Journal: Frontiers in Molecular Biosciences

Article Title: Aged Callus Skeletal Stem/Progenitor Cells Contain an Inflammatory Osteogenic Population With Increased IRF and NF-κB Pathways and Reduced Osteogenic Potential

doi: 10.3389/fmolb.2022.806528

Figure Lengend Snippet: Callus SSPCs are composed of osteogenic, proliferating, and adipogenic clusters. Analysis was performed on clusters 1–3. Cluster 4 was excluded from further analysis due to possible contamination of B cells during cell harvest, which is described in . (A) Heatmap of the top 10 DEGs in clusters 1–3 showing cluster 1 expressed inflammatory and matrix related genes, cluster 2 expressed proliferating genes, and cluster 3 expressed a miscellany of inflammatory, stem cell-related, and oncogenes. (B) Violin plot of the expression level of surface markers, Cxcr2 and Ccr2, within clusters 1–3. (C) Isolation of clusters 1–3 using FACS sorting: Cluster 1: Dapi-TER119-CD45-CD31-CXCR2+CCR2-; Cluster 2: Dapi-TER119-CD45-CD31-CXCR2-CCR2-; Cluster 3: Dapi-TER119-CD45-CD31-CXCR2-CCR2+. (D) RT-qPCR of cluster 1–3 cells to assess their osteogenic, proliferative, and adipogenic capacity. Data represent mean ± SD. One-way ANOVA followed by Tukey post-hoc. * p < 0.05 for significant difference from any other clusters.

Article Snippet: CXCR2 low and CXCR2 high callus mesenchymal progenitor cells were purified using CXCR2 antibody (Biolegend cat#149304, clone SA044G4) by fluorescence-activated cell sorting (FACS) or magnetic beads sorting (Miltenyl cat#130-048-801).

Techniques: Expressing, Isolation, Quantitative RT-PCR

Journal: Frontiers in Molecular Biosciences

Article Title: Aged Callus Skeletal Stem/Progenitor Cells Contain an Inflammatory Osteogenic Population With Increased IRF and NF-κB Pathways and Reduced Osteogenic Potential

doi: 10.3389/fmolb.2022.806528

Figure Lengend Snippet: Validation of increased CXCR2 high cells in the callus of aged mice with increased IRF and NF-κB pathways and reduced osteogenic potential. The tibial fracture was performed on 4-month- (young) and 21-month-old (aged) C57BL/6J male mice and fracture callus was harvested 10 days after the procedure. (A) Immunostaining with anti-CXCR2 antibody in callus sections for CXCR2+ cells. Images show numerous CXCR2+ cells (arrows) localize on the surface of woven bone. The percentage of CXCR2+ cells in the callus of young and aged mice was quantified by Image J software. n = 4 mice/young and 3 mice/aged. Data represent mean ± SD. Unpaired two-tailed t-test. * p < 0.05. WB: Woven bone. Scale bar = 1 mm. (B) Western blot to assess IRF-response protein IFITM1 and NF-κB-response protein S100A6 in young and aged callus tissues. (C) Violin plot of the expression level of Cxcr2 in cluster 1.1 and cluster 1.2. (D) CXCR2 low and CXCR2 high cells were isolated from callus-derived mesenchymal progenitor culture. (E) The expression levels of target genes for IRF and NF-κB were measured by RT-qPCR. n = 3 mice/group. Data represent mean ± SD. Unpaired two-tailed t-test. * p < 0.05. (F) CXCR2 low and CXCR2 high cells isolated from callus-derived mesenchymal progenitor culture were cultured in osteoblast inducing medium for 7 days and stained for ALP. The percentage area of ALP+ was measured in Image J software. Expression level of Alp and Runx2 was measured with RT-qPCR. Data represent mean ± SD. Unpaired two-tailed t -test. * p < 0.05.

Article Snippet: CXCR2 low and CXCR2 high callus mesenchymal progenitor cells were purified using CXCR2 antibody (Biolegend cat#149304, clone SA044G4) by fluorescence-activated cell sorting (FACS) or magnetic beads sorting (Miltenyl cat#130-048-801).

Techniques: Biomarker Discovery, Immunostaining, Software, Two Tailed Test, Western Blot, Expressing, Isolation, Derivative Assay, Quantitative RT-PCR, Cell Culture, Staining