Single-Cell Gene Expression Analysis Identifies Chronic Alcohol-Mediated Shift in Hepatocyte Molecular States After Partial Hepatectomy

Abstract

The analysis of molecular states of individual cells, as defined by their mRNA expression profiles and protein composition, has gained widespread interest in studying biological phenomena ranging from embryonic development to homeostatic tissue function and genesis and evolution of cancers. Although the molecular content of individual cells in a tissue can vary widely, their molecular states tend to be constrained within a transcriptional landscape partly described by the canonical archetypes of a population of cells. In this study we sought to characterize the effects of an acute (partial hepatectomy) and chronic (alcohol consumption) perturbation on the molecular states of individual hepatocytes during the onset and progression of liver regeneration. We analyzed the expression of 84 genes across 233 individual hepatocytes acquired using laser capture microdissection. Analysis of this single cell data revealed that hepatocyte molecular states can be considered as distributed across a set of four states irrespective of perturbation, with the proportions of hepatocytes in these states being dependent on the perturbation. In addition to the quiescent, primed, and replicating hepatocytes, we identified a fourth molecular state lying between the primed and replicating subpopulations. Comparison of the proportions of hepatocytes from each experimental condition in these four molecular states suggested that, in addition to aberrant priming, a slower transition from primed to replication state could contribute towards ethanol-mediated suppression of liver regenerative response to partial hepatectomy.

Cellular plasticity balances the metabolic and proliferation dynamics of a regenerating liver

“…Lately, single-cell transcriptomic methods are being employed to probe cellular heterogeneities of tissues and reconstruct developmental trajectories of individual organs (Haghverdi et al, 2016;Saelens et al, 2019;Schiebinger et al, 2019). In the case of the liver, this has led to the identification of previously unknown subpopulations of hepatocytes, cholangiocytes, endothelial cells, scar-associated macrophages, stellate cells, and myofibroblasts from healthy and diseased conditions (Aizarani et al, 2019;Dobie et al, 2019;Hyun et al, 2019;MacParland et al, 2018;Pepe-Mooney et al, 2019;Ramachandran et al, 2019;Xiong et al, 2019;Achanta et al, 2019;Cook et al, 2018). The studies revealed that more than 50% of hepatocyte genes follow a discrete zonated expression pattern in a liver lobule, and surprisingly, similar metabolic zonation exists for LSECs and stellate cells Halpern et al, 2018;2017).…”

Epithelial splicing regulatory protein 2–mediated alternative splicing reprograms hepatocytes in severe alcoholic hepatitis

“…is inactivated in some individuals with alcohol-induced liver injury, causing extensive repopulation of SAH livers with immature, fetal-like cells. The results identify the mechanism for liver failure in SAH by revealing that TNF-α and IL-1β ( 19), proinflamma-prevented maturation of hepatocytes (8), which explains why liver regeneration is inhibited by alcohol (17,18). However, the mechanisms involved were not defined.…”

“…Single-cell gene expression analysis recently demonstrated that feeding alcohol to adult rats increased the proportion of hepatocytes that are either becoming proliferative or proliferat-ing (8), suggesting that excessive alcohol drinking might promote some degree of hepatocyte dedifferentiation. Maintaining hepatocytes in a differentiated state is a critical function of the Hippo/ YAP signaling pathway (9).…”

Single cell high-throughput qRT-PCR protocol

“…This protocol has been developed over the past decade in our lab to enable reliable high-throughput detection of gene expression by qRT-PCR in low input samples starting from 10 pg total RNA (single cells). We have used this protocol to measure gene expression in a wide variety of samples types, tissue treatments and disease contexts (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). The present version of this protocol was developed as part of the National Institutes of Health SPARC project efforts to construct a comprehensive anatomical, molecular and functional map of the peripheral nervous system at the visceral organs.…”