Inflammatory macrophages reduce liver organoid formation in a TGF-β–dependent manner. (A) Immunofluorescent staining of the liver sections with TGF-β1 (green) and F4/80 (red) on day 28. Bar, 100 μm. (B) Intracellular staining of TGF-β was performed on day 28. Absolute numbers of TGF-β–producing Kupffer cells (KCs) and infiltrating macrophages (Mφs) were shown (n = 9-10/group). (C-D) Liver organoids derived from naïve BDF1 were cultured in the presence or absence of LMCs from syngeneic (Syn) or allogeneic (Allo) recipients. (D) Organoids were enumerated 4 days later, and the fold changes from controls were calculated (n = 7/group). (E) LMCs were separated into T cells and non-T cells, and then cocultured with liver organoids. Organoids were enumerated 4 days later (n = 8/group). (F) Organoids were cultured in the presence or absence of LMCs from syngeneic or allogeneic recipients with or without SB-431542 for 4 days. The fold changes from controls were calculated (n = 8-14/group). Data from 2 (B-E) or 3 (F) similar experiments were combined and shown as mean ± standard error.