Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes.
Bottom Line: Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes.The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes.This current phenotype mimics human fetal rather than adult hepatocytes.
Affiliation: Centre for Endocrinology & Diabetes, Institute of Human Development, Faculty of Medical & Human Sciences, University of Manchester, Manchester Academic Health Science Centre, AV Hill Building, Oxford Road, Manchester, UK.Show MeSH
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Mentions: While these data were encouraging of liver-specific differentiation, it is an assumption based on limited user-selected proteins and assays and ignores potential similarity to cell types from other organs. We performed unbiased proteomic assessment of whole cell extracts from undifferentiated H9 cells and their HLCs at the end of stage 3. 61 proteins showed significant upregulation (>2-fold) including known liver markers, such as AAT/SERPINA1 and phase 1 (e.g. aldehyde dehydrogenases) and phase 2 enzymes (e.g., nicotinamide N-methyltransferase [NNMT] and glutathione S-transferase [GST] Mu3) (Supplementary Table 2). Cytokeratin (KRT) 8 and KRT18, both upregulated, function together as heteropolymers to protect hepatocytes from mechanical and non-mechanical stress . KRT7, apparent in <10% of cells by immunocytochemistry (data not shown), may reflect slight permissiveness in our protocol to cholangiocyte differentiation but has also been reported as a hepatocyte progenitor cell marker [31,32]. Some upregulated proteins were not recognized as hepatocyte markers, such as KRT5 and KRT6A. Therefore to assess the broad proteome phenotype, we compared the upregulated protein dataset against data from 30 other human organs and tissues in the EBI Gene Expression Atlas (Fig. 3). By heatmap, the upregulated HLC proteome most closely resembled the liver, followed by another anterior derivative of foregut endoderm, the thyroid. Other foregut endoderm derivatives (stomach, small intestine and pancreas) showed recognizable similarity, in contrast to a marked divergence of HLCs from mesodermal and ectodermal derivatives such as bone marrow, skeletal muscle and brain.
Affiliation: Centre for Endocrinology & Diabetes, Institute of Human Development, Faculty of Medical & Human Sciences, University of Manchester, Manchester Academic Health Science Centre, AV Hill Building, Oxford Road, Manchester, UK.