Introduction Liver injury is associated with changes in the biochemical and physical properties of the extracellular matrix (ECM). Hepatic progenitor cell (HPC) activation occurs in the context of severe liver injury. ECM stiffness has been shown to direct differentiation in mesenchymal stem cells. However, the effect of mechanical factors, such as ECM stiffness on HPC responses is poorly characterised. We examined the effect of ECM stiffness on HPC proliferation and differentiation.
Methods Experiments were undertaken using a murine HPC line (BMOL) and primary murine HPCs. Cell culture experiments were performed using a system of laminin-coated polyacrylamide (PA) gel supports of variable stiffness. The stiffness of the PA supports (expressed as shear modulus) was altered across a physiological range (1–12 kPa) corresponding to values encountered in normal and fibrotic livers.
Results Increasing matrix stiffness is associated with enhanced cell spreading. BMOL cells cultured on stiff (12kPa) supports develop prominent actin stress fibres. The projected surface area (mean±SEM) of BMOL cells culture on soft (1kPa) supports was 378±21 μm2 compared to 687±47 μm2 for BMOL cells cultured on stiff (12 kPa) supports (p<0.001). Cell proliferation (Ki67 positivity) increased as a function of increasing matrix stiffness. The proliferative index (PI) of BMOL cells cultured on 2.5 kPa and 12 kPa supports was 7.1-fold (p<0.01) and 11.8-fold higher (p<0.001), respectively, than cells cultured on 1kPa supports. Similarly, in experiments with primary cells, the PI of murine HPCs was 1.7-fold higher (p<0.05) when cells were cultured on stiff (12 kPa) vs soft (1 kPa) supports. Quantitative PCR revealed that BMOL cells cultured on soft (1 kPa) supports up-regulate hepatocyte markers, including; albumin (1.5-fold, p<0.01) and CYP7A1 (1.6-fold, p<0.01), and down-regulate the HPC/biliary marker cytokeratin-19 (0.6-fold, p<0.01), relative to cells on stiff (12kPa) supports. There was no significant change in expression of the biliary epithelial cell markers aquaporin-1 and γ-glutamyl-transferase.
Conclusion Physiological changes in ECM stiffness lead to alterations in HPC morphology, proliferation and differentiation. Increased ECM stiffness (as would be encountered in an injured or fibrotic liver) promotes HPC proliferation and expression of the HPC/biliary marker cytokeratin-19. In contrast, a low-stiffness environment is associated with a reduction in cell proliferation and up-regulation of hepatocyte-specific markers. These results suggest that mechanical factors, such as ECM stiffness might regulate HPC responses following liver injury.
Competing interests None declared.
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