Journal: The Journal of Experimental Medicine
Article Title: Hematopoietic stem and progenitor cells are present in healthy gingiva tissue
doi: 10.1084/jem.20200737
Figure Lengend Snippet: A unique monocyte population is present in the gingiva. (A) Gating strategy to identify monocytes in the gingiva and GI tract (numbers indicate frequencies expressed as mean ± SEM). (B) Quantification of monocytes as a percentage of live CD45 + Lin − CD11b + Ly6C +/− CD64 +/− cells (excludes Ly6C − CD64 − cells) in the gingiva of C57BL/6 and Balb/c mice and GI tract and skin of C57BL/6 mice. n = 6–13 mice per group. Lin = CD3ε, TCR-β, CD19, B220, NK1.1, Ter119, Siglec F, and Ly6G. (C and D) Representative staining of gingiva tissue for Ly6G + , Ly6C bright , and F4/80 + cells. (C) Images show locations of cells staining positive for each marker. (D) Representative sections stained for Ly6G, Ly6C, and F4/80. Solid white line indicates edge of tooth. Scale bar = 200 µm. Staining from three separate experiments with n = 2–3 per experiment. (E) Representative histograms showing staining for CD44, CD68, CCR2, and CX3CR1 by gingival Ly6C hi monocytes (Mo). Data from seven experiments with two to three mice per experiment. FMO, fluorescence minus one. (F) Cytospins of sorted gingival Ly6C hi monocytes and macrophages (Mϕ) stained with H&E. Scale bar = 10 µm. Images are representative of two independent experiments. (G) Representative FACS plots of sorted BM and gingiva monocytes that were cultured with M-CSF for 7 d and analyzed by FACS. Data from two independent experiments. (H) Monocytes were FACS purified from the blood, BM, gingiva, skin, and GI tract and analyzed by RNA-seq. n = 2–3 biological replicates per group. Heatmap of the expression profile of canonical monocyte and macrophage-associated genes. FPKM, fragments per kilobase per million mapped reads. (I) Representative FACS plots showing host- and donor-derived Ly6C hi monocytes in the blood and gingiva of head-shielded chimeras 20 wk after reconstitution. Numbers indicate percentage of cells in the gate. (J) Quantification of donor-derived (white bar) and host-derived (black bar) Ly6C hi gingival monocytes 12 and 20 wk after reconstitution in head-shielded chimeras. n = 6–11 mice per group from two to three experiments. (K) Chimerism of Ly6C hi monocytes in the lungs of torso-shielded chimeras (left) and GI tract of abdomen-shielded chimeras (right). The frequency of donor-derived Ly6C hi gingiva monocytes was normalized to that of blood Ly6C hi monocytes to determine percent chimerism. Data from one to two experiments with n = 3–9 mice per group. (L) Proportions of innate cells presented as the percentage of all CD45 + cells in the gingiva, GI tract, and skin. n = 3 from three independent experiments. Asterisks indicate significant differences compared with gingiva. (M) tSNE map of CD45 + Lin − Ly6G + gingival cells that were subjected to dimensional reduction based on Sca-1, cKit, CXCR2, CD45, Ly6G, CXCR4, MHCII, CD11b, Ly6C, CD11c, CD101, and Lin. Identified subpopulations are highlighted in the tSNE plot. Data representative of two experiments with n = 2–3 mice. Data are presented as mean ± SEM. Statistical comparisons were performed using a one-way ANOVA with a post hoc Tukey’s test (B) and a two-way ANOVA with a Tukey’s (L) and Holm–Šídák post hoc test (J); ****, P < 0.0001; **, P < 0.01; *, P < 0.05.
Article Snippet: Where appropriate, dimensional reduction was performed using the t-distributed stochastic neighbor embedding (tSNE) algorithm in FlowJo.
Techniques: Staining, Marker, Fluorescence, Cell Culture, Purification, RNA Sequencing Assay, Expressing, Derivative Assay
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