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

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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 MatrigelTM, 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 MatrigelTM 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 α1 (IV), α2(IV), α4(IV), and α6(IV) chains, whereas cirrhotic LBLM contains only α1(IV) and α2(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.

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Abbreviations

BM:

basement membrane

DTT:

dithiothreitol

ECM:

extracellular matrix

EDTA:

ethylenediaminetetraacetic acid

ELISA:

enzyme-linked immunoabsorbent assay

HCl:

hydrochloric acid

HSPG:

heparan sulfate proteoglycans

LBML:

liver basement membrane-like matrix

NC1 domain:

non-collagenous domain

PMSF:

phenylmethylsulphonylfluoride

PHH:

primary human hepatocytes

SMA:

smooth-muscle actin

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Authors and Affiliations

  1. Center for Matrix Biology, Department of Medicine and the Liver Center, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, 02215, USA

    Michael Zeisberg, Kyle Kramer, Nazia Sindhi, Pradip Sarkar & Raghu Kalluri

  2. Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA

    Melissa Upton

  3. Harvard-MIT Division of Health Sciences and Technology, Boston, MA, 02215, USA

    Raghu Kalluri

  4. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02215, USA

    Raghu Kalluri

  5. Department of Medicine, Harvard Medical School and Center for Matrix Biology, Beth Israel Deaconess Medical Center, Dana 514, 330 Brookline Avenue, Boston, MA, 02215

    Raghu Kalluri

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  1. Michael Zeisberg
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  2. Kyle Kramer
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  3. Nazia Sindhi
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  4. Pradip Sarkar
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  6. Raghu Kalluri
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Correspondence to Raghu Kalluri.

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Zeisberg, M., Kramer, K., Sindhi, N. et al. De-differentiation of primary human hepatocytes depends on the composition of specialized liver basement membrane. Mol Cell Biochem 283, 181–189 (2006). https://doi.org/10.1007/s11010-006-2677-8

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  • DOI: https://doi.org/10.1007/s11010-006-2677-8

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