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Cellular network modeling and single cell gene expression analysis reveals novel hepatic stellate cell phenotypes controlling liver regeneration dynamics.

ABSTRACT: BACKGROUND:Recent results from single cell gene and protein regulation studies are starting to uncover the previously underappreciated fact that individual cells within a population exhibit high variability in the expression of mRNA and proteins (i.e., molecular variability). By combining cellular network modeling, and high-throughput gene expression measurements in single cells, we seek to reconcile the high molecular variability in single cells with the relatively low variability in tissue-scale gene and protein expression and the highly coordinated functional responses of tissues to physiological challenges. In this study, we focus on relating the dynamic changes in distributions of hepatic stellate cell (HSC) functional phenotypes to the tightly regulated physiological response of liver regeneration. RESULTS:We develop a mathematical model describing contributions of HSC functional phenotype populations to liver regeneration and test model predictions through isolation and transcriptional characterization of single HSCs. We identify and characterize four HSC transcriptional states contributing to liver regeneration, two of which are described for the first time in this work. We show that HSC state populations change in vivo in response to acute challenges (in this case, 70% partial hepatectomy) and chronic challenges (chronic ethanol consumption). Our results indicate that HSCs influence the dynamics of liver regeneration through steady-state tissue preconditioning prior to an acute insult and through dynamic control of cell state balances. Furthermore, our modeling approach provides a framework to understand how balances among cell states influence tissue dynamics. CONCLUSIONS:Taken together, our combined modeling and experimental studies reveal novel HSC transcriptional states and indicate that baseline differences in HSC phenotypes as well as a dynamic balance of transitions between these phenotypes control liver regeneration responses.

SUBMITTER: Cook D

PROVIDER: S-EPMC6171157 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Cellular network modeling and single cell gene expression analysis reveals novel hepatic stellate cell phenotypes controlling liver regeneration dynamics.

Cook Daniel D Achanta Sirisha S Hoek Jan B JB Ogunnaike Babatunde A BA Vadigepalli Rajanikanth R

BMC systems biology 20181003 1

<h4>Background</h4>Recent results from single cell gene and protein regulation studies are starting to uncover the previously underappreciated fact that individual cells within a population exhibit high variability in the expression of mRNA and proteins (i.e., molecular variability). By combining cellular network modeling, and high-throughput gene expression measurements in single cells, we seek to reconcile the high molecular variability in single cells with the relatively low variability in ti ...[more]

PMID: 30285726

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Project description:BackgroundAs a critical cellular component in the hepatic stem cell niche, hepatic stellate cells (HSCs) play critical roles in regulating the expansion of hepatic stem cells, liver regeneration, and fibrogenesis. However, the signaling of HSCs, particularly that involved in promoting hepatic stem cell expansion, remains unclear. While the overexpression of galectins has been identified in regenerating liver tissues, their involvement in cell-cell interactions between HSCs and hepatic stem cells remains to be elucidated.MethodsTo generate a liver regeneration rat model and establish a hepatic oval cell microenvironment as a stem cell niche, 2-acetylaminofluorene treatment plus partial hepatectomy was performed. Immunofluorescence staining was conducted to detect the emergence of hepatic stem cells and their niche. Liver parenchymal cells, non-parenchymal cells, and HSCs were isolated for gene and protein expression analysis by qPCR or western blotting. To evaluate the effect of galectins on the colony-forming efficiency of hepatic stem cells, c-Kit-CD29+CD49f+/lowCD45-Ter-119- cells were cultured with recombinant galectin protein, galectin antibody, galectin-producing HSCs, and galectin-knockdown HSCs.ResultsFollowing liver injury, the cytokeratin 19+ ductal cells were robustly induced together with the emergence of OV6+CD44+CD133+EpCAM+ hepatic stem cells. The activated desmin+ HSCs were recruited around the periportal area and markedly enriched in the galectin-positive domain compared to the other non-parenchymal cells. Notably, the HSC fraction isolated from regenerating liver was accompanied by dramatically elevated gene and protein expression of galectins. Hepatic stem cells co-cultured with HSCs significantly enhanced colony-forming efficiency. Conversely, single or double knockdown of galectin-1 and galectin-3 led into a significant function loss, impaired the co-cultured hepatic stem cells to attenuated colony size, inhibited colony frequency, and reduced total cell numbers in colonies. On the other hand, the promotive function of galectins was further confirmed by recombinant galectin protein supplementation and galectins blocking antibodies.ConclusionsOur findings, for the first time, demonstrated that galectins from activated HSCs contribute to hepatic stem cell expansion during liver regeneration, suggesting that galectins serve as important stem cell niche components.

Project description:IntroductionHepatic stellate cells (HSC) become activated, differentiate to myofibroblasts and produce extracellular fibrillar matrix during liver fibrosis. The hepatic fibrogenic response is orchestrated by reciprocal interactions between HSCs and macrophages and their secreted products. SOCS1 can regulate several cytokines and growth factors implicated in liver fibrosis. Here we investigated the role of SOCS1 in regulating HSC activation.MethodsMice lacking SOCS1 in HSCs (Socs1ΔHSC) were generated by crossing Socs1fl/fl and LratCre mice. Liver fibrosis was induced by carbon tetrachloride and evaluated by Sirius red staining, hydroxyproline content and immunostaining of myofibroblasts. Gene expression of pro-fibrogenic factors, cytokines, growth factors and chemokines were quantified by RT-qPCR. The phenotype and the numbers of intrahepatic leukocyte subsets were studied by flow cytometry. The impact of fibrosis on the development of diethyl nitrosamine-induced hepatocellular carcinoma was evaluated.ResultsSocs1ΔHSC mice developed more severe liver fibrosis than control Socs1fl/fl mice that was characterized by increased collagen deposition and myofibroblast differentiation. Socs1ΔHSC mice showed a significant increase in the expression of smooth muscle actin, collagens, matrix metalloproteases, cytokines, growth factors and chemokines in the liver following fibrosis induction. The fibrotic livers of Socs1ΔHSC mice displayed heightened inflammatory cell infiltration with increased proportion and numbers of Ly6ChiCCR2+ pro-inflammatory macrophages. This macrophage population contained elevated numbers of CCR2+CX3CR1+ cells, suggesting impaired transition towards restorative macrophages. Fibrosis induction following exposure to diethyl nitrosamine resulted in more numerous and larger liver tumor nodules in Socs1ΔHSC mice than in Socs1fl/fl mice.DiscussionOur findings indicate that (i) SOCS1 expression in HSCs is a critical to control liver fibrosis and development of hepatocaellular carcinoma, and (ii) attenuation of HSC activation by SOCS1 regulates pro-inflammatory macrophage recruitment and differentiation during liver fibrosis.

Project description:Background & aimsThe multiple vital functions of the human liver are performed by highly specialised parenchymal and non-parenchymal cells organised in complex collaborative sinusoidal units. Although crucial for homeostasis, the cellular make-up of the human liver remains to be fully elucidated. Here, single-cell RNA-sequencing was used to unravel the heterogeneity of human liver cells, in particular of hepatocytes (HEPs) and hepatic stellate cells (HSCs).MethodThe transcriptome of ~25,000 freshly isolated human liver cells was profiled using droplet-based RNA-sequencing. Recently published data sets and RNA in situ hybridisation were integrated to validate and locate newly identified cell populations.ResultsIn total, 22 cell populations were annotated that reflected the heterogeneity of human parenchymal and non-parenchymal liver cells. More than 20,000 HEPs were ordered along the portocentral axis to confirm known, and reveal previously undescribed, zonated liver functions. The existence of 2 subpopulations of human HSCs with unique gene expression signatures and distinct intralobular localisation was revealed (i.e. portal and central vein-concentrated GPC3 + HSCs and perisinusoidally located DBH + HSCs). In particular, these data suggest that, although both subpopulations collaborate in the production and organisation of extracellular matrix, GPC3 + HSCs specifically express genes involved in the metabolism of glycosaminoglycans, whereas DBH + HSCs display a gene signature that is reminiscent of antigen-presenting cells.ConclusionsThis study highlights metabolic zonation as a key determinant of HEP transcriptomic heterogeneity and, for the first time, outlines the existence of heterogeneous HSC subpopulations in the human liver. These findings call for further research on the functional implications of liver cell heterogeneity in health and disease.Lay summaryThis study resolves the cellular landscape of the human liver in an unbiased manner and at high resolution to provide new insights into human liver cell biology. The results highlight the physiological heterogeneity of human hepatic stellate cells.

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Cellular network modeling and single cell gene expression analysis reveals novel hepatic stellate cell phenotypes controlling liver regeneration dynamics.

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Cellular network modeling and single cell gene expression analysis reveals novel hepatic stellate cell phenotypes controlling liver regeneration dynamics.

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