Proteome-Wide Analyses of Human Hepatocytes During Differentiation and Dedifferentiation

Abstract

Failure to predict hepatotoxic drugs in preclinical testing makes it imperative to develop better liver models with a stable phenotype in culture. Stem cell-derived models offer promise, with differentiated hepatocyte-like cells currently considered to be “fetal-like” in their maturity. However, this judgment is based on limited biomarkers or transcripts and lacks the required proteomic datasets that directly compare fetal and adult hepatocytes. Here, we quantitatively compare the proteomes of human fetal liver, adult hepatocytes, and the HepG2 cell line. In addition, we investigate the proteome changes in human fetal and adult hepatocytes when cultured in a new air-liquid interface format compared to conventional submerged extracellular matrix sandwich culture. From albumin and urea secretion, and luciferase-based cytochrome P450 activity, adult hepatocytes were viable in either culture model over 2 weeks. The function of fetal cells was better maintained in the air-liquid interface system. Strikingly, the proteome was qualitatively similar across all samples but hierarchical clustering showed that each sample type had a distinct quantitative profile. HepG2 cells more closely resembled fetal than adult hepatocytes. Furthermore, clustering showed that primary adult hepatocytes cultured at the air-liquid interface retained a proteome that more closely mimicked their fresh counterparts than conventional culture, which acquired myofibroblast features. Principal component analysis extended these findings and identified a simple set of proteins, including cytochrome P450 2A6, glutathione S transferase P, and alcohol dehydrogenases as specialized indicators of hepatocyte differentiation. Conclusion: Our quantitative datasets are the first that directly compare multiple human liver cells, define a model for enhanced maintenance of the hepatocyte proteome in culture, and provide a new protein “toolkit” for determining human hepatocyte maturity in cultured cells. (Hepatology 2013;58:799–809)

Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile

“…For example, whilst CYP2A6 and CYP2C8 are the most down-regulated proteins, CYP2C18 and CYP2J2 remain unaltered by culture conditions. Of further interest, CYP2A6 has been reported to be the largest discriminant between foetal and adult hepatocytes (Rowe et al 2013); here, we show CYP2A6 to be the largest discriminator between freshly isolated and 168 h cultured hepatocytes.…”

“…The labelled samples were then pooled and made up to 5 mL with 10 mM potassium dihydrogen phosphate/25 % w/v acetonitrile. The pH was then adjusted using concentrated phosphoric acid to <pH 3, before cation-exchange chromatography, followed by identification with mass spectrometry, as described previously (Rowe et al 2010, 2013). Samples were run across three 8-plex iTRAQ runs (table S1), and results obtained relative to each donor’s fresh sample to control for inter-donor variation.…”

Decellularized human liver scaffold‐based three‐dimensional culture system facilitate hepatitis B virus infection

“…To investigate cell repopulate in hDLS, PHHs cells were seeded into the hDLS and cultured for 14 days continuously. Since PHH dedifferentiation is featured as the change of cell morphology, loss of liver-specific gene (such as p450 cytochromes, and HNF4A) expression and specific protein secretion, 17,18 Albumin (ALB) secreting and liver-specific genes were determined to evaluate whether scaffold-based 3D culture could retain PHH phenotype and physiology. Since PHH dedifferentiation is featured as the change of cell morphology, loss of liver-specific gene (such as p450 cytochromes, and HNF4A) expression and specific protein secretion, 17,18 Albumin (ALB) secreting and liver-specific genes were determined to evaluate whether scaffold-based 3D culture could retain PHH phenotype and physiology.…”

The Importance of Patient-Specific Factors for Hepatic Drug Response and Toxicity

“…However, their physiological phenotypes are lost in conventional 2D monolayer cultures due to the lack of necessary biochemical cues and cell–cell interactions as well as non-physiological biophysical properties of the culture substratum, e.g., with regards to stiffness [195,196]. As a consequence, PHH lose expression of genes characteristic for mature hepatocytes within hours of culture and acquire fetal-like phenotypes [197,198,199]. To prevent this dedifferentiation a variety of advanced 3D hepatocyte culture methodologies have been developed (extensively reviewed in reference [200]).…”