growth of these structures after attachment is CSF1R-ligand dependent, as they arrested and devolved in absence of CSF1 and IL-34 (not shown).
