A new method for obtaining bankable and expandable adult-like microglial cells

Abstract Background The emerging role of microglia in neurological disorders requires a novel method for obtaining massive amounts of adult microglia. We aim to develop a new method for obtaining bankable and expandable adult-like microglial cells.Methods The head neuroepithelial layer (NEL) that composed of microglial progenitor and neuroepithelial cells at mouse E13.5 was dissected and then cultured or banked. CD11b-positive cells (NEL-MG) were isolated from the cultured NEL by magnetic-activated cell sorting system (MACS).Results The NEL included microglia progenitors that proliferate and ramify over time with neuroepithelial cells as feeder. Functional validation with a MACS using the NEL showed that the NEL-MG exhibited microglial functions, such as phagocytosis (microbeads, amyloid β, synaptosome), migration, and inflammatory changes following lipopolysaccharide (LPS) stimulation. NEL was subcultured and the NEL-MG exhibited a higher expression of microglia signature genes than the neonatal microglia, a widely used in vitro surrogate. Banking or long-term subculture of NEL did not affect NEL-MG characteristics. Transcriptome analysis revealed that NEL-MG exhibited better conservation of microglia signature genes with a closer fidelity to freshly isolated adult microglia than neonatal microglia. NEL-MG could be re-expandable when they were plated again on neuroepithelial cellsConclusions This new method effectively contributes to obtaining adult-like microglial cells, even when only a small number of experimental animals are available, leading to a broad application in neuroscience-associated fields.