Mesenchymal stromal cells prevent progression of liver fibrosis in a novel zebrafish embryo model

Abstract

Chronic liver damage leads to the onset of fibrogenesis. Rodent models for liver fibrosis have been widely used, but are less suitable for screening purposes. Therefore the aim of our study was to design a novel model for liver fibrosis in zebrafish embryos, suitable for high throughput screening. Furthermore, we evaluated the efficacy of mesenchymal stromal cells (MSCs) to inhibit the fibrotic process and thereby the applicability of this model to evaluate therapeutic responses. Zebrafish embryos were exposed to TAA or CCL4 and mRNA levels of fibrosis-related genes (Collagen-1α1, Hand-2, and Acta-2) and tissue damage-related genes (TGF-β and SDF-1a, SDF-1b) were determined, while Sirius-red staining was used to estimate collagen deposition. Three days after start of TAA exposure, MSCs were injected after which the fibrotic response was determined. In contrast to CCL4, TAA resulted in an upregulation of the fibrosis-related genes, increased extracellular matrix deposition and decreased liver sizes suggesting the onset of fibrosis. The applicability of this model to evaluate therapeutic responses was shown by local treatment with MSCs which resulted in decreased expression of the fibrosis-related RNA markers. In conclusion, TAA induces liver fibrosis in zebrafish embryos, thereby providing a promising model for future mechanistic and therapeutic studies.

Contributions of biliary epithelial cells to hepatocyte homeostasis and regeneration in zebrafish

“…As previously described ( Huang et al., 2014 , Huang et al, 2016 ), the zebrafish developed hepatic steatosis with strong collagen expression in response to acute EtOH exposure ( Figure 4 B, arrows and S5 B). The Sirius Red staining which is used to detect fibrosis in the liver ( van der Helm et al., 2018 ) also showed fibrosis after extreme injury of EtOH-exposed liver ( Figure S5 C). After extreme injury of the fibrotic liver by Mtz, the 2F11-positive BECs started to co-express the hepatocyte marker Dendra2 from 8 hr post-Mtz treatment (R8h) on and maintained through R48h ( Figure 4 C).…”

Using Zebrafish as a Disease Model to Study Fibrotic Disease

“…However, liver fibrosis was not detected in zebrafish larvae following CCl4 treatment using gene expression as a read-out [23]. In contrast, three days of TAA exposure induced ECM accumulation in the liver as visualized by Sirius-red staining and upregulated fibrosisrelated genes (col1a1, acta-2, hand-2, tgfb) in zebrafish larvae [23]. Interestingly, the ECM accumulation in zebrafish larvae was visibly different from that found in human or rodent liver.…”

“…However, one drawback of zebrafish is that, in contrast to most human organs (i.e., heart, pancreas, and kidney), their counterparts have been found to be regenerative and proliferative [20]. Despite this, zebrafish fibrotic models have been generated that closely mimic mammalian fibrotic models and clinical symptoms, especially with regard to the accumulation of extracellular matrix (ECM) [21][22][23]. Currently, there are only a handful of established zebrafish models of fibrosis, which highlights the opportunity for innovation and the possibility of developing and investigating mechanistically new models in detail.…”

“…Carbontetrachloride (CCl4) and thioacetamide (TAA) are the most commonly used toxic chemicals to generate fibrotic liver disease through repetitive injection in rodents [88][89][90][91]. However, liver fibrosis was not detected in zebrafish larvae following CCl4 treatment using gene expression as a read-out [23]. In contrast, three days of TAA exposure induced ECM accumulation in the liver as visualized by Sirius-red staining and upregulated fibrosisrelated genes (col1a1, acta-2, hand-2, tgfb) in zebrafish larvae [23].…”

“…Although fibrogenesis of the liver has been detected in larval zebrafish, sensitive techniques (e.g., IHC, qPCR, etc.) were required due to the lower abundancy of accumulated ECM [23,67,68]. Future model optimization should be directed towards a balance between the time length of treatment and ECM occurrence.…”