Journal: JCI Insight
Article Title: Live-cell imaging of human liver fibrosis using hepatic micro-organoids
doi: 10.1172/jci.insight.187099
Figure Lengend Snippet: ( A ) Top : CRISPaint system used to insert P2A-Clover at the 3′ end of COL1A1 in iPSCs. After blasticidin selection, cells were cloned to generate COL1A1-P2A-Clover iPSCs. Bottom : Self-cleaving P2A peptide enables COL1A1 -expressing cell labeling with fluorescent intracellular protein (Clover). ( B ) Collagen-producing cells in an HO produced from COL1A1 -P2A-Clover iPSCs labeled as in A . ( C ) Clover + cells increase between days 3 and 12. ( D ) The Clover + cell population is absent in iPSCs (day 0) but appears in (day 9) hepatoblasts in differentiating COL1A1 -P2A-Clover HO cultures. ( E ) Location of antibody-stained fluorescent Clover + or EPCAM + cells in day 20 COL1A1 -P2A-Clover organoids. Yellow dashed circles indicate areas with EPCAM + (i.e., hepatocytes or cholangiocytes) cells; Clover + cells are absent from those areas but present elsewhere in the HO. ( F ) Day 21 COL1A1 -P2A-Clover HOs treated on day 13 with no addition (NC), 50 ng/mL TGFB, or 50 ng/mL PDGFB. Both growth factors induced a marked increase in COL1A1 + cells. ( G ) Clover + cells are increased in day 21 COL1A1 -P2A-Clover HOs after TGFB or PDGFB exposure and distinct from EPCAM + (hepatocytes or cholangiocytes) cells in HOs. ( H ) Left : Clover protein expression in differentiating HO cultures (iPSC, day 9 hepatoblasts [HB]) and in day 21 control (NC), PDGFB-treated, or TGFB-treated HOs. Clover and β-actin (ACTB) proteins are indicated. Right : Fold-change in normalized Clover protein expression (relative to control HOs). Each data point is the average of 5 experimental repeats. ( I ) SHG analysis of collagen fibers in human HOs. Top: Cross-sectional view of collagen fibers within day 21 control organoids and organoids treated with TGFB or PDGFB on day 13. Control organoids (left) have isolated regions with relatively thin collagen fibers (blue). TGFB- or PDGFB-treated organoids form a network of thick collagen fibers extending throughout the organoid. Collagen-producing cells (green). Bottom: Quantitative comparison of collagen fiber area in SHG images for control, TGFB-treated, or PDGFB-treated hepatic organoids ( n = 5/measurement; day 21). Increase in total collagen abundance in organoids after TGFB (* P < 0.05) or PDGFB exposure (** P < 0.01, Welch’s t test) and in abundance of thick collagen fibers (i.e., >3 μm diameter) in TGFB- and PDGFB-treated hepatic organoids. Scale bars, 50 mm ( C and E ); 50 μm ( F and I ).
Article Snippet: The CRISPaint ( ) plasmid (pCRISPR-HOT-Clover-BlastR) was obtained from Addgene (plasmid 138569; http://n2t.net/addgene:138569 ).
Techniques: Selection, Clone Assay, Expressing, Labeling, Produced, Staining, Control, Isolation, Comparison
Addgene inc is a verified supplier 
![( A ) Top : <t>CRISPaint</t> system used to insert P2A-Clover at the 3′ end of COL1A1 in iPSCs. After blasticidin selection, cells were cloned to generate COL1A1-P2A-Clover iPSCs. Bottom : Self-cleaving P2A peptide enables COL1A1 -expressing cell labeling with fluorescent intracellular protein (Clover). ( B ) Collagen-producing cells in an HO produced from COL1A1 -P2A-Clover iPSCs labeled as in A . ( C ) Clover + cells increase between days 3 and 12. ( D ) The Clover + cell population is absent in iPSCs (day 0) but appears in (day 9) hepatoblasts in differentiating COL1A1 -P2A-Clover HO cultures. ( E ) Location of antibody-stained fluorescent Clover + or EPCAM + cells in day 20 COL1A1 -P2A-Clover organoids. Yellow dashed circles indicate areas with EPCAM + (i.e., hepatocytes or cholangiocytes) cells; Clover + cells are absent from those areas but present elsewhere in the HO. ( F ) Day 21 COL1A1 -P2A-Clover HOs treated on day 13 with no addition (NC), 50 ng/mL TGFB, or 50 ng/mL PDGFB. Both growth factors induced a marked increase in COL1A1 + cells. ( G ) Clover + cells are increased in day 21 COL1A1 -P2A-Clover HOs after TGFB or PDGFB exposure and distinct from EPCAM + (hepatocytes or cholangiocytes) cells in HOs. ( H ) Left : Clover protein expression in differentiating HO cultures (iPSC, day 9 hepatoblasts [HB]) and in day 21 control (NC), PDGFB-treated, or TGFB-treated HOs. Clover and β-actin (ACTB) proteins are indicated. Right : Fold-change in normalized Clover protein expression (relative to control HOs). Each data point is the average of 5 experimental repeats. ( I ) SHG analysis of collagen fibers in human HOs. Top: Cross-sectional view of collagen fibers within day 21 control organoids and organoids treated with TGFB or PDGFB on day 13. Control organoids (left) have isolated regions with relatively thin collagen fibers (blue). TGFB- or PDGFB-treated organoids form a network of thick collagen fibers extending throughout the organoid. Collagen-producing cells (green). Bottom: Quantitative comparison of collagen fiber area in SHG images for control, TGFB-treated, or PDGFB-treated hepatic organoids ( n = 5/measurement; day 21). Increase in total collagen abundance in organoids after TGFB (* P < 0.05) or PDGFB exposure (** P < 0.01, Welch’s t test) and in abundance of thick collagen fibers (i.e., >3 μm diameter) in TGFB- and PDGFB-treated hepatic organoids. Scale bars, 50 mm ( C and E ); 50 μm ( F and I ).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0020/pmc11790020/pmc11790020__jciinsight-10-187099-g071.jpg)