Fig 1: Human iPSC-microglia and blood-derived monocytes fully engraft within the brains of adult hFIRE mice but adopt differing morphologies.(a) Microglia arise from primitive hematopoietic stem cells within the yolk sac, whereas monocytes arise later in development from bone marrow derived definitive hematopoietic stem and progenitor cells. Lineage tracing studies have shown that very few monocytes infiltrate the healthy brain, but increased numbers migrate into the brain in response to injury, irradiation-induced loss of microglia, or neurodegeneration. (b) Schematic depicting transplantation paradigm of 2-month-old xenotolerant FIRE mice with iPSC-microglia (Mg) or human CD14+ monocytes (Mo) derived from 4 male subjects then sacrificed at 5.5-months of age. (c-d) Brain wide human (Ku80, human-specific nuclei, white) xenografted iMG (xMG) and monocytes (IBA1, green), scale bar: 500μm. (e-f) Quantification of xenografted microglia and monocytes in half brain coronal sections of xenotolerant hFIRE mice; p values from unpaired, two-sided t test. Half brain (Bregma AP −1.94): t(16)= 0.6068; ns=0.5525. Forebrain (Bregma AP +0.14): t(16)= 0.01849; ns=0.9855. (g-h) Representative confocal microscopy combined with IMARIS-based branching analysis of xenografted microglia and monocytes, scale bar: 20μm. (i-j) Quantification of xenografted microglia and monocytes branch complexity within the cortex and hippocamps; p values from unpaired, two-sided t test (Cortex: t(16)= 8.087; ****P<0.0001) (Hippocampus: t(16)=3.446; **P=0.0033). Data represented as average mean value ± SEM (Mg, n = 7; Mo, n = 11). ns, not significant; **p < 0.01, and ****p < 0.0001.
Fig 2: Proteomic analysis reveals increased levels of many proinflammatory proteins in monocyte engrafted brains.(a)Schematic depicting frozen hemibrain tissue and protein processing for TMT Mass Spec Analysis (n=8 mice per group, engrafted with n=4 Monocyte or Microglia lines). (b) Heatmap showing log2-transformed intensities of the top 70 differentially expressed proteins (DEPs) between microglia and monocytes engrafted brains (n=8 mice/group). (c) Expression levels of P2RY12, LGALS3, CD14, GPNMB, CX3CR1, CD74, ITGAX, OAS2, ACY3, CTSD, MX1, CD9, APOE, IFIT3, BIN1, and CSF1R protein. (d) Principal component analysis reveals microglia or monocyte identity as the primary source of variation between xenografted brains. (e) Representative immunostaining for LGALS3 positive (blue) microglia or monocytes (IBA1, green) in the cortex of xenografted mice; scale bar: 40μm. (f-g) Quantification of LGALS3 within the cortex and hippocampus, p values from unpaired, two-sided t test (Cortex t(16)= 7.568; ****P<0.0001) (Hippocampus: t(16)=4.571; ***P=0.0003). (h-k) Representative immunostaining for human nuclei (Ku80, white), P2RY12 (red), GPNMB (purple), HLA-DR (orange), and CD9 (pastel red) positive microglia or monocytes (IBA1, green) in the cortex of xenografted mice; scale bar: 40μm. Quantification of P2RY12 (I,m), GPNMB (n,o), HLA-DR (p,q), and CD9 (r,s) immunoreactivity in the cortex and hippocampus of xenografted mice, p values from unpaired, two-sided t test (P2RY12 Cortex: t(16)= 20.83; ****P<0.0001) (P2RY12 Hippocampus t(16)= 9.605; ****P<0.0001) (GPNMB Cortex: t(16)= 2.904; *P=0.0104) (GPNMB Hippocampus t(16)= 2.424; *P=0.0276) (HLA-DR Cortex: t(16)= 7.592; ****P<0.0001) (HLA-DR Hippocampus t(16)= 6.299; ****P<0.0001) (CD9 Cortex: t(16)= 5.448 ****P<0.0001) (CD9 Hippocampus t(16)= 4.079; ***P=0.009). Data represented as average mean value ± SEM. Immunohistological analysis examined n=7 microglia and n=11 monocyte engrafted mice. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
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