Differential CSF1R signaling enables widespread integration of genetically unmodified human microglia into the mouse brain

Microglia are long-lived myeloid cells in the central nervous system that are implicated in many neurological diseases. The differentiation of pluripotent stem cells provides an opportunity to develop in vitro human cellular models carrying disease gene mutations complementing existing animal models of disease. Microglia are particularly sensitive to their cellular environment and can adopt a variety of reactive states depending on different pathological conditions which may be difficult to mimic in vitro. Therefore, to best investigate human microglia in vivo, it would be helpful to generate mice whose endogenous microglia are exchanged with human cells without the need of genetic manipulation of donor cells which could alter microglia function. Colony stimulating factor 1 receptor (CSF1R) signaling is critical for microglial survival in mice, and humans with CSF1R mutations are born with fewer microglia. We made the surprising discovery that transplanted human pluripotent stem cell-derived microglia (hMG) survive pharmacological CSF1R inhibition unlike endogenous mouse microglia. Cellular assays confirmed that CSF1R signaling is necessary for human microglia survival and revealed differential CSF1R signaling with species-specific ligands. Moreover, receptor ligands and small molecule inhibitors acted in a competitive fashion. Based on these insights, we found that transient CSF1R inhibition after cell transplantation led to near-complete and wide-spread repopulation of hMGs into the mouse brain of immunodeficient mice with humanized CSF1 ligand. This approach allows the facile generation of mice whose brain microglia are replaced with genetically unmodified human cells.