GVHD targets organoid-forming BDSCs in a TGF-β–dependent manner. Lethally irradiated BDF1 mice were transplanted from BDF1 (Syn) or B6 (Allo) donors as in Figure 1A, and the liver right lobes were subjected to organoid-forming assay. A schematic overview (A) of the organoid-forming assay and the number of liver organoids derived from the right lobe of the livers at indicated time points are shown. (B; n = 5-15/group). Time trends from days 7 to 21 to days 28 to 42 within Allo group were significantly different. The average value from days 28 to 42 in Allo group was significantly less than the combined average of the naïve and syngeneic groups during the same period. (C) Representative images of liver organoids generated from recipients’ livers on day 35. Bar, 500 μm. Quantitative polymerase chain reaction (Q-PCR) targeting IFN-γ (D), TNF-α (E), and TGF-β (H) was performed using total RNA extracted from recipient’s liver on day 28 (n = 6-11/group). (F,G,I) Liver organoids produced from naïve BDF1 mice were dissociated into small pieces, seeded at a concentration of 200 fragments/well on the 24-well plate in the presence or absence of IFN-γ (F), TNF-α (G), or TGF-β (I). Organoids were enumerated 4 days later, and the fold changes from controls were calculated (n = 6/group). (J) Dissociated organoids were cultured in the presence or absence of TGF-β and an SMAD2/3 inhibitor, SB-431542, for 4 days. The fold changes from controls were calculated. (K) The total RNA extracted from BECs from naïve BDF1 mice, syngeneic or allogeneic recipients, or liver organoids generated from naïve BDF1 mice was subjected to Q-PCR targeting the TGF-β receptor (n = 6-10/group). (B,D-K) Data from 2 independent experiments were combine and shown as mean ± standard error. ∗P < .05, ∗∗P < .01, ∗∗∗P < .005.