Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Continuous intraosseous administration of SCS prevents glucocorticoid-induced bone degeneration. ( A ) Schematic illustration of the glucocorticoid (GC; MPS)-induced bone deterioration and intraosseous SCS treatment. ( B-D ) Representative H&E staining images of the femur at 6 weeks (B). Magnified views of the cortical bone and trabecular bone in the marrow cavity are shown on the right. Solid arrows indicate normal osteocytes, while hollow arrows indicate empty osteocyte lacunae. Quantification of empty lacunae ratios in cortical bone (C) and trabecular bone (D). n = 6 biological replicates. (Scale bars, 500 μm and 25 μm) ( E-H ) Representative immunofluorescence staining of OPN + mature osteoblasts, osteolectin + osteoprogenitors, and VE-cadherin + endothelial cells (ECs) in femur at 6 weeks (E), and corresponding quantifications (F–H). n = 6 biological replicates. (Scale bars, 100 μm and 20 μm) ( I and J ) Representative flow cytometry plots of capillary subtypes in the femur (I), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (J). n = 6 biological replicates. ( K and L ) Flow cytometry plots showing Sca-1 hi CD31 hi arteriolar ECs (K), and corresponding quantification (L). n = 6 biological replicates. ( M and N ) Representative micro-CT 3D images of the femur (M). Quantitative analysis of percent bone volume (BV/TV) (N). n = 6 biological replicates. (Scale bars, 1.5 mm, 600 μm and 545 μm) ( O and P ) ELISA analysis of VEGF (O) and PDGF-BB (P) levels in bone marrow supernatant and peripheral serum from PBS- and SCS-treated groups at week 6. n = 6 biological replicates. ( Q ) ELISA quantification of the osteogenic factor osteocalcin in peripheral serum at week 6. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, F, G, H, J, L, N, O, P and Q ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Staining, Immunofluorescence, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS attenuates full-blown bone marrow senescence during GC-induced skeletal degeneration. ( A ) Schematic illustration of the experimental design for assessing bone marrow senescence at 4 weeks after combined SCS and MPS treatment. ( B ) Representative images of SA-β-Gal–positive cells (green) in femur after MPS treatment. BM indicates bone marrow; TBM indicates trabecular bone matrix. (Scale bars, 100 μm and 25 μm) ( C – E ) Representative immunofluorescence images at week 4 showing Emcn + sinusoidal ECs, ALP + osteoblasts, and p16 + senescent cells (C), with corresponding quantification of Emcn + p16 + (D) and ALP + p16 + cells (E). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) ( F – H ) Flow cytometry analysis of CD45 − Ter119 − CD31 + arteriolar ECs in the femur after PBS or SCS treatment (F). Ki-67 + proliferative status was further analyzed within this population (G), and corresponding double-positive cell quantification is shown in (H). n = 6 biological replicates. ( I – K ) Representative flow cytometry plots of CD45 − Ter119 − CD31 − leptin receptor + (LepR + ) mesenchymal stem cells (MSCs) in the bone marrow at 4 weeks (I), with analysis of the proportion of SA-β-Gal–positive cells (J) and corresponding quantification (K). n = 6 biological replicates. ( L ) Representative flow cytometry plots of CD45 − Ter119 − CD144 + cells (including endothelial cells and endothelial progenitors) in the bone marrow at week 4 post-MPS treatment. ( M and N ) Gating and analysis of CD45 − Ter119 − CD144 + HMGB1 + ECs by flow cytometry (M), and corresponding quantification (N). n = 6 biological replicates. ( O and P ) Representative immunofluorescence images showing OPN + osteoblasts and γ-H2A.X + DNA damage marker–positive cells in the femur at 4 weeks (O), with quantification of senescent osteoblasts (P). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) Data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Statistical significance was determined using an unpaired two-tailed Student's t -test ( D, E, H, K, N and P ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Immunofluorescence, Flow Cytometry, Marker, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS suppresses senescence cascade amplification by attenuating secondary spread from GC-induced primary senescent adipocytes. ( A ) Schematic illustration of SCS intervention exclusively during the fully developed senescent phase of MPS-induced bone marrow. ( B ) qPCR analysis of senescence-associated markers ( Cdkn1b , Cdkn1a , and Cdkn2c ) in bone tissues at 4 weeks following combined SCS and MPS treatment. n = 3 biological replicates. ( C ) ELISA analysis of bone marrow senescence-associated factors (IL-1β, IL-18, TNF-α, IL-6, CXCL1, and CCL3) after 4 weeks of combined treatment with SCS and MPS. n = 4 biological replicates. ( D ) Quantification of the maximal compressive load of the isolated distal femur and femoral diaphysis. n = 6 biological replicates. ( E ) Schematic diagram depicting isolation of bone marrow adipocytes from mice treated with SCS and MPS for 14 days using mature adipocyte-specific fast centrifugation and construction of a senescence propagation model in vitro . ( F and G ) Representative flow cytometry plots (D) and quantification (E) of EdU-positive (proliferating) CD45 − Ter119 − CD31 − LepR + MSCs cultured for 3 days with adipocyte conditioned medium (CM). n = 6 biological replicates. ( H and I ) Representative ALP staining images (F) and corresponding quantification of ALP activity (G) in CD45 − Ter119 − CD31 − LepR + MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 30 μm) ( J and K ) Representative Oil Red O staining (H) and quantification (I) of adipogenic differentiation in MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) ( L and M ) Representative images (J) and quantification (K) of crystal violet-stained fibroblast colony-forming units (CFU-F) in MSCs cultured with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 400 μm) ( N ) qPCR analysis of senescence-related markers ( Cdkn2a and Cdkn1a ) in MSCs treated with different adipocyte CMs. n = 3 biological replicates. ( O and P ) Representative immunofluorescence-FISH images (M) and quantification (N) showing colocalization of γ-H2A.X with telomere-associated foci (TAF) in MSCs cultured with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 7 μm and 1 μm) ( Q and R ) Representative images (O) and quantification (P) of 2D tube formation assays in HUVECs cultured for 3 days with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( S and T ) Representative images (Q) and quantification (R) of SA-β-Gal–positive HUVECs (green) following 3-day treatment with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( U ) qPCR analysis of the senescence-related gene LMNB1 in HUVECs treated with various adipocyte CMs. n = 3 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B, C, D, G, I, K, M, N, R, T and U ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Amplification, Enzyme-linked Immunosorbent Assay, Isolation, Centrifugation, In Vitro, Flow Cytometry, Cell Culture, Staining, Activity Assay, Immunofluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS reprograms the lineage commitment of MSCs after GC treatment and inhibits the generation of primary senescent adipocytes. ( A ) Schematic illustration of the in vitro investigation of SCS targeting the prostaglandin/PPARγ/INK positive feedback loop in MPS-induced primary senescent adipocytes. ( B ) Representative flow cytometry plot showing p16 + senescent cells in adipocytes derived from bone marrow after 14 days of in vivo MPS induction and subsequently treated with SCS in vitro . ( C ) qPCR analysis of 12 senescence-associated markers in primary senescent adipocytes after in vitro SCS treatment. n = 3 biological replicates. ( D ) ELISA analysis of IL-1β levels in adipocyte supernatant following in vitro SCS treatment. n = 6 biological replicates. ( E ) ELISA analysis of secreted prostaglandins PGD2 and PGE2 in adipocytes under different treatment conditions. D-PBS: bone marrow adipocytes isolated from mice treated in vivo with the solvent control DMSO, followed by in vitro treatment with PBS; M-PBS: bone marrow adipocytes isolated from mice treated in vivo with MPS, followed by in vitro treatment with PBS. M-SCS: bone marrow adipocytes isolated from mice treated in vivo with MPS, followed by in vitro treatment with SCS. ( F ) Western blot analysis of intracellular COX-2 protein levels in adipocytes across the three treatment conditions. ( G ) Schematic illustration of competitive osteogenic–adipogenic differentiation of CD45 − Ter119 − CD31 − LepR + MSCs after 7 days of in vivo SCS and MPS co-treatment. ( H ) qPCR analysis of pan-adipocyte markers ( Fabp4 , Adipoq , Plin1 , Cd36 , and Lep ) in CD45 − Ter119 − CD31 − LepR + MSCs after 14 days of in vitro competitive lineage differentiation. n = 3 biological replicates. ( I and J ) Representative immunofluorescence images (I) and quantification (J) of perilipin + adipocytes and osteopontin + mature osteoblasts derived from lineage-committed MSCs. n = 6 biological replicates. (Scale bars, 30 μm, 15 μm and 15 μm). ( K ) Western blot analysis of adipogenesis-related markers C/EBPα, PPARγ, and C/EBPβ in the lineage-mixed cells after in vitro competitive differentiation of CD45 − Ter119 − CD31 − LepR + MSCs. ( L ) qPCR analysis of lipogenesis-related markers Fasn , Scd1 , Srebf1 , Acaca , and Acacb . n = 3 biological replicates. ( M and N ) Representative H&E staining images (M) of the femurs at day 14 following SCS and MPS co-treatment. Yellow arrows indicate bone marrow adipocytes. Magnified images show hypertrophic adipocyte morphology, with quantification of adipocyte diameter (N). n = 19 biological replicates. (Scale bars, 200 μm, 50 μm and 20 μm). Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( C, D, H, J, L and N ), or one-way ANOVA with Tukey's post hoc test ( E ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: In Vitro, Flow Cytometry, Derivative Assay, In Vivo, Enzyme-linked Immunosorbent Assay, Isolation, Solvent, Control, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Gene expression profiles of bone marrow-derived LepR + MSCs after 7-day in vivo co-treatment with SCS and MPS. ( A ) Heatmap showing DEGs in CD45 − Ter119 − CD31 − LepR + MSCs sorted from bone marrow at day 7 post-treatment with SCS versus PBS ( P < 0.05, |log fold change| > 1.5). n = 3 biological replicates. ( B ) Representative GO biological process enrichment analysis of downregulated DEGs. ( C ) Top 20 enriched KEGG pathways of downregulated DEGs in SCS versus PBS. ( D ) GSEA plots of biological processes positively enriched in the SCS group (|NES| > 1, nominal P < 0.05, FDR <0.25). ( E ) Representative downregulated DEGs associated with adipogenesis and lipogenesis identified through KEGG pathway analysis. n = 3 biological replicates. ( F ) Top 20 enriched KEGG pathways of upregulated DEGs in SCS versus PBS. ( G ) Representative GO biological process enrichment analysis of upregulated DEGs. ( H ) Representative upregulated DEGs identified through biological process enrichment analysis. n = 3 biological replicates. ( I and J ) GSEA plots of KEGG pathways negatively enriched in the SCS group (|NES| > 1, nominal P < 0.05, FDR <0.25).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Gene Expression, Derivative Assay, In Vivo

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS targets downstream senescent lineage commitment of bone marrow MSCs to mitigate GC-induced bone deterioration. ( A ) Schematic diagram illustrating the experimental design: CD45 − Ter119 − CD31 − LepR + MSCs isolated from mice co-treated with SCS and MPS for 7 days were subjected to in vitro lineage-competitive differentiation, followed by DEX-induced senescence in lineage-mixed cells. These cells were then adoptively transplanted into healthy bone marrow cavity to assess bone deterioration development. ( B ) Representative H&E-stained images of the femur 12 weeks after adoptive transfer. PBS-DEX group: LepR + MSCs from PBS and MPS co-treated mice subjected to in vitro lineage differentiation and DEX-induced senescence, followed by transplantation. SCS-DEX group: LepR + MSCs from SCS and MPS co-treated mice processed similarly. PBS group: solvent control without cell transplantation. Solid arrows indicate intact osteocytes; hollow arrows indicate empty lacunae. (Scale bars, 250 μm and 25 μm) ( C – E ) Quantitative analysis of marrow hypertrophic adipocyte diameter (C), proportion of empty osteocyte lacunae in trabecular bone (D), and adipocyte number (E) in the metaphysis 12 weeks post-transplantation. n = 19 biological replicates (C), n = 6 biological replicates (D), n = 8 biological replicates (E). ( F ) Quantification of empty lacunae in epiphysis at 12 weeks post-transplantation. n = 6 biological replicates. ( G – I ) Representative flow cytometry plots of capillary ECs subtypes in the femur at 12 weeks (G), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (H) and CD45 − Ter119 − CD31 lo Emcn lo ECs (I). n = 6 biological replicates. ( J and K ) Representative flow cytometry plots (J) and corresponding quantification (K) of CD45 − Ter119 − Sca-1 hi CD31 hi arteriolar ECs in the femur at 12 weeks post-transplantation. n = 6 biological replicates. ( L ) Representative micro-CT images of the femur at 12 weeks post-transplantation across different treatment groups. (Scale bars, 1.5 mm and 500 μm) ( M – P ) Quantitative analysis of bone parameters in the metaphysis: bone mineral density (BMD) (M), percent bone volume (BV/TV) (N), trabecular separation (Tb.Sp) (O), and trabecular number (Tb.N) (P). n = 6 biological replicates. ( Q ) Serum ELISA analysis of the osteogenic marker osteocalcin at 12 weeks post-transplantation. n = 6 biological replicates. ( R and S ) ELISA analysis of PDGF-BB (R) and VEGF (S) in both bone marrow supernatant and peripheral serum at 12 weeks post-transplantation. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, E, F, H, I, K, M, N, O, P, Q, R and S ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Isolation, In Vitro, Staining, Adoptive Transfer Assay, Transplantation Assay, Solvent, Control, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay, Marker

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS modulates mesenchymal stem cell lineage bias via activation of the IGF-1/PI3K/Akt/mTOR signaling pathway. ( A ) Quantitative analysis of osteocyte morphology in the trabecular bone matrix of the bone marrow at week 6 after MPS treatment with or without SCS, in the presence of various neutralizing antibodies (NAbs) and antagonistic proteins. ( B ) ELISA analysis of IGF-1 and BMP-2 levels in the femoral bone marrow and peripheral serum at day 7 following SCS treatment under MPS conditions. ( C and D ) Western blot analysis of phospho-PI3K, phospho-Akt, and phospho-mTOR (C), as well as phospho-Smad1/5/8, phospho-ERK, and phospho-p38 (D), in CD45 − Ter119 − CD31 − LepR + MSCs after 15-min stimulation with conditioned medium (CM) derived from bone marrow fluid at day 7 following SCS treatment. ( E – G ) Representative flow cytometry plots (E, F) and quantitative analysis (G) of CD45 − CD31 − Sca-1 + CD24 − adipocyte progenitor cells (APCs), CD45 − CD31 − Sca-1 + CD24 + MSCs (E), and CD45 − CD31 − Sca-1 − PDGFRα + (Pα + ) osteoprogenitor cells (OPCs) (F) from femoral bone marrow at day 14 post-MPS induction with or without combined treatment using SCS and IGF-1 NAb or Noggin. ( H and I ) Representative SA-β-Gal staining images (green) of the femur (H), and corresponding quantification (I), at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. Insets show magnified views of bone marrow (BM) and trabecular bone matrix (TBM) regions. (Scale bars, 100 μm and 25 μm) ( J ) qPCR analysis of 12 senescence-associated markers in ex vivo femoral bone tissues at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. ( K ) Representative Oil Red O staining images of CD45 − Ter119 − CD31 − LepR + MSCs sorted from femurs at day 7 following MPS treatment with SCS in combination with LY294002 or LDN-193189, after in vitro adipogenic induction. (Scale bars, 50 μm and 25 μm) ( L and M ) γ-H2A.X and telomere-associated DNA damage foci (TAFs) co-localization analysis (L), and corresponding quantification (M), in CD45 − Ter119 − CD31 + arteriolar ECs sorted from femurs at day 28 following MPS treatment with SCS in combination with rapamycin or LDN-193189, using immuno-FISH staining. (Scale bars, 7 μm and 1 μm) ( N and O ) Sequential fluorescent labeling using calcein (N) and quantification of mineral apposition rate (O) in femurs treated with SCS and MPS for 4 weeks, with or without LY294002 and/or GW9662. (Scale bars, 50 μm) ( P ) ELISA analysis of five senescence-associated cytokines in femoral bone marrow at day 28 following MPS treatment with SCS in combination with rapamycin and/or T0070907. ( Q and R ) Representative t-distributed stochastic neighbor embedding (t-SNE) plots (Q) from flow cytometric analysis of CD45 − CD31 − Sca-1 + CD24 − APCs, CD45 − CD31 − Sca-1 + CD24 + MSCs, CD45 − CD31 − Sca-1 − Pα + OPCs, CD45 − Ter119 − CD31 + arteriolar ECs, and CD45 − Ter119 − Emcn + sinusoidal ECs at day 14 following MPS treatment with SCS in combination with IGF-1 and/or rosiglitazone, and quantitative analysis of APCs (R) ( S ) Heatmap showing the fluorescent intensity distribution of Lamin-B1 expression across five cellular subpopulations as identified in the t-SNE clustering plot. ∗ P < 0.05 vs. IgG (empty lacunae); # P < 0.05 vs. IgG (filled lacunae). ∗ P < 0.05 vs. SCS; # P < 0.05 vs. SCS + IGF-1 NAb. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B ), or one-way ANOVA with Tukey's post hoc test ( A, G, I, J, O, P and R ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Flow Cytometry, Staining, Ex Vivo, In Vitro, Labeling, Expressing, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Sensing of metabolic signals via GPR183 promotes occupation of lung macrophage niches by monocytes

doi: 10.1084/jem.20252667

Figure Lengend Snippet: Lung monocytes express GPR183 and migrate toward 7α,25-dihydroxycholesterol. (A) Gpr183 -GFP expression by the indicated mouse lung myeloid cells. Green and grey histograms show Gpr183 -GFP expression of lung cells from Gpr183 GFP/+ and Gpr183 +/+ mice, respectively. Gating strategy for monocytes, neutrophils, and eosinophils is shown in . Dendritic cells were gated as live CD45 + lineage (CD3/CD19/NK1.1) − CD64 − Ly6G - CD11c + MHCII + single cells. Data are representative of greater than or equal to three independent experiments with a total of n ≥ 6 mice per genotype. (B) Gpr183 -GFP expression by lung lymphocytes from Gpr183 GFP/+ (green histograms) and Gpr183 +/+ mice (grey histograms). After gating on live CD45 + F4/80 − single cells, B cells were gated as B220 + CD3 − cells, CD4 T cells as B220 - CD3 + TCRβ + CD4 + cells, and CD8 T cells as B220 − CD3 + TCRβ + CD8α + cells. Data are representative of two independent experiments with a total of n = 6 mice per genotype. (C) Chemotaxis of mouse bone marrow (BM) monocytes toward 7α,25-dihydroxycholesterol (7α,25-OHC). Left panel shows the transwell migration of bone marrow monocytes from B6 mice to the indicated concentrations of 7α,25-dihydroxycholesterol. Right panel shows the transwell migration of bone marrow monocytes from Gpr183 +/+ and Gpr183 −/− mice to 7α,25-dihydroxycholesterol. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by one-way ANOVA with Tukey’s multiple comparison post hoc test. Data are pooled from two (left panel) or three (right panel) independent experiments. Each experiment was performed with cells from one mouse per genotype and with two technical replicates per condition (total n = 4–6).

Article Snippet: Single-cell suspensions were obtained after enzymatic digestion and non-hematopoietic CD45 − niche cells were isolated by MACS using CD45 MicroBeads (Miltenyi Biotec).

Techniques: Expressing, Chemotaxis Assay, Migration, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Sensing of metabolic signals via GPR183 promotes occupation of lung macrophage niches by monocytes

doi: 10.1084/jem.20252667

Figure Lengend Snippet: Gating strategy for immune cells in the mouse lung. (A) Gating strategy to identify lung myeloid cells in Gpr183 GFP/+ reporter mice (see and ). Lung cells were first gated on live single CD45 + lineage (CD3/CD19/NK1.1) − cells before separating myeloid cells into the indicated CD64 + macrophage and CD64 − cell populations as shown. Alveolar macrophages in BAL fluid were gated as CD64 + Ly6G − CD11c + SiglecF + cells as shown here for alveolar macrophages in the lung. AMs, alveolar macrophages; Eosino, eosinophils; FSC-A, forward scatter area; FSC-H, forward scatter height; FSC-W, forward scatter width; IMs, interstitial macrophages; PMNs, polymorphonuclear neutrophils; SSC-A, side scatter area. Data are representative of at least three independent experiments with a total of n = 5–6 mice. (B) Gating strategy to identify lung macrophage populations in chimeras 3 wk after bone marrow reconstitution (see and ). Cells were pre-gated as live single CD45 + lineage − CD64 + Ly6G − cells as in panel A and then separated into the different macrophage populations. Mono-mac, monocyte-macrophages. Data are representative of at least two independent experiments with a total of n = 4–13 mice. (C) Gating strategy to identify lung immune cells in the steady-state lung of Gpr183 +/+ and Gpr183 −/− mice and in the lung of bone marrow chimeras ( ; ; and ). After pre-gating on live single CD45 + lineage − CD64 − , CD45 + lineage − CD64 + , or CD45 + lineage − cells as in panel A, the indicated cell populations were gated as shown. Data are representative of at least two independent experiments with a total of n = 4–19 mice.

Article Snippet: Single-cell suspensions were obtained after enzymatic digestion and non-hematopoietic CD45 − niche cells were isolated by MACS using CD45 MicroBeads (Miltenyi Biotec).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Sensing of metabolic signals via GPR183 promotes occupation of lung macrophage niches by monocytes

doi: 10.1084/jem.20252667

Figure Lengend Snippet: Anatomical niche impacts GPR183 expression by lung macrophages. (A) Gpr183 -GFP expression by interstitial and alveolar macrophages from lung and BAL fluid of Gpr183 GFP/+ (green histograms) and Gpr183 +/+ mice (grey histograms), respectively. Gating strategy is shown in . Data are representative of three independent experiments with a total of n = 5–6 mice per genotype. (B) Gpr183 -GFP expression by subsets of interstitial lung macrophages from Gpr183 GFP/+ (green histograms) and Gpr183 +/+ mice (grey histograms). Gating strategy is shown in . Data are representative of three independent experiments with a total of n = 6 mice per genotype. (C) Number of lung monocytes and macrophages in Gpr183 +/+ and Gpr183 −/− mice. Gating strategy is shown in . Data are represented as mean ± SEM. ns, not significant by unpaired Student’s t test. Lung data are pooled from three independent experiments with a total of n = 8 mice per genotype. BAL data are pooled from two independent experiments with a total of n = 5 mice per genotype. (D) Generation of Gpr183 GFP/+ or Gpr183 GFP/GFP (CD45.2 + ) → B6 (CD45.1 + ) bone marrow chimeras. Gpr183 +/+ or Gpr183 +/− (CD45.2 + ) → B6 (CD45.1 + ) chimeras were used as negative controls. (E) Gpr183 -GFP expression by the indicated CD45.2 + monocyte–derived macrophage populations from the lung of Gpr183 GFP/GFP (CD45.2 + ) → B6 (CD45.1 + ) bone marrow chimeras (green histograms) 3 wk after bone marrow transfer. CD45.2 + macrophages from Gpr183 +/− (CD45.2 + ) → B6 (CD45.1 + ) bone marrow chimeras (grey histograms) were used as a control. Gating strategy is shown in . Data are representative of two independent bone marrow chimera experiments with a total of n = 4 mice per genotype. (F) Gpr183 -GFP expression by CD45.2 + monocyte–derived alveolar macrophages from the BAL fluid of Gpr183 GFP/+ (CD45.2 + ) → B6 (CD45.1 + ) bone marrow chimeras (green histograms) 5 and 12 wk after bone marrow transfer. Macrophages from Gpr183 +/+ (CD45.2 + ) → B6 (CD45.1 + ) bone marrow chimeras (grey histograms) were used as a control. Gating strategy is shown in . Data are representative of two independent bone marrow chimera experiments with a total of n = 2–4 mice per genotype and time point. (G) Experimental setup to generate bone marrow–derived macrophages in vitro . Bone marrow cells from Gpr183 GFP/+ or Gpr183 +/+ mice were cultured with either M-CSF or GM-CSF and TGFβ 1 to generate generic macrophages or alveolar macrophage-like cells, respectively. (H) Gpr183 -GFP expression by in vitro –generated macrophages from Gpr183 GFP/+ bone marrow cells (green histograms). Macrophages differentiated from Gpr183 +/+ bone marrow were used as a control (grey histograms). Gpr183 -GFP expression by Ly6C hi monocytes (bone marrow input) is shown on the left. Data are representative of two independent experiments with a total of n = 2 mice per genotype and two technical replicates per experiment. Panels D and G were adapted from Servier Medical Art.

Article Snippet: Single-cell suspensions were obtained after enzymatic digestion and non-hematopoietic CD45 − niche cells were isolated by MACS using CD45 MicroBeads (Miltenyi Biotec).

Techniques: Expressing, Derivative Assay, Control, In Vitro, Cell Culture, Generated

Journal: The Journal of Experimental Medicine

Article Title: Sensing of metabolic signals via GPR183 promotes occupation of lung macrophage niches by monocytes

doi: 10.1084/jem.20252667

Figure Lengend Snippet: Trajectory of monocyte-to-macrophage differentiation and GPR183 expression after experimental emptying of the alveolar niche. (A) Overview of SPAM deleter mice. In Siglecf Lox-DTR-Lox mice, SiglecF + alveolar macrophages and eosinophils express DTR. In Epx Cre Siglecf Lox-DTR-Lox mice (SPAM deleter mice), Epx Cre activity excises the floxed DTR cassette in eosinophils, resulting in alveolar macrophage–specific DTR expression. This allows specific depletion of alveolar macrophages in an inducible manner after DT administration. (B and C) Frequency of alveolar macrophages and other immune cells in the BAL fluid (BALF) of SPAM deleter mice after intratracheal administration of 100 pg DT as determined by flow cytometry. BAL cells in panel B were gated as live CD45 + single cells. Myeloid cells in panel C were gated by excluding CD11b − CD11c − cells and were further separated into alveolar macrophages (CD11c + SiglecF + ), CD64 + non-alveolar macrophages (CD11c − SiglecF − CD64 + ), and neutrophils (CD11c − SiglecF − CD11b + Ly6G + ). Nonmyeloid cells (CD11b − CD11c − ) were separated into T (CD3 + B220 − ) and B cells (CD3 - B220 + ). Alveolar macs, alveolar macrophages. Data are from a single experiment with n = 5–6 mice per time point. (D) Experimental setup for single-cell RNA sequencing of lung cells from SPAM deleter mice. Lungs were harvested at the indicated time points before and after alveolar macrophage depletion with 40 ng intratracheal DT. Purified CD45 − non-hematopoietic cells as well as resident CD45 + CD64 + and CD45 + CD64 − hematopoietic cells were used for single-cell RNA sequencing as described in the Materials and methods. Resident hematopoietic cells were isolated based on being protected from intravascular cell labeling after intravenous injection of anti-mouse CD45 antibody. AM, alveolar macrophages. (E) Combined UMAP of 117,715 lung cells from SPAM deleter mice at 0, 12, 24, and 48 h and on day 5, 8, and 14 after alveolar macrophage depletion with DT as determined by single-cell RNA sequencing. See for markers used to annotate the cell clusters. (F) UMAP showing monocyte and macrophage clusters (26,817 cells) in the lung of SPAM deleter mice before and after alveolar macrophage depletion with DT. See for markers used to annotate the monocyte-macrophage clusters. ( G) Gpr183 expression in the monocyte and macrophage clusters from panel F. IMs, interstitial macrophages; MoMac, monocyte-macrophages; MoAM or mono-AMs, monocyte-derived alveolar macrophages. Data in panels E–G are from one single-cell RNA-sequencing experiment with n = 4 mice per time point. Panels A and D were adapted from Servier Medical Art.

Article Snippet: Single-cell suspensions were obtained after enzymatic digestion and non-hematopoietic CD45 − niche cells were isolated by MACS using CD45 MicroBeads (Miltenyi Biotec).

Techniques: Expressing, Activity Assay, Flow Cytometry, Single Cell, RNA Sequencing, Purification, Isolation, Labeling, Injection, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Sensing of metabolic signals via GPR183 promotes occupation of lung macrophage niches by monocytes

doi: 10.1084/jem.20252667

Figure Lengend Snippet: GPR183 promotes the development of monocyte-derived macrophages in the lung. (A and B) Chimerism of monocytes, alveolar macrophages, interstitial macrophages, and other lung immune cells in competitive Gpr183 +/+ / Gpr183 −/− chimeras 9–13 wk after bone marrow transfer. Injected bone marrow input is shown in the upper left flow cytometry dot plot of panel A. Gating strategy is shown in . The ratio of the indicated Gpr183 +/+ / Gpr183 −/− cells in panel B was normalized to the bone marrow input of each independent chimera experiment. AMs, alveolar macrophages; IMs, interstitial macrophages; PMNs, polymorphonuclear neutrophils. Data are represented as mean ± SEM. ****P < 0.0001 by one-way ANOVA with Tukey’s multiple comparison post hoc test. Data are pooled from three independent bone marrow chimera experiments with a total of n = 17–19 mice. (C) Chimerism of Ly6C hi monocytes and alveolar macrophages in the lung of competitive Gpr183 +/+ / Gpr183 −/− chimeras 19–23 wk after bone marrow transfer. Gating strategy is shown in . The ratio of the indicated Gpr183 +/+ / Gpr183 −/− cells was normalized to the bone marrow input of each independent chimera experiment. Data are represented as mean ± SEM. ns, not significant; ****P < 0.0001 by one-way ANOVA with Tukey’s multiple comparison post hoc test. Data are pooled from two independent bone marrow chimera experiments with a total of n = 14–15 mice. (D) Number of CD45.2 + lung monocytes and macrophages in single-transfer Gpr183 +/+ (CD45.2 + ) → B6 (CD45.1 + ) and Gpr183 −/− (CD45.2 + ) → B6 (CD45.1 + ) chimeras 7–8 wk after bone marrow transfer. Gating strategy is shown in . Data are represented as mean ± SEM. ns, not significant by unpaired Student’s t test. Data are pooled from two independent experiments from a single bone marrow chimera experiment with a total of n = 4–5 mice per genotype. (E) Chimerism of Ly6C hi monocytes and macrophages in the indicated tissues of competitive Gpr183 +/+ / Gpr183 −/− chimeras 9–29 wk after bone marrow transfer. Macs, macrophages; monos, monocytes. Gating strategy is shown in . Data are represented as mean ± SEM. ns, not significant; ***P < 0.001; ****P < 0.0001 by one-way ANOVA with Tukey’s Multiple Comparison post hoc test (spleen, peritoneal cavity, and liver) or by unpaired Welch’s t test (brain). Data are pooled from one (peritoneal cavity), three (brain), four (liver), or five (spleen) independent bone marrow chimera experiments with a total of n = 6–32 mice per tissue.

Article Snippet: Single-cell suspensions were obtained after enzymatic digestion and non-hematopoietic CD45 − niche cells were isolated by MACS using CD45 MicroBeads (Miltenyi Biotec).

Techniques: Derivative Assay, Injection, Flow Cytometry, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Sensing of metabolic signals via GPR183 promotes occupation of lung macrophage niches by monocytes

doi: 10.1084/jem.20252667

Figure Lengend Snippet: Fibroblasts are the main source of GPR183 ligand-producing enzymes in the lung. (A) Dot plot showing expression of Ch25h , Cyp7b1 , and Hsd3b7 in the indicated non-hematopoietic single-cell RNA-sequencing clusters from SPAM deleter mice as in . AT1, alveolar type I cells; AT2, alveolar type II cells. See for markers used to annotate the cell clusters. (B) Time course of Ch25h , Cyp7b1 , and Hsd3b7 expression in the lung of SPAM deleter mice after DT-induced alveolar macrophage depletion as determined by single-cell RNA sequencing. (C) Feature plots showing Ch25h and Cyp7b1 expression in the SPAM deleter cell clusters from . (D) Time course of Ch25h and Cyp7b1 expression in the indicated SPAM deleter cell populations corresponding to the lung clusters in . Data in panels A–D are from one single-cell RNA-sequencing experiment with n = 4 mice per time point. (E) Experimental setup for single-cell RNA sequencing of lung niche cells from IM-DTR mice. CD45 − non-hematopoietic cells (fibroblasts, epithelial cells, and endothelial cells) were purified from the lungs of IM-DTR mice as described in the Materials and methods. IM, interstitial macrophage. (F) UMAP of lung niche cells at the indicated time points after interstitial lung macrophage depletion with 50 ng intraperitoneal DT. Markers used to annotate lung fibroblasts, epithelial cells, and endothelial cells are shown in . (G) Dot plot of Ch25h , Cyp7b1 , and Hsd3b7 expression in lung fibroblasts, epithelial cells, and endothelial cells from IM-DTR mice. (H) Time course of Ch25h , Cyp7b1 , and Hsd3b7 expression in lung fibroblasts after DT-induced interstitial macrophage depletion. Data in panels F–H are from one single-cell RNA-sequencing experiment with n = 3–4 mice per time point. Panel E was adapted from Servier Medical Art.

Article Snippet: Single-cell suspensions were obtained after enzymatic digestion and non-hematopoietic CD45 − niche cells were isolated by MACS using CD45 MicroBeads (Miltenyi Biotec).

Techniques: Expressing, Single Cell, RNA Sequencing, Purification

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Human OvCa) (A) Imaris 3D surface rendering of autofluorescence (cyan) and CD326 positive cells (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) with target plane in yellow. (C) Light sheet guided target plane selection representing CD326 positive cell (purple), CD45 positive cells (red), and CD3 positive cells (green). (D) DAPI overview image of selected tissue slice for 2D MACSima™ imaging. (E) Magnified merged six color multiparameter MICS image with CD45 (green), CD326 (cyan), FOLR1 (purple), Collagen III (red), Collagen IV (red), and CD31 (yellow). (F–L) Single staining MICS images (white) of DAPI (F), CD45 (G), CD326 (H), FOLR1 (I), Collagen III (J), Collagen IV (K), and CD31 (L) (gray) (see “Antibodies”). Scale bars: (A–F) 1 mm; (E) 250 μm; (F–L) 500 μm.

Article Snippet: CD45 antibody, anti-human, Vio R667, REAfinity , Miltenyi Biotec B.V. & Co. KG , Cat# 130-128-742 RRID: AB_2905022.

Techniques: Imaging, Comparison, Selection, Staining

Journal: STAR Protocols

Article Title: Protocol for generating megakaryocytes from patient induced pluripotent stem cells for disease modeling and compound screening

doi: 10.1016/j.xpro.2026.104393

Figure Lengend Snippet: tSNE analysis of megakaryocytes (A) Gating strategy post concatenation. Segregation of samples based on treatment function, gate for red blood cells (CD45 vs CD235), megakaryocytes (CD45 vs CD41/CD61) and subsequently gated for maturation marker CD42. (B) Selection of parameters for generating a tSNE plot. (C) Example of the two samples files generated into tSNE plot. (D) Automatic overlay of markers of interest in the panel. (E) Legend for changing colors. (F) Interpretation of results for the samples.

Article Snippet: Anti-human CD45 antibody (APC-vio 770); 1:50 , Miltenyi Biotec , CAT#130-110-773, RRID: AB_2905022.

Techniques: Marker, Selection, Generated