Fig 1: 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.