RESUMO
Hematopoietic stem cell transplantation (HSCT) can replace endogenous microglia with circulation-derived macrophages but has high mortality. To mitigate the risks of HSCT and expand the potential for microglia replacement, we engineered an inhibitor-resistant CSF1R that enables robust microglia replacement. A glycine to alanine substitution at position 795 of human CSF1R (G795A) confers resistance to multiple CSF1R inhibitors, including PLX3397 and PLX5622. Biochemical and cell-based assays show no discernable gain or loss of function. G795A- but not wildtype-CSF1R expressing macrophages efficiently engraft the brain of PLX3397-treated mice and persist after cessation of inhibitor treatment. To gauge translational potential, we CRISPR engineered human-induced pluripotent stem cell-derived microglia (iMG) to express G795A. Xenotransplantation studies demonstrate that G795A-iMG exhibit nearly identical gene expression to wildtype iMG, respond to inflammatory stimuli, and progressively expand in the presence of PLX3397, replacing endogenous microglia to fully occupy the brain. In sum, we engineered a human CSF1R variant that enables nontoxic, cell type, and tissue-specific replacement of microglia.
Assuntos
Microglia , Engenharia de Proteínas , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos , Animais , Humanos , Camundongos , Aminopiridinas/farmacologia , Encéfalo/metabolismo , Microglia/metabolismo , Engenharia de Proteínas/métodos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Terapia Baseada em Transplante de Células e Tecidos/métodosRESUMO
We present a high-resolution miniature, light-weight fluorescence microscope with electrowetting lens and onboard CMOS for high resolution volumetric imaging and structured illumination for rejection of out-of-focus and scattered light. The miniature microscope (SIMscope3D) delivers structured light using a coherent fiber bundle to obtain optical sectioning with an axial resolution of 18 µm. Volumetric imaging of eGFP labeled cells in fixed mouse brain tissue at depths up to 260 µm is demonstrated. The functionality of SIMscope3D to provide background free 3D imaging is shown by recording time series of microglia dynamics in awake mice at depths up to 120 µm in the brain.
