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.

FIG. 1

Matrigel enhances cellular morphology of primary human hepatocyte cultures for donor A, donor B, and donor C. Primary human hepatocytes from donor A (A and B), donor B (C and D), and donor C (E and F) were cultured in the presence (B, D, F) and absence (A, C, E) of a Matrigel overlay. Photomicrographs were taken under ×20 magnification using phase contrast imaging. Arrows indicate compromised morphology in the absence of a Matrigel overlay.

FIG. 2

Matrigel overlay exhibits minimal impact on cellular morphology for primary human hepatocyte cultures obtained from donor D or donor E. Primary human hepatocytes from donor D (A and B) and donor E (C and D) were cultured in the presence (B and D) and absence (A and C) of a Matrigel overlay. Photomicrographs were taken under ×20 magnification using phase contrast imaging.

FIG. 3

Effects of Matrigel addition on differentiation status of primary human hepatocyte cultures. Total RNA was isolated from primary human hepatocytes cultured for 5 days in the presence or absence of a Matrigel overlay. (A) Transcript levels for the hepatocyte differentiation markers, albumin, transferrin, and transthyretin, were assessed −/+ Matrigel additions using quantitative RT-PCR and the ΔΔC_T method. (B) Transcript levels for the hepatocyte dedifferentiation markers, AFP and GSTPi, were similarly ascertained −/+ Matrigel additions and were normalized to each differentiation marker. (C) Primary human hepatocytes were cultured in the absence (control) or presence of Matrigel (MG). Cultures of primary human hepatocytes and HepG2 cells (indicated by arrows) were treated on day 4 with phenobarbital (PB and PB + MG) or left untreated (control) for 24 h prior to RNA isolation. Relative fold changes in transcript levels for the PB-inducible marker genes, CYP2B6 and CYP3A4, are indicated. (D) Total RNA was isolated from HepG2 cells, a section of human liver #154, as well as three different donor samples of primary human hepatocytes that were cultured with a Matrigel overlay. Relative expression analyses for a panel of differentiation and dedifferentiation markers were determined by quantitative RT-PCR analysis, and the results are graphically depicted.

FIG. 4

Hierarchical clustering analyses of interindividual differences and treatment regimens of primary human hepatocyte samples. Total RNA was isolated from cultures of primary human hepatocytes from five different donors −/+ Matrigel overlay. RNA transcript profiles were analyzed by microarray hybridizations, as described in “Materials and Methods” section. Results were hierarchically clustered (ArrayAssist, Stratagene) before (A) and after (B) filtering for twofold changes in gene expression level between Matrigel-treated samples and untreated controls.

FIG. 5

Biological impact of Matrigel additions in primary human hepatocyte cultures. Microarray analyses were performed on total RNA collected from primary human hepatocytes cultured in the presence or absence of a Matrigel overlay for either 4 or 5 days. Raw signal intensities for every probe set were plotted for control and Matrigel samples for each donor (A–E). A correlation coefficient was generated for each scatterplot. Total numbers of changed genes (change call p ≤ 0.00267) were reported for each donor (F).

FIG. 6

Protein expression profiling of primary human hepatocytes and HepG2 cells. Total cellular protein was isolated from primary human hepatocyte cultures and cultures of HepG2 cells, and equal quantities of protein from the respective samples were applied to 10% denaturing gels, separated by SDS-PAGE and then transferred to PVDF membranes and assessed by immunoblotting analyses, as described in “Materials and Methods” section.