Mouse iPSC-derived microglia replacement as a platform for elucidating microglia biology and therapeutic potential

Microglia, the primary immune cells of the central nervous system, are essential for maintaining brain homeostasis. Their dysregulation is implicated in numerous neurodegenerative and neuroinflammatory disorders, making them important therapeutic targets. Induced pluripotent stem cells (iPSCs) oMer a promising platform for generating microglia progenitors capable of replacing dysfunctional cells. However, optimizing diMerentiation protocols to produce in vivo engrafted microglia that closely resemble their embryonic counterparts remains challenging. Previous eMorts have primarily focused on generating human iPSC-derived microglia for use in immune-deficient mouse models. However, syngeneic mouse studies remain important due to species-specific ligand-receptor incompatibilities in xenotransplantation, which may fail to fully capture the cellular interactions and signalling pathways that shape microglial activation. Additionally, the importance of adaptive immunity in brain diseases, including neurodegenerative conditions, underscores the need for studies in immune-competent models. Therefore, developing optimized mouse iPSC-derived microglia replacement paradigms represents a powerful tool for elucidating microglia biology and disease mechanisms in vivo.
Here, we describe the development of an in vitro diMerentiation protocol for generating large numbers of mouse iPSC-derived
microglia progenitors capable of engrafting the mouse brain. After depleting endogenous microglia through a combination of genetic and pharmacological approaches, these progenitors were transplanted into the brains of neonatal mice. Our optimized protocol successfully generated mouse iPSC-derived microglia progenitors that eMiciently engrafted the mouse brain and exhibited phenotypic characteristics similar to embryonic microglia, as demonstrated by their highly ramified morphology and expression of key markers, including P2RY12, TMEM119, and IBA1. This refined in vitro and in vivo protocol sets the stage for advancing our understanding of microglia biology and developing proof-of-concept studies for microglia replacement therapies to treat brain disease.