Gene Expression Profiling of Extracellular Matrix as an Effector of Human Hepatocyte Phenotype in Primary Cell Culture

Abstract

Previously, we demonstrated that primary cultures of rat hepatocytes evidence higher levels of differentiated function when cultured in the presence of a dilute overlay of extracellular matrix (Matrigel). In this investigation, we used DNA microarrays, quantitative RT-PCR, immunoblotting, and cell morphology analyses to evaluate the biological responses imparted by Matrigel overlays on primary cultures of human hepatocytes from five independent donors. Although interindividual variability in responses was evident, our results demonstrated that Matrigel additions typically improved hepatocyte morphology and differentiation character. Results from RNA-profiling experiments indicated that Matrigel additions enhanced hepatocyte RNA expression levels associated with a battery of differentiated features, to levels comparable to those seen in vivo, for genes such as the cytochrome P450s, solute carrier family members, sulfotransferases, certain nuclear transcription factors, and other liver-specific markers, such as albumin, transferrin, and response to the inducer, phenobarbital. In contrast, Matrigel additions were generally associated with reduced RNA expression levels for several cytokeratins, integrins, and a number of stress-related pathways. Decreases in integrin protein expression were similarly detected, although enhanced levels of the gap junction-associated protein, connexin 32, were detected in Matrigel-treated cultures. These data support the concept that ECM functions mechanistically to augment the differentiation character of primary human hepatocytes in culture by mediating a reduction in cellular stress response signaling and by enhancing gap junctional cell-cell communication.

Differentiation of Monkey Embryonic Stem Cells into Hepatocytes and mRNA Expression of Cytochrome P450 Enzymes Responsible for Drug Metabolism: Comparison of Embryoid Body Formation Conditions and Matrices

“…5). Matrigel influenced gene expression in adult rat hepatocytes 45,46) as well as in three-dimensional culture of mouse [47][48][49] and human 50,51) mammary epithelial cells. Three-dimensional culture is important not only in accommodating an organ-like mass but also in enhancing physical cell-to-cell contact, accumulation of extracellular matrices, and local growth factor delivery, with a much greater biochemical effect on the maturation of fetal mouse and human hepatocytes than those in monolayer cultures.…”

Hepatic Aryl Hydrocarbon Receptor Attenuates Fibroblast Growth Factor 21 Expression

“…Enriched normal primary human hepatocytes were obtained through the Liver Tissue Cell Distribution System (University of Pittsburgh, PA). The isolated hepatocytes were seeded at Ͼ90% confluence in 24-well collagen-coated dishes and cultured as previously described, with minor modifications (28). Briefly, the cells were overlaid with 225 g/ml of BD Matrigel TM Basement Membrane Matrix (Corning, NY) and cultured in serum-free Leibovitz's L-15 media (Life Technologies), supplemented with 10 mM HEPES, 100 units/ml of penicillin, 100 g/ml of streptomycin, 25 nM dexamethasone, 10 nM insulin, 5 ng/ml of selenium, 5 g/ml of transferrin, 1% linoleic acid, 1% albumin, and 1% sodium bicarbonate.…”

The Expression of Human Microsomal Epoxide Hydrolase Is Predominantly Driven by a Genetically Polymorphic Far Upstream Promoter

“…Likewise, the E1-b variant was detected using the forward primer (5Ј-GATCGCGCGCCTGC-3Ј), reverse primer (5Ј-GTGAGGAGGATTTCTAGCCACATG-3Ј), and probe (5Ј-CTCGCAGGCTCCGGC-3Ј). Real-time reverse transcription-PCR data were analyzed using methods described previously (Page et al, 2007). Plasmids containing cloned E1 or E1-b full-length sequences were diluted to create standard curves, ranging from 30 copies to 3 ϫ 10 7 copies.…”