Extended Data Fig. 9: Characterization of periportal vein hepatocytes organoids.
From: Generation of human adult hepatocyte organoids with metabolic functions
a. Glycogen visualization in pre-fasted (top) and post-fasted (bottom) dHHOs by Periodic acid-Schiff (PAS) staining. Serial sections treated with α-amylase are shown as negative controls. Scale bar: 50 μm. b. Glycogen measurement in dHHOs using glycogen bioluminescent assay. Data from two organoid lines derived from different donors (biological replicates). Each dot represents one technical replicate. Three technical replicates were shown for each condition. Statistics, Welch’s two-sided t-test. c. Isotope-tracing of 13C-labeled substrates in HHOs using LC-MS/MS (top) and CE-TOFMS (bottom). During gluconeogenesis, dHHOs were treated with the indicated 13C-labeled glucose substrates. Non-labeled glucose substrate-treated dHHOs are used as control. Data are shown as mean ± SD. One bar represents one experiment. (top) n = 6 replicates for each isotope for PY30, n = 5, 6, 5, 5 and 5 replicates (from the left) for PY39a, n = 5, 5, 5, 4 and 5 replicates (from the left) for PY39b. (bottom) Data of 4 (pyruvate) and 5 (non-labeled) replicates for PY12, 6 for PY30, and five replicates for PY39b. Error bar shows mean ± SD. Each dot represents one technical replicate. d. Quantification of Seahorse assay results (Related to Fig. 5f). 1) basal respiration, 2) Proton leak, 3) Maximal respiration and 4) Spare respiratory capacity were calculated. Data are from three HHOs derived from different donors. Each dot shows one technical replicate. Data and error bars show mean ± SD of three technical replicates. e. HHOs were cultured according to the workflow before analysis. Glucose and urea production (left) and CYP activity (right) of cells cultured in 3D and 2D were assessed. Data are from three HHOs derived from different donors (biological replicates), with each dot representing one technical replicate. For the gluocose data, n = 3 technical replicates for conditions 1, 2, and n = 5 replicates for condition 3, and for the urea data,n = 5 technical replicates for each condition. Data and error bars show mean ± SD. N.A., Not applicable. f. (Top) Comparison of expansion efficiency of PHHs between eHHO conditions and the protocol described by Hendriks et al. Cell numbers were estimated using passage ratios and ATP-based cell number. Each dot represents a passage event. (Bottom) Glucose production and urea synthesis under the indicated expansion condition. After expansion, both conditions were differentiated with our differentiation medium (DM) and subjected to fasting. Each dot represents one well. Data and error bars show mean ± SD of four technical replicates. Total culture days are shown in parentheses. g. The gene expression of the indicated secretory proteins in PHHs, eHHOs, and dHHOs based on RNA-seq data. h, i, j. The abundance of factor IX (h), complement C3 (i) and α1-antitrypsin (j) in the supernatant of PHHs, eHHOs, and dHHOs. Data from three HHO lines derived from independent donors (biological replicates). Each dot shows one well. Data and error bars show mean ± SD of three technical replicates.
