Single-cell metabolic profiling reveals subgroups of primary human hepatocytes with heterogeneous responses to drug challenge

Background: Xenobiotics are primarily metabolized by hepatocytes in the liver, and primary human hepatocytes are the gold standard model for the assessment of drug efficacy, safety, and toxicity in the early phases of drug development. Recent advances in single‐cell genomics demonstrate liver zonation and ploidy as main drivers of cellular heterogeneity. However, little is known about the impact of hepatocyte spe‐ cialization on liver function upon metabolic challenge, including hepatic metabolism, detoxification, and protein synthesis. Results: Here, we investigate the metabolic capacity of individual human hepatocytes in vitro. We assess how chronic accumulation of lipids enhances cellular heterogene‐ ity and impairs the metabolisms of drugs. Using a phenotyping five‐probe cocktail, we identify four functional subgroups of hepatocytes responding differently to drug challenge and fatty acid accumulation. These four subgroups display differential gene expression profiles upon cocktail treatment and xenobiotic metabolism‐related specialization. Notably, intracellular fat accumulation leads to increased transcriptional variability and diminishes the drug‐related metabolic capacity of hepatocytes. Conclusions: Our results demonstrate that, upon a metabolic challenge such as expo‐ sure to drugs or intracellular fat accumulation, hepatocyte subgroups display different and heterogeneous transcriptional responses.