Journal: iScience

Article Title: Differentiation of mesenchymal stem/stromal cells into CD45 + macrophage-like cells: expanding insights into MSC plasticity

doi: 10.1016/j.isci.2026.114906

Figure Lengend Snippet: Functional validation of M-CSF-induced macrophage differentiation from PαS and human MSCs (A) Visual summary for blocking M-CSF-induced macrophage differentiation. PαS cells isolated from the bone marrow and cultured in M-CSF-containing medium with or without CSF1R-blocking antibody or small molecule inhibitor (GW2580). Representative immunofluorescence images show CD206 (green), CD45 (red), and CD68 (gray) expression. Scale bars, 200 μm. (B) Experiment process for M-CSF injection. M-CSF was injected into the tail vein 7 days and again 3 days before collecting PαS cells, and the PαS cell yield was analyzed by flow cytometry. (C) CD45 − TER119-cells after M-CSF tail vein injection (PBS, n = 5, MCSF, n = 5) (ns, not significant, Student’s t tests). (D) PαS cells in the bone in response to M-CSF injection (PBS, n = 5, MCSF, n = 5) (∗ p < 0.05, Student’s t tests). (E) CSF1R expressing PαS cells in response to M-CSF injection (PBS, n = 5, MCSF, n = 5) (ns, not significant, Student’s t tests). (F) Experimental setup for human cell analysis. CD90 + CD271 + human MSCs (huMSCs) and human bone marrow cells (huBMCs) were cultured under DMEM or M-CSF-containing RPMI (RM) conditions. (G) Expression of CSF1R in freshly isolated huMSCs from the BM (CD271 + CD90 + cells). (H) Immunofluorescence staining of huBMCs and huMSCs after culture in DMEM or RM. Cells were isolated from the bone marrow and stained for CD206 (green), CD45 (red), and CD68 (gray). All results were confirmed in three independent experiments. Scale bars, 200 μm.

Article Snippet: GW2580 , Selleck Chemicals LLC , Cat# S8042.

Techniques: Functional Assay, Biomarker Discovery, Blocking Assay, Isolation, Cell Culture, Immunofluorescence, Expressing, Injection, Flow Cytometry, Cell Analysis, Staining

Journal: The Journal of Immunology Author Choice

Article Title: Csf1r -mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System

doi: 10.4049/jimmunol.1701488

Figure Lengend Snippet: Distribution of the Csf1r -mApple and Csf1r -EGFP transgenes is similar throughout tissues. Whole-mount imaging of freshly isolated liver ( A ), lung ( B ), skin epidermis ( C ), cardiac muscle ( D ), jejunum ( E ), and cross-section of spleen ( F ) from WT (left panel), Csf1r -mApple (central), and Csf1r -EGFP (right panel) mice, or from muscularis ( G ) of Csf1r -mApple mice treated with PBS (left panel) or CSF1-Fc (right panel). In (C), a region of interest has been further magnified to better show Langerhans cells (white arrows), and hair follicles can be detected as highly autofluorescent structures (white circles). Scale bars in skin represent 100 μm; 200 μm in liver, spleen, and cardiac muscle; 500 μm in lung and jejunum; and 10 μm in the muscularis.

Article Snippet: The CSF1R kinase inhibitor GW2580 (LC Laboratories) was suspended in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 20 using a Teflon glass homogenizer.

Techniques: Imaging, Isolation

Journal: The Journal of Immunology Author Choice

Article Title: Csf1r -mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System

doi: 10.4049/jimmunol.1701488

Figure Lengend Snippet: Csf1r -mApple transgene expression in blood leukocytes. ( A ) Flow cytometric strategy to identify blood leukocytes. ( B ) Expression of Csf1r -mApple in venous blood leukocytes from a representative Csf1r- mApple (right) and WT littermate control (left) mouse. ( C ) Frequency of cells positive for Csf1r- mApple and ( D ) geometric mean fluorescence intensity (GeoMFI) of Csf1r- mApple gated on Csf1r- mApple + cells for different blood leukocytes. (B–D) Representative data from one of three experiments. Data are presented as mean ± SD of four mice (C and D).

Article Snippet: The CSF1R kinase inhibitor GW2580 (LC Laboratories) was suspended in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 20 using a Teflon glass homogenizer.

Techniques: Expressing, Control, Fluorescence

Journal: The Journal of Immunology Author Choice

Article Title: Csf1r -mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System

doi: 10.4049/jimmunol.1701488

Figure Lengend Snippet: Csf1r transgene and CSF1R protein expression in the peritoneal cavity. ( A ) Flow cytometric strategy to identify peritoneal cavity myeloid cells as recently described . ( B – D ) Expression of Csf1r- mApple in peritoneal cavity myeloid populations determined by flow cytometry, showing a representative WT littermate control (left) and Csf1r -mApple (right) mouse (B), and graphs depicting the mean frequency of Csf1r- mApple + cells in each population (C) and geometric mean fluorescence intensity (GeoMFI) of Csf1r- mApple for different peritoneal leukocytes gated on Csf1r -mApple + cells (D). ( E and F ) Expression of Csf1r -EGFP in peritoneal cavity myeloid populations from Csf1r -EGFP mice, showing a representative flow cytometric overlay (E) and graphs depicting the frequency and GeoMFI of Csf1r -EGFP + cells in each population (F) across multiple mice. Representative data from one of three experiments (B–D) or a single experiment (E and F). Data are presented as mean ± SD of four mice (C, D, and F).

Article Snippet: The CSF1R kinase inhibitor GW2580 (LC Laboratories) was suspended in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 20 using a Teflon glass homogenizer.

Techniques: Expressing, Flow Cytometry, Control, Fluorescence

Journal: The Journal of Immunology Author Choice

Article Title: Csf1r -mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System

doi: 10.4049/jimmunol.1701488

Figure Lengend Snippet: Lineage-restricted Csf1r -mApple transgene expression in the lung. ( A ) Flow cytometric strategy to identify lung leukocytes. ( B ) Frequency of donor cells within each lung population from tissue protected BM chimeric mice 8 wk after receiving BM from WT (white) or Ccr2 −/− (black) mice. Mean donor chimerism for circulating Ly6C + monocytes is presented as short- or long-dashed lines for recipients of WT or Ccr2 −/− BM, respectively. ( C ) Expression of Csf1r -mApple in lung leukocytes from a representative WT littermate control (left) and Csf1r -mApple (right) mouse. ( D ) Frequency of cells expressing Csf1r- mApple and ( E ) geometric mean fluorescence intensity (GeoMFI) of Csf1r -mApple gated on Csf1r- mApple + cells for different lung leukocytes. Data are from one of two independent experiments. Data presented as mean ± SD of four mice (D and E) or mean ± SEM of five mice (B). The asterisk (*) indicates significant differences using multiple t tests corrected for multiple comparisons using the Holm–Sidak method.

Article Snippet: The CSF1R kinase inhibitor GW2580 (LC Laboratories) was suspended in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 20 using a Teflon glass homogenizer.

Techniques: Expressing, Control, Fluorescence

Journal: The Journal of Immunology Author Choice

Article Title: Csf1r -mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System

doi: 10.4049/jimmunol.1701488

Figure Lengend Snippet: Csf1r -mApple transgene expression in the liver. ( A ) Flow cytometric strategy to identify liver leukocytes. ( B ) Expression of Csf1r -mApple in liver leukocytes with histograms from a representative Csf1r -mApple (right) and WT littermate control (left) mouse. ( C ) Frequency of cells expressing Csf1r -mApple and ( D ) geometric mean fluorescence intensity (GeoMFI) of Csf1r -mApple gated on Csf1r -mApple + cells for different liver leukocytes. ( E ) Frequency of donor cells within each hepatic population from tissue protected BM chimeric mice 8 wk after receiving BM from WT (white) or Ccr2 −/− (black) mice. Mean donor chimerism for blood Ly6C + monocytes is presented as short- or long-dashed lines for recipients of WT or Ccr2 −/− BM, respectively. ( F ) Representative histograms showing forward scatter area (FSC-A) characteristics and CD26, CD11c, CD64, and PDCA1 expression (tinted) overlayed with FMO controls (open) for liver leukocytes and blood monocytes. (B–D) Representative data from one of three or (E) two experiments, with data presented as mean ± SD of four mice (C and D) or mean ± SEM of five (E) mice. The asterisk (*) indicates significant differences using multiple t tests corrected for multiple comparisons using the Holm–Sidak method.

Article Snippet: The CSF1R kinase inhibitor GW2580 (LC Laboratories) was suspended in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 20 using a Teflon glass homogenizer.

Techniques: Expressing, Control, Fluorescence

Journal: The Journal of Immunology Author Choice

Article Title: Csf1r -mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System

doi: 10.4049/jimmunol.1701488

Figure Lengend Snippet: Tissue-specific consumption of CSF1 by cDC2 in vivo. ( A ) Histograms of AF647 fluorescence of leukocyte populations from lung and liver following i.v. injection of anti-CSF1R mAb AF-S98 or PBS vehicle before subsequent i.v. delivery of CSF1-Fc AF647 or PBS vehicle. ( B ) Geometric mean fluorescence intensity (GeoMFI) of AF647 of cells from mice in (A) (left graph) and change in MFI between mice given CSF1-Fc AF647 alone or after AFS-98 pretreatment (right graph), with data presented as mean ± SEM of four mice per group (left), or with data points for individual mice shown (right). Data are from one of two repeat independent experiments. ( C ) Representative plots showing CSF1-Fc AF647 uptake versus CD11c expression or FMO control on hepatic cDC2 from mice in (A). ( D ) PE fluorescence of liver leukocytes from C57BL/6 mice injected i.v. with anti–CD45-PE mAb or PBS vehicle 2 min prior to necropsy, showing data from one representative mouse of two per group. ( E ) Histograms of AF647 fluorescence of Ly6C + and Ly6C − blood monocytes from mice treated as in (A) but given CSF1 AF647 , and a graph showing change in GeoMFI of blood monocytes and liver KC between mice given CSF1 AF647 alone or after pretreatment with AFS98, and a dot plot showing Ly6C versus AF647 uptake on all CD3 − CD19 − Ly6G − blood cells with data points representing individual mice. ( F ) Conventional surface CSF1R (CD115) and Ly6C staining on CD3 − CD19 − Ly6G − blood cells from naive mice. Data are from one representative experiment of two (B–D and F) or three (E) independent repeats. The asterisk (*) indicates significant differences between CSF1-Fc AF647 alone and AFS98 + CSF1-Fc AF647 using t tests corrected for multiple comparisons with the Holm–Sidak method (B, left graph), whereas lines indicate significant differences using one-way ANOVA (B, right graph).

Article Snippet: The CSF1R kinase inhibitor GW2580 (LC Laboratories) was suspended in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 20 using a Teflon glass homogenizer.

Techniques: In Vivo, Fluorescence, Injection, Expressing, Control, Staining

Journal: The Journal of Immunology Author Choice

Article Title: Csf1r -mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System

doi: 10.4049/jimmunol.1701488

Figure Lengend Snippet: Consumption of CSF1 differs between monocytes and resident peritoneal macrophage populations. ( A ) Representative histograms of AF647 fluorescence in cavity leukocytes 10 min after i.p. injection of CSF1 AF647 or PBS vehicle alone or in combination with pretreatment with AFS98, and graphs showing MFI of AF647 (right of histogram) and change in MFI between mice given CSF1 AF647 alone or after AFS98 pretreatment (below histogram), with data presented as mean ± SEM of three to four mice per group. Data are from one of three independent repeat experiments. ( B ) Representative histograms of Texas Red fluorescence in cavity leukocytes 10 min after i.p. injection of OVA–Texas Red conjugate from a single experiment. ( C ) Total number of cavity leukocytes and proportion expressing high levels of Ki67 from mice treated for 4 d with GW2580 (open) or vehicle control (closed), with data points depicting individual mice. Data representative of two independent experiments. The asterisk (*) indicates significant differences using multiple t tests corrected for multiple comparisons using the Holm–Sidak method.

Article Snippet: The CSF1R kinase inhibitor GW2580 (LC Laboratories) was suspended in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 20 using a Teflon glass homogenizer.

Techniques: Fluorescence, Injection, Expressing, Control

Journal: The Journal of Immunology Author Choice

Article Title: Csf1r -mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System

doi: 10.4049/jimmunol.1701488

Figure Lengend Snippet: Csf1r -mApple and Δ Csf1r -ECFP transgenes allow imaging of distinct lineages of hepatic myeloid cells. Confocal image of the surface of the left lobe of the liver of a Δ Csf1r -ECFP ( A ), Csf1r -mApple ( B ), WT ( C ), and Csf1r- mApple/Δ Csf1r -ECFP ( D ) mouse imaged ex vivo. FITC-Lectin I was injected i.v. to reveal liver sinusoidal endothelium. Scale bars represent 20 μm (A–C) or 50 μm (D).

Article Snippet: The CSF1R kinase inhibitor GW2580 (LC Laboratories) was suspended in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 20 using a Teflon glass homogenizer.

Techniques: Imaging, Ex Vivo, Injection

Journal: The Journal of Immunology Author Choice

Article Title: Csf1r -mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System

doi: 10.4049/jimmunol.1701488

Figure Lengend Snippet: Csf1r- mApple and Δ Csf1r -ECFP transgenes allow imaging of distinct lineages of pulmonary myeloid cells. Confocal image of a transverse section of lung from a Δ Csf1r -ECFP ( A ), Csf1r -mApple ( B ), WT ( C ), and Csf1r -mApple/Δ Csf1r -ECFP ( D ) mouse imaged ex vivo. FITC-Lectin was injected i.v. to reveal pulmonary vasculature. Scale bars in all panels represent 50 μm.

Article Snippet: The CSF1R kinase inhibitor GW2580 (LC Laboratories) was suspended in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 20 using a Teflon glass homogenizer.

Techniques: Imaging, Ex Vivo, Injection

Journal: The Journal of Immunology Author Choice

Article Title: Csf1r -mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System

doi: 10.4049/jimmunol.1701488

Figure Lengend Snippet: The expression of Csf1r -mApple and Δ Csf1r -ECFP transgenes in regional brain homogenates. Cerebellum, cortex, hippocampus, and striatum were processed to generate a single-cell suspension. ( A ) Compared are CD11b + CD45 lo microglia and CD11b + CD45 hi cells of the cerebellum and cortex regarding their expression of Csf1r -mApple and ECFP. ( B ) Percentage of double transgene positive CD11b + CD45 lo and CD11b + CD45 hi cell populations across all selected regions.

Article Snippet: The CSF1R kinase inhibitor GW2580 (LC Laboratories) was suspended in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 20 using a Teflon glass homogenizer.

Techniques: Expressing, Suspension

Journal: Immunity

Article Title: Airway Epithelial Cell-Derived Colony Stimulating Factor-1 Promotes Allergen Sensitization

doi: 10.1016/j.immuni.2018.06.009

Figure Lengend Snippet: (A) Surface plasmon resonance (SPR) binding analysis for CSF1R inhibitors (GW2580 & PLX647). Experimental sensorgrams (colored lines) were fitted by using a two-state reaction model, and the fitted curves are shown in the black dotted line. Phosphorylation of CSF1R at tyrosine 546 was measured at 20 minutes after CSF1 (10 ng/ml) stimulation with or without pre-treatment of CSF1 inhibitors (GW2580 and PLX647; each 1 μM) in RAW 264.7 cells. (B) Increasing doses of GW2580 were injected during the DRA challenge period (0.01, 0.1, 1 to 10 mg/kg daily, i.p). Eosinophil counts in BAL fluid were decreased in a dose-dependent manner. (C-E) GW2580 (10 mg/kg, i.p.) was treated during challenge period of the DRA model and the degree of lung inflammation, the concentrations of BAL IL-4 & IL-13, total and DRA reactive serum IgE were measured. The colors in quantification of lung pathology indicates the following, green; inflammatory cells, yellow; area of the airway, purple; void space (n=5–6). Scale bars are 5mm in middle and 500μm in right panels. (F-G) Experimental scheme of adoptive transfer (i.t) of DRA-pulsed DCs or macrophages to MAFIA mice (n=2–5). CSF1R+ cells in recipient mice were depleted by AP20187 injection after the DRA sensitization. Either the cultured DCs or macrophages were transferred intratracheally before the challenge phase. AP20187 abolished the eosinophilic recruitment and total and DRA reactive IgE production, which was partially restored by DC transfer, but not by macrophage transfer. Data in A is representative of three independent experiments and data in B-G are two independent experiments. *p<.05, **p<.01.

Article Snippet: GW2580 , Tocris , cat#5673.

Techniques: SPR Assay, Binding Assay, Phospho-proteomics, Injection, Adoptive Transfer Assay, Cell Culture

Journal: Immunity

Article Title: Airway Epithelial Cell-Derived Colony Stimulating Factor-1 Promotes Allergen Sensitization

doi: 10.1016/j.immuni.2018.06.009

Figure Lengend Snippet: (A-B) Mass cytometry of human BAL cells before (Pre) and at 48 hours (Post) after allergen challenge. From the cDC gated population, the numbers of cDC1, cDC2 and CSF1R+ cells were calculated. (C) The percentage of CSF1R+ cells among alveolar macrophages (Amac), cDC1 and cDC2 were measured with or without DRA challenges in the BAL fluids at 6, 24 h after last challenge. (D & E) Number of CSF1R+ cDC1 and cDC2 in BAL fluid and mediastinal LNs with DRA challenge or vehicle in Scgb1a1-creERT;Csf1fl/fl with or without Tm injection. (F-G) Fluorophore ZW800-conjugated HDM (ZW-HDM) was measured in the mediastinal LNs in the DRA asthma model. After gating to the CD172a+ cDC2 population, the ZW-HDM+ cells were counted in Scgb1a1-creERT;Csf1fl/fl mice with or without Tm injection in the presence or absence of GW2580 (10 mg/kg, i.p). Data in C-G are at least two of independent experiments. *p<.05, **p<.01. Please also see figure S2–S3.

Article Snippet: GW2580 , Tocris , cat#5673.

Techniques: Mass Cytometry, Injection

Journal: Immunity

Article Title: Airway Epithelial Cell-Derived Colony Stimulating Factor-1 Promotes Allergen Sensitization

doi: 10.1016/j.immuni.2018.06.009

Figure Lengend Snippet: (A) Experimental scheme of adoptive transfer of EGFP+DCs. After intranasal sensitization, EGFP+ BMDCs were adoptively transferred to the DRA sensitized mice on day 11 with or without concomitant intranasal anti-CSF1 antibody (100 ng/mouse). The number of migratory DC to mLN was measured by flow cytometry. (B-C) Single cell suspension from lung was cultured and stimulated with CSF1 or/and DRA for 24 hours. The expressions of CSF1R and CCR7 on cDC2 were measured by flow cytometry. However, GW2580 treatment effectively blocked the increase of CSF1R+CCR7+cells in a dose-responsive manner in the combined treatment with DRA and CSF1. (D) After intranasal sensitization, myeloid cells in recipient lung were depleted by clodronate liposome. The intratracheal adoptive transfer of EGFP+DCs was performed with or without GW2580 treatment for 1 hour. The number of migratory EGFP+ DCs was measured at mLN by flow cytometry. Data represent at least two A-D) independent experiments. *p<.05, **p<.01. Please also see figure S4–S6.

Article Snippet: GW2580 , Tocris , cat#5673.

Techniques: Adoptive Transfer Assay, Flow Cytometry, Suspension, Cell Culture

Journal: Immunity

Article Title: Airway Epithelial Cell-Derived Colony Stimulating Factor-1 Promotes Allergen Sensitization

doi: 10.1016/j.immuni.2018.06.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: GW2580 , Tocris , cat#5673.

Techniques: Antibody Labeling, Virus, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Software