Extended Data Fig. 4: OSM-STAT3 promotes stable expansion of HHOs OSM and YAP signaling synergistically promote expansion of HHOs.
From: Generation of human adult hepatocyte organoids with metabolic functions
a. Representative images of YAP staining (green) in single eHHO cells cultured with the indicated medium and duration. Nuclear counterstaining, Hoechst 33342. Scale bar: 10 μm. b. Percentage of YAP-positive cells at different time points in the OSM+ or OSM− condition after passaging. Each dot shows the proportion of nuclear YAP-positive cells of four biological replicates (donors), and error bars show mean ± SD of the replicates. The OSM− and OSM+ data was compared using two-way ANOVA followed by Tukey’s two-way post-hoc (p = 0.0025475). For the comparison at each time point, Welch’s two-sided t-test and Bonferroni’s correction were used. c. The effect of OSM and/or TEAD inhibitor (TEADi; MYF-01-37, 20 μM) on HHO expansion. 1,000 cells were plated per well, and the number of cells in each well was analyzed at day 14. Each dot shows one well (each three wells per sample). Statistics, Welch’s two-sided t-test. d. Nuclear YAP localization of HHOs after administration of LATS inhibitor (LATSi; TDI-011536, 3 μM). The organoids were fixed and stained 24 h after LATSi administration. Representative images of YAP localization of HHOs (left). Percentage of nuclear YAP+ cells in LATSi− and LATSi+ conditions (right). Each dot represents the mean of technical replicates of one donor. Data and error bars are shown as mean ± SD. N = 5 HHO lines (biological replicates) derived from different donors. For LATSi− and LATSi+ conditions, 86 and 84 (PY12), 44 and 65 (PY30), 74 and 72 (PY31), 82 and 126 (PY39a), 51 and 50 (PY53) organoids were analyzed, respectively. Statistics, Welch’s two-sided t-test. e. The effect of LATSi with or without a TEAD inhibitor (TEADi; MYF-01-37, 20 or 40 μM) on CYR61 mRNA expression. RNA was harvested 24 h after treatment. For each condition, n = 3 biological replicates. Data are shown as mean ± SD. Statistics, Welch’s two-sided t-test. f. The effect of LATSi with or without a TEADi on the growth of HHO lines. Data from two HHO lines derived from different donors. Each dot shows one well. Statistics, Welch’s two-sided t-test. g. Growth curves of HHOs cultured with or without OSM and LATSi. Each dot denotes passaging. Data from three HHO lines derived from different donors. h. Percentage of EdU + cells in HHOs cultured with or without LATSi and TEADi. Data from two HHO lines derived from different donors. n indicates the number of cells analyzed. i. Comparative transcriptome analysis of PHH-derived organoids, PHHs and ICOs generated based on RNA-seq data. Genes differentially expressed in PHHs versus ICOs (top 20 upregulated and downregulated genes) are used for visualization. j. Expression of EMT markers SNAI2 and ZEB1 in eHHOs with or without OSM and in 3D or 2D culture format. n = 3 biological replicates. Data are shown as mean ± SD. Statistics, Welch’s two-sided t-test.
