Extended Data Fig. 7: Fatty acid metabolism and bile acid synthesis in HHOs.
From: Generation of human adult hepatocyte organoids with metabolic functions
a. Representative ZO-1 (green) and MRP2 (red) immunostaining of dHHOs are shown by a 3D-reconstructed image. Scale bar; 50 μm. Similar results were obtained from three donors. b. 3D-reconstructed visualization of BSEP-mediated efflux of CLF-labeled bile acid in a dHHO. Scale bar: 50 μm. c. BSEP-mediated efflux of CLF-labeled bile acid (green) in an eHHO and dHHO. F-actin was visualized with a silicon rhodamine F-actin probe (red). Scale bar, 50 μm. Similar results were obtained from two donors. d. The bile acid metabolism pathway (top) and bile acid secretion from hepatocytes to the bile canaliculus (bottom). HOC; hydroxycholesterol, C4; hydroxy-4-cholesten-3-one, CA; cholic acid, CDCA; 7α-hydroxy-chenodeoxy cholic acid, GCA; glyco-cholic acid, TCA; tauro-cholic acid, GCDCA; glyco-chenodeoxy cholic acid, TCDCA; tauro-chenodeoxy cholic acid, CDFDA; 5(6)-Carboxy-2′,7′-dichloro-fluorescein diacetate, and CLF; cholyl-lysine fluorescein. e. The expression of genes related to bile acid metabolism in PHHs and HHOs generated based on RNA-seq data. f. FXR agonist (INT747, 10 μM) and FGF19 (100 ng/ml) suppress bile acid secretion from dHHOs. Data from two HHO lines derived from different donors (biological replicates). Data and error bars show mean ± SD of three technical replicates. Each dot shows one technical replicate. Statistics, Welch’s two-sided t-test. g. Suppression of CYP7A1 mRNA expression by INT747 and FGF19 in dHHOs. The gene expression was measured using quantitative real-time PCR. Data from two HHO lines derived from different donors. n = 3 technical replicates. Data are shown as mean ± SD. Statistics, Welch’s two-sided t-test. h. Lipid volume in HHOs cultured with the indicated conditions after short and long culture maintenance. Lipids were measured using 3D-reconstructed images of organoids and averaged by the cell (nuclei) count. Data from two HHO lines derived from different donors (biological replicates). For each line, data was collected at two time points as indicated. Each dot shows one organoid. Whiskers; 1.5 × interquartile ranges, box; 25th and 75th percentiles and the median. Statistics, Welch’s two-sided t-test. From the left, n = 7, 6, 6 and 6 replicates for PY31 (day 69), n = 8, 8, 8 and 7 replicates for PY31 (day 132), n = 6, 5, 6 and 6 replicates for PY39a (day 72), and n = 11, 11, 7 and 7 replicates for PY39a (day 275). The upper whisker, box top, center, box bottom, and lower whisker of the boxplot show –(1.5-fold IQR)), 25, 50, 75 percentiles, and 1.5-fold IQR of organoids, respectively. i. Representative bright field of an eHHO and dHHO+WR. Scale bar: 50 μm. Similar results were obtained from three donors. j. Representative H&E staining images of an eHHO and dHHO+WR. dHHO+WR undergoes cell ballooning by lipid accumulation. Scale bar: 50 μm. Similar results were obtained from three donors.
