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: For blocking experiments either 5 μL/mL of Ultra-LEAF Purified anti-mouse CD115 (CSF-1R) blocking antibody (clone: AFS98, Biolegend, San-Diego, CA, USA), 5 μg/mL GW2580 (Selleck Chemicals LLC, Cologne, Germnay) or a combination of both.

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

Journal: Leukemia

Article Title: CSF1R marks a subset of foetal haematopoietic multipotent progenitor cells with acute myeloid leukaemia propagation properties

doi: 10.1038/s41375-025-02856-4

Figure Lengend Snippet: A Experimental layout. B Type 1 (T1), Type 2 (T2), and Type 3 (T3) colony output of CSF1R + /- KMT2A::MLLT3+ LMPPs in CFU-lymphoid assays ( N = 4, from 2 experiments/litters); right panel showing representative pictures of Type 1 (T1), Type 2 (T2), Type 3 (T3) colonies. All pictures were acquired at 4X magnification. C Phenotype of CSF1R- and CSF1R+ colonies at the end of the CFU-lymphoid assay. Data shown as percent of B220+ lymphoid, CD11b+ myeloid cells in the CD45+ population ( N = 3). D Representative flow cytometry plots showing the cell phenotype of CSF1R- and CSF1R+ colonies at the end of the CFU-lymphoid assay. E BLAST, CFU-GM, CFU-M, CFU-G colony output of CSF1R + /- KMT2A::MLLT3+ LMPPs in CFU-myeloid assay ( N = 3, from 2 experiments/litters). F Phenotype of CSF1R- and CSF1R+ colonies at the end of the CFU-myeloid assay. Data shown as percent of B220+ lymphoid, CD11b+ myeloid and B220 + CD11b+ mixed-phenotype cells in the CD45+ population. G Representative flow cytometry plots showing the cell phenotype of CSF1R- and CSF1R+ colonies at the end of the CFU-myeloid assay (N = 3). Data are presented as mean with SD, and statistical analysis was calculated with 2-way ANOVA test: p < 0.01 (**) p < 0.001 (***), p < 0.0001 (****). CFU-GM (colony-forming unit-granulocytes/macrophages), CFU-M (colony-forming unit macrophages), CFU-G (colony-forming unit granulocytes).

Article Snippet: Where stated, 50 ng/mL IL7 (cat# 17-17-10uG, PEPROTECH EC LTD), 10 μM GW2580, a chemical inhibitor of CSF1R (SELLECK CHEMICALS GMBH, cat# S8042) and Doxycycline at 1 μg/mL were added to the coculture media.

Techniques: Flow Cytometry

Journal: Leukemia

Article Title: CSF1R marks a subset of foetal haematopoietic multipotent progenitor cells with acute myeloid leukaemia propagation properties

doi: 10.1038/s41375-025-02856-4

Figure Lengend Snippet: A Experimental design for the in vivo studies. Freshly sorted CSF1R- KMT2A::MLLT3+ LMPPs and CSF1R+ KMT2A::MLLT3+ LMPPs were injected via tail-vein into NSG mice (CSF1R- LMPPs, n = 7 recipients; CSF1R+ LMPPs n = 4 recipients). B Peripheral blood reconstitution of NSG recipients injected with CSF1R+ /- KMT2A::MLLT3+ LMPPs. Blood was analysed monthly by flow cytometry, as percentage of CD45.2+ engrafted cells in the live population. 12-week timepoint for CSF1R+ mice was not analysed since mice had already succumbed to leukaemia. C Peripheral blood reconstitution of CSF1R- recipients, assessed by flow cytometry as percentage of B220+ and CD11b+ cells within CD45.2+ cells. D Representative flow cytometry plots of Pro-B phenotype of engrafted cells from CSF1R- recipient at week 12 post-transplant. E Pro-B phenotype of peripheral blood from CSF1R- recipients, assessed by flow cytometry as percentage of engrafted B220+ CD19+ CD43+ CD24+ cells within CD45.2+ cells at week 12 post-transplant. F Phenotype of peripheral blood donor cells in NSG recipients injected with KMT2A::MLLT3+ CSF1R- LMPPs ( n = 4) at the end point of the experiment (120–150 days post-transplant), assessed by flow cytometry as percentage of engrafted B220+ and CD11b+ cells within the CD45.2+ population. G Peripheral blood reconstitution of NSG recipients injected with KMT2A::MLLT3+ CSF1R+ LMPPs. Blood was analysed monthly by flow cytometry, as a percentage of CD45.2+ B220+ , and CD45.2+ CD11b+ engrafted cells in the live population. The 12-week timepoint was not analysed since the mice had already succumbed to leukaemia. H Survival curve of NSG primary recipients of CSF1R- LMPPs ( n = 7) and CSF1R+ LMPPs ( n = 4) (both KMT2A::MLLT3+ ). I Spleen weights in control (ctrl, n = 4), CSF1R- LMPPs ( n = 5) and CSF1R+ LMPPs ( n = 4) mice (ctrl mice were injected with vehicle solution: PBS + 2% FCS 1% P/S). J Representative picture of CNS histological sections of control (CSF1R- recipient that did not develop disease; left image) and leukaemia infiltration in CSF1R- and CSF1R+ primary recipients (scale bar 200 µm, 500 µm). K Bone marrow engraftment of NSG recipients injected with CSF1R+ /- KMT2A::MLLT3+ LMPPs. Blood was analysed at the end point of the experiment by flow cytometry, as percentage of CD45.2+ engrafted cells in the live population (CSF1R+ n = 3; CSF1R- n = 5). Survival differences were analysed with the Gehan-Breslow-Wilcoxon test. Data are presented as mean with SD, and statistical analysis was calculated with Mann-Whitney U test: p = 0.0006 (***), p = 0.0022 (**), p = 0.0286 (*). CNS (central nervous system).

Article Snippet: Where stated, 50 ng/mL IL7 (cat# 17-17-10uG, PEPROTECH EC LTD), 10 μM GW2580, a chemical inhibitor of CSF1R (SELLECK CHEMICALS GMBH, cat# S8042) and Doxycycline at 1 μg/mL were added to the coculture media.

Techniques: In Vivo, Injection, Flow Cytometry, Control, MANN-WHITNEY

Journal: Leukemia

Article Title: CSF1R marks a subset of foetal haematopoietic multipotent progenitor cells with acute myeloid leukaemia propagation properties

doi: 10.1038/s41375-025-02856-4

Figure Lengend Snippet: A Experimental layout of secondary transplants. 200.000 total bone marrow cells from CSF1R- primary recipients ( n = 2) and CSF1R+ primary recipients ( n = 3) were intravenously injected into NSG mice (6 recipients each). CSF1R- donor cells failed to engraft secondary recipients, while all CSF1R+ recipients showed similar chimerism. B Survival curve of NSG secondary recipients that received total bone marrow cells from CSF1R- and CSF1R+ primary recipients. C Percentages of donor CD11b+ Gr1+ and CD11b+ Gr1+ cKIT+ cells in the bone marrow, peripheral blood, and spleens of CSF1R+ secondary recipients. Percentages of CD11b+ Gr1+ cells are shown within CD45.2+ cells; percentages of cKIT+ cells are shown within CD11b+ Gr1+ cells. D Representative picture of May-Grünwald Giemsa staining of peripheral blood smear (PB, left) and pale bones (BM, right) from secondary CSF1R+ NSG recipients. Black arrow indicates the presence of myeloblast cells; scale bar = 0.5 µm. E Representative picture of spleen enlargement and leukaemia infiltration (black arrow) in secondary CSF1R+ NSG recipients. F Liver histological sections (Haematoxylin-Eosin staining) of CSF1R- secondary recipients, and leukaemic liver from secondary CSF1R+ NSG recipients (left panel scale bar = 200 µm, right panel scale bar = 20 µm). White box area shown at higher magnification on the right. G Representative picture of CNS histological sections of leukaemia infiltration in CSF1R+ secondary recipients (lower panel) compared to CSF1R- secondary recipients (upper panel); scale bar 500 µm. Survival differences were analysed with the Gehan-Breslow-Wilcoxon test. CNS (central nervous system).

Article Snippet: Where stated, 50 ng/mL IL7 (cat# 17-17-10uG, PEPROTECH EC LTD), 10 μM GW2580, a chemical inhibitor of CSF1R (SELLECK CHEMICALS GMBH, cat# S8042) and Doxycycline at 1 μg/mL were added to the coculture media.

Techniques: Injection, Staining

Journal: Leukemia

Article Title: CSF1R marks a subset of foetal haematopoietic multipotent progenitor cells with acute myeloid leukaemia propagation properties

doi: 10.1038/s41375-025-02856-4

Figure Lengend Snippet: A Principal component analysis (PCA) of all 7 samples. B Volcano plot showing all 2400 differentially expressed genes between the two populations (log2-fold-change threshold = 1, corrected p-value threshold = 0.05). Top 10 upregulated genes were: Zfyve27, Zfp763, Nelfb, Ccdc91, Hlf, Chil5, Armcx1, Arhgap6, Cers4, Mtmr3 . Top 10 downregulated genes were: Adgre4, 9430069I07Rik, Pmaip1, Ms4a6c, Hpf1, Ly86, Il13ra1, Anxa3, Gm13772, Gm19880 . C Heatmap of the most overrepresented haematopoiesis-associated genes (red asterisks indicate upregulated genes in CSF1R+ LMPPs associated with definitive haematopoiesis) in KMT2A::MLLT3+ CSF1R+ LMPPs versus KMT2A::MLLT3+ CSF1R- LMPPs; gene list obtained from DAVID. D GSEA of genes overrepresented in KMT2A::MLLT3+ CSF1R+ LMPPs versus KMT2A::MLLT3+ CSF1R- LMPPs. Upper panel indicates enriched genes for haematopoietic stem cell homoeostasis, lower panel indicated enriched genes for IFNγ response. NES= normalised enrichment score; FDR= false discovery rate. E Enrichment for transcription factors (TFs)/target genes obtained from ChIP Enrichment Analysis (ChEA) 2016 dataset (FDR = 0.05), data obtained from ShinyGO 0.77. F GO terms for molecular functions of DEGs KMT2A::MLLT3+ CSF1R+ LMPPs.

Article Snippet: Where stated, 50 ng/mL IL7 (cat# 17-17-10uG, PEPROTECH EC LTD), 10 μM GW2580, a chemical inhibitor of CSF1R (SELLECK CHEMICALS GMBH, cat# S8042) and Doxycycline at 1 μg/mL were added to the coculture media.

Techniques:

Journal: Leukemia

Article Title: CSF1R marks a subset of foetal haematopoietic multipotent progenitor cells with acute myeloid leukaemia propagation properties

doi: 10.1038/s41375-025-02856-4

Figure Lengend Snippet: A Heatmap showing a list of the most overrepresented KMT2A complex genes in KMT2A::MLLT3+ CSF1R+ versus KMT2A::MLLT3+ CSF1R- LMPPs. B Heatmap of the most overrepresented histone-binding genes in KMT2A::MLLT3+ CSF1R+ LMPPs versus KMT2A::MLLT3+ CSF1R- LMPPs. C Gene set enrichment analysis of the DEGs in KMT2A::MLLT3+ CSF1R+ LMPPs showing as most enriched term acute myelomonocytic leukaemia ( p = 0.0006); data obtained from Rare Diseases GeneRIF Gene Lists. Gene lists for the heatmaps were obtained from DAVID, Gene Ontology terms were obtained from RStudio, and Gene Set Enrichment analysis from EnrichR. D Bar graph showing Log2 fold change of OxPhos-related genes ( Slc25a1, Cdk1, Timm13, Timm21, Mrpl12, Mrps12, Mki67 ) and leukaemia stem cell (LSC) genes ( Hpox, Gata2, Erg, Mllt3 ) in KMT2A::MLLT3+ CSF1R+ LMPPs. Genes were selected from Zhang et al. paediatric AML sc-RNA sequencing dataset . E List of upregulated genes in KMT2A::MLLT3+ CSF1R- LMPPs related to KMT2A complex and histone-binding, and pathways in cancer annotations. Data are shown as gene name with log2FC and padj. values. F Gene set enrichment analysis of the upregulated genes in KMT2A::MLLT3+ CSF1R- LMPPs indicating acute lymphoblastic leukaemia as most enriched term ( p = 0.0001). Data obtained from Rare Diseases GeneRIF Gene Lists, and Gene Set Enrichment analysis from EnrichR.

Article Snippet: Where stated, 50 ng/mL IL7 (cat# 17-17-10uG, PEPROTECH EC LTD), 10 μM GW2580, a chemical inhibitor of CSF1R (SELLECK CHEMICALS GMBH, cat# S8042) and Doxycycline at 1 μg/mL were added to the coculture media.

Techniques: Binding Assay, RNA Sequencing

Journal: Leukemia

Article Title: CSF1R marks a subset of foetal haematopoietic multipotent progenitor cells with acute myeloid leukaemia propagation properties

doi: 10.1038/s41375-025-02856-4

Figure Lengend Snippet: A Gene Ontology (GO) term enrichment analysis for biological processes of the DEGs in KMT2A::MLLT3+ CSF1R+ LMPPs. B Heatmap showing a list of the most overrepresented autophagy-related genes in KMT2A::MLLT3+ CSF1R+ versus KMT2A::MLLT3+ CSF1R- LMPPs. C CD36 and CD84 expression assessed by flow cytometry in KMT2A::MLLT3+ CSF1R-/+ LMPPs ( n = 3). Data are presented as median fluorescence intensity (MFI), and statistical analysis was calculated with Unpaired T-test: p < 0.01 (*). D BLAST, CFU-M, CFU-G and CFU-GEMM colony output of KMT2A::MLLT3+ CSF1R+ LMPPs in CFU-myeloid assay ( N = 3). Doxycycline (1 μg/ml), GW2580 (10 μM), and Chloroquine (10 μM) were added to the media as indicated. Data are presented as mean with SD, and statistical analysis was calculated with Dunnett’s multiple comparison test: p < 0.01 (*), p < 0.001 (**), p < 0.0001 (****). E Flow cytometry assessment of lymphoid (B220+ cKIT+ ) and myeloid (CD11b+ cKIT+ ) cell output of CSF1R+ LMPPs at 7 days after coculturing with and without 10 µM GW2580 (chemical inhibitor of CSF1R). 50 ng/ml IL7 was added to both experimental conditions ( N = 4 biological replicates, 3 experiments/litters). Data are presented as mean with SD, and statistical analysis was calculated with Mann-Whitney U test: p = 0.0286 (*). CFU-M (colony-forming unit macrophages), CFU-G (colony-forming unit granulocytes), CFU-GEMM (colony-forming unit granulocyte/erythrocyte/monocyte/ megakaryocyte).

Article Snippet: Where stated, 50 ng/mL IL7 (cat# 17-17-10uG, PEPROTECH EC LTD), 10 μM GW2580, a chemical inhibitor of CSF1R (SELLECK CHEMICALS GMBH, cat# S8042) and Doxycycline at 1 μg/mL were added to the coculture media.

Techniques: Expressing, Flow Cytometry, Fluorescence, Comparison, MANN-WHITNEY

Journal: Leukemia

Article Title: CSF1R marks a subset of foetal haematopoietic multipotent progenitor cells with acute myeloid leukaemia propagation properties

doi: 10.1038/s41375-025-02856-4

Figure Lengend Snippet: A Pseudo-time analysis originated with Monocle of clustered AGM, liver and cord blood from CS14 to 40 weeks embryos/foetuses. B CSF1R , LIN28B , MECOM and TAL1 pseudo-time expression. C Uniform Manifold Approximation and Projection (UMAP) analysis of haematopoietic clusters from AGM and liver from CS14 to 15 weeks embryos. D Feature plots showing the expression of LMPP marker genes (Lin- CD34+ CD38- CD45RA+) and CSF1R expression in the LMPP-containing area. PTPRC : gene encoding CD45RA protein marker. Black circles indicate LMPP-containing area.

Article Snippet: Where stated, 50 ng/mL IL7 (cat# 17-17-10uG, PEPROTECH EC LTD), 10 μM GW2580, a chemical inhibitor of CSF1R (SELLECK CHEMICALS GMBH, cat# S8042) and Doxycycline at 1 μg/mL were added to the coculture media.

Techniques: Expressing, Marker