Fig 1.
Construction of liver organoids.
(A) shows aggregation and compaction of cells at 24 hr. intervals. Organoids were fully formed after 4 days undisturbed culture. (B) Direct viability visualization of organoids over a 28-day period using calcein AM to stain live cells green and ethidium homodimer to stain dead cells red. Even by 28 days, the percentage of dead cells was <10%.
Fig 2.
Liver Organoid characterization.
(A) whole mount staining shows co-expression of p450 reductase (green) and serum albumin (red). Expression of hepatocyte marker serum albumin and stellate cell marker vimentin indicates that the latter cells are present tightly interspersed with hepatocytes. (B) Transmission electron microscopy of newly formed liver organoids (day 2 after formation) shows, even at this early stage, the presence of rudimentary bile canaliculi in between adjacent hepatocytes indicating the establishment of a primitive biliary system.
Fig 3.
Expression of liver markers during long-term culture.
(A) Cellular ATP expression indicates that viability only drops by approximately 10% over 28days. (B) Although albumin levels excreted into the medium are initially relatively low, expression triples by 7 days and thereafter remains high over the entire 28 day period. (C) Urea synthesis dropped somewhat but remained at over 50ug/ml at day 28. (D) Hepatocyte alpha-1 antitrypsin levels remain constant over the entire 28 day period.
Fig 4.
Liver organoid live/dead cell assay.
Liver organoids infected with either control lentivirus with MOI = 5 (A-C) or with lentivirus expressing miR-122 inhibitor at different weeks following infection. Organoids were fixed, with calcein Blue and Ethidium homodimer and images were taken using a Leica confocal microscope. Blue = cell nuclei, Red = dead cells, Green = lentivirus GFP expression. W denotes week.
Fig 5.
H&E staining of liver organoids.
IHC sections of liver organoids stained with H&E at 4 days post-infection (A-C), 11 days post-infection (D-F), and 18 days post-infection (G-I). Left column pictures (A, D, G) show control organoids, middle column show miR-122 inhibited organoids with MOI = 2 (B, E, H), right column show miR-122 inhibited organoids with MOI = 5 (C, F, I). Arrows indicate ballooned hepatocytes, arrowhead indicate Mallory’s hyaline.
Fig 6.
Sirius red staining of liver organoids.
IHC sections of liver organoids stained with Sirius Red at 4 days post-infection (A-C), 11 days post-infection (D-F), and 18 days post-infection (G-I). Left column pictures (A, D, G) show control organoids, middle column show miR-122 inhibited organoids with MOI = 2 (B, E, H), right column show miR-122 inhibited organoids with MOI = 5 (C, F, I). Arrows indicate fibrosis.
Fig 7.
Key genes in fibrosis pathways differentially expressed in liver organoids with miR-122 inhibition vs control organoids.
A. Scatterplot of gene expression in fibrosis pathway in miR-122 inhibited organoids after 4 days (group 1) vs control organoids infected with control lentivirus after 4 days (control group). Over-expressed genes are depicted in red while under-expressed genes depicted in green. B. Over-expressed genes (>1.5 fold) at day 4 post-infection. C. Under-expressed genes (<1.5 fold) at day 4 post-infection. D. Scatterplot of gene expression in fibrosis pathway in miR-122 inhibited organoids after 11 days (group 2) vs control organoids infected with control lentivirus after 11 days (control group). Over-expressed genes are depicted in red while under-expressed genes depicted in green. B. Over-expressed genes (>1.5 fold) at day 11 post-infection. C. Under-expressed genes (<1.5 fold) at day 11 post-infection.
Fig 8.
Key genes in lipid metabolism and insulin signaling pathways differentially expressed in liver organoids with miR-122 inhibition vs control organoids.
A. Scatterplot of gene expression in lipid metabolism and insulin signaling pathway in miR-122 inhibited organoids after 4 days (group 1) vs control organoids infected with control lentivirus after 4 days (control group). Over-expressed genes are depicted in red while under-expressed genes depicted in green. B. Over-expressed genes (>1.5 fold) at day 4 post-infection. C. Under-expressed genes (<1.5 fold) at day 4 post-infection. D. Scatterplot of gene expression in lipid metabolism and insulin signaling pathway in miR-122 inhibited organoids after 11 days (group 2) vs control organoids infected with control lentivirus after 11 days (control group). Over-expressed genes are depicted in red while under-expressed genes depicted in green. B. Over-expressed genes (>1.5 fold) at day 11 post-infection. C. Under-expressed genes (<1.5 fold) at day 11 post-infection.