Protective effects of lipocalin-2 (LCN2) in acute liver injury suggest a novel function in liver homeostasis. on day 15, n = 2 biological replicates. (F) Primary hepatocytes were cultured with or Anemoside A3 without TNF, or with IL-6 and colony count Anemoside A3 was evaluated on day 20, n Rabbit Polyclonal to B-RAF = 2 biological replicates. (G) Phase-contrast images of cultures at early and late passages. TNF was either maintained or withdrawn from culture media at indicated passage numbers. Arrowheads indicate lipid droplets. Related to Figure 1. Figure S2. 3D hepatocytes express functional hepatocyte markers and modulation of functional genes. (A) Confocal images of 3D hepatocyte showing the presence of lumen within the organoid (arrowheads; left). Mitotic cell observed by DAPI stain (dashed circle; right). (B)Gene expression analysis by q-PCR. Data are represented as mean SEM of three independent measurements for each biological replicate. Data are an extension of Figure 2. Related to Figure 2. Figure S3. 3D hepatocytes perform hepatocyte functions. (A) Confocal images (z-stack projection) of multiple hepatocyte organoids, showing fluorescein diacetate (green) secretion into bile canaliculi structures (arrowheads) and lumen (asterisk). Red = mTdTomato, blue = DAPI. (B) Overlay of BF- and fluorescence images of a representative hepatocyte colony. At day 5 after second lentiviral infection, 3D hepatocytes were transferred from ultra-low attachment plate to geltrex coated plate, for visualization of GFP-expressing hepatocytes. (C)Co-culture of 3D hepatocytes with GFP-HUVEC, two days (top) and two weeks (bottom) after Anemoside A3 the initiation of co-culture. Figure S4. Single-cell RNA-seq reveals broad expression of hepatocyte markers and the presence of a subset of cycling cells. (A) A list of the 20 most highly expressed genes in the hepatocyte and cycling clusters and their average UMI counts (left). Mitochondrial and ribosomal genes are omitted for clarity. Heatmap showing the 30 most differentially expressed genes between the hepatocyte and cycling clusters (right). DEGs are determined by the top Wilcoxon rank sum test scores. (B) GO term analysis for the 1000 most highly expressed genes in the hepatocyte cluster (left) and the top 150 DEGs in the cycling cluster (right). Figure S5. Expression of regeneration- associated markers in expanding media and upregulation of functional genes in induction media. (A) Heatmap of differentially expressed genes for exp hepatocytes relative to primary hepatocytes, showing the most upregulated and downregulated genes. Mitochondrial and ribosomal genes are omitted for clarity. (B) Heatmap of differentially expressed genes for hepatocytes relative to positive cells express the endothelial cell marker CD31/Pecam. Inset represents a higher magnification of central vein endothelial cells to show that both CD31 and transcripts do not co-localize with CD45. Inset shows a dense cluster of cells positive for the CD45 signal. does not overlap with this cell population. FAH+ clone boundary is marked with a dashed line. (6) Rspo1 is not expressed in the liver but is found in the mesothelial layer surrounding the liver. Scale bar = 20 m (7) Rspo2 is found in dense clusters of cells interspersed among FAH? hepatocytes. Inset shows a close-up view of transcript localization. (8) Rspo4 is also found in dense clusters of cells that often reside near vascular structures. Inset shows Rspo4 signal at higher magnification. Anemoside A3 (9) TNF Anemoside A3 is expressed in dense clusters of cells that are found near the vasculature. Inset shows a higher magnification look at of transcript localization to these cells. Level bars = 100 m, unless stated otherwise. NIHMS1008587-product-1.pdf (7.2M) GUID:?05D1099B-6B6F-4851-B6BF-6CA4F26CA174 SUMMARY In the healthy adult liver, most hepatocytes proliferate minimally..