De-differentiation of primary human hepatocytes depends on the composition of specialized liver basement membrane

Abstract

Basement membrane (BM) is a highly specialized extracellular matrix (ECM), which is associated with epithelia and endothelia. BM provide epithelia with structural support and also regulate cell behavior. The liver contains a unique ECM within the space of Disse, which consists of basement membrane constituents as well as fibrillar ECM molecules. Changes in composition of this ECM are considered detrimental for viability of hepatocytes during progression of liver disease. Mouse tumor-derived BM preparations, such as Matrigel, which are commonly used as a model for BM in vitro, differ significantly in their composition from liver BM present in vivo. In order to gain further insights into the role of BM in the regulation of hepatocyte behavior in health and disease, we generated a liver-derived basement membrane matrix (LBLM). LBLM allowed investigation of BM-hepatocyte interactions in vitro. Here we report a novel approach of generating a liver-derived basement membrane matrix by separate isolation of type IV collagen, laminin, nidogen, and heparan sulfate proteoglycans, and subsequent reconstitution into a matrix-like gel. Adhesion of primary human hepatocytes to LBLM was increased and the rate of de-differentiation was decreased compared to hepatocyte cultivation on Matrigel or type I collagen matrix. Primary human hepatocytes maintained their differentiated epithelial phenotype on LBLM isolated from normal human livers for more than 21 days, whereas they de-differentiated rapidly on LBLM isolated from cirrhotic human livers. Normal human LBLM contains a unique isoform composition of type IV collagen, namely alpha1 (IV), alpha2(IV), alpha4(IV), and alpha6(IV) chains, whereas cirrhotic LBLM contains only alpha1(IV) and alpha2(IV) isoforms, albeit present in increased amounts. These findings suggest that the composition of liver basement membrane is important for the maintenance of hepatocyte viability and provide anti-de-differentiation clues.