Live-cell imaging of human liver fibrosis using hepatic micro-organoids
Yuan Guan, Zhuoqing Fang, Angelina Hu, Sarah Roberts, Meiyue Wang, Wenlong Ren, Patrik K. Johansson, Sarah C. Heilshorn, Annika Enejder, Gary Peltz
Yuan Guan, Zhuoqing Fang, Angelina Hu, Sarah Roberts, Meiyue Wang, Wenlong Ren, Patrik K. Johansson, Sarah C. Heilshorn, Annika Enejder, Gary Peltz
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Research Article Hepatology

Live-cell imaging of human liver fibrosis using hepatic micro-organoids

Abstract

Due to the limitations of available in vitro systems and animal models, we lack a detailed understanding of the pathogenetic mechanisms of and have minimal treatment options for liver fibrosis. Therefore, we engineered a live-cell imaging system that assessed fibrosis in a human multilineage hepatic organoid in a microwell (i.e., microHOs). Transcriptomic analysis revealed that TGFB converted mesenchymal cells in microHOs into myofibroblast-like cells resembling those in fibrotic human liver tissue. When pro-fibrotic intracellular signaling pathways were examined, the antifibrotic effect of receptor-specific tyrosine kinase inhibitors was limited to the fibrosis induced by the corresponding growth factor, which indicates their antifibrotic efficacy would be limited to fibrotic diseases solely mediated by that growth factor. Based upon transcriptomic and transcription factor activation analyses in microHOs, glycogen synthase kinase 3β and p38 MAPK inhibitors were identified as potential new broad-spectrum therapies for liver fibrosis. Other new therapies could subsequently be identified using the microHO system.

Authors

Yuan Guan, Zhuoqing Fang, Angelina Hu, Sarah Roberts, Meiyue Wang, Wenlong Ren, Patrik K. Johansson, Sarah C. Heilshorn, Annika Enejder, Gary Peltz

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Figure 7

GSK3β inhibitors block TGFB- and PDGFB-induced fibrosis.

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GSK3β inhibitors block TGFB- and PDGFB-induced fibrosis. (A) The PDGFB-i...
(A) The PDGFB-induced increase in COL1A1+ cells is blocked by PDGFRβ and GSK3β inhibitors. COL1A1-P2A-Clover HOs were formed by adding 10,000 cells from day 9 organoid cultures to each microwell. Then, nothing (NC), 50 ng/mL PDGFB (P), or 50 ng/mL PDGFB with 10 μM imatinib, 10 μM SB431542 (SB), 10 μM CHIR99021 (CHIR), 100 ng/mL Wnt3a, or 100 ng/mL R-spondin 1 was added to each microHO on day 13. (B) The TGFB-induced increase in COL1A1+ cells is blocked by TGFBR1 and GSK3β inhibitors. Nothing (NC), 50 ng/mL TGFB (T), 50 ng/mL TGFB with 50 ng/mL PDGFB (T+P), or 50 ng/mL TGFB and 10 μM imatinib, 10 μM SB431542 (SB), or 10 μM CHIR99021 (CHIR) was added to each microHO on day 13. microHO fluorescence, which indicates the number of COL1A1+ cells, was serially measured on days 18 through 25. Each dot represents a measurement made on a microHO, the thick line is the median for 8 microHOs that were assessed, and the box plot shows the 25% to 75% range for the measurements. In the panels, *, P < 0.05; **, P < 0.01; or ***, P < 0.001 for the measurement relative to the value in the NC; when GFs or inhibitors were added, the P values were calculated relative to those of the PDGFB- or TGFB-treated cultures. A 2-way ANOVA indicates that drug treatment and time are 2 variables that have a significant interaction on the fluorescence measurements (Supplemental Table 9).