[en] Background & Aims: The 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).
Method: The 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.
Results: In 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.
Conclusions: This 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 summary: This 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.
Disciplines :
Gastroenterology & hepatology
Author, co-author :
Payen, Valéry L ✱; Laboratory of Pediatric Hepatology and Cell Therapy (PEDI), IREC Institute, Université catholique de Louvain, Brussels, Belgium ; Laboratory of Advanced Drug Delivery and Biomaterials (ADDB), LDRI Institute, Université catholique de Louvain, Brussels, Belgium
Lavergne, Arnaud ✱; Université de Liège - ULiège > Département de gestion vétérinaire des Ressources Animales (DRA) > Génomique animale
Alevra Sarika, Niki; Laboratory of Pediatric Hepatology and Cell Therapy (PEDI), IREC Institute, Université catholique de Louvain, Brussels, Belgium ; Laboratory of Advanced Drug Delivery and Biomaterials (ADDB), LDRI Institute, Université catholique de Louvain, Brussels, Belgium
This research was funded by grants from La Wallonie (SPW Recherche DGO6 WALInnov program, convention no. 1710035) and the Brussels-Capital Region (Innoviris, RBC/2020-SPIN-206), together with the support of Promethera Biosciences SA . Promethera Biosciences SA had no influence on the design, the collection, analysis and interpretation of data, the writing of the report, or the decision to submit the article for publication.
El Taghdouini, A., Najimi, M., Sancho-Bru, P., Sokal, E., van Grunsven, L.A., In vitro reversion of activated primary human hepatic stellate cells. Fibrogenesis Tissue Repair, 8, 2015, 14.
El Taghdouini, A., Sorensen, A.L., Reiner, A.H., Coll, M., Verhulst, S., Mannaerts, I., et al. Genome-wide analysis of DNA methylation and gene expression patterns in purified, uncultured human liver cells and activated hepatic stellate cells. Oncotarget 6 (2015), 26729–26745.
Mederacke, I., Dapito, D.H., Affo, S., Uchinami, H., Schwabe, R.F., High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers. Nat Protoc 10 (2015), 305–315.
D'Ambrosio, D.N., Walewski, J.L., Clugston, R.D., Berk, P.D., Rippe, R.A., Blaner, W.S., Distinct populations of hepatic stellate cells in the mouse liver have different capacities for retinoid and lipid storage. PLoS One, 6, 2011, e24993.
Macosko, E.Z., Basu, A., Satija, R., Nemesh, J., Shekhar, K., Goldman, M., et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161 (2015), 1202–1214.
Baron, M., Veres, A., Wolock, S.L., Faust, A.L., Gaujoux, R., Vetere, A., et al. A single-cell transcriptomic map of the human and mouse pancreas reveals inter- and intra-cell population structure. Cell Syst 3 (2016), 346–360.
Liao, J., Yu, Z., Chen, Y., Bao, M., Zou, C., Zhang, H., et al. Single-cell RNA sequencing of human kidney. Sci Data, 7, 2020, 4.
Schiller, H.B., Montoro, D.T., Simon, L.M., Rawlins, E.L., Meyer, K.B., Strunz, M., et al. The human lung cell atlas: a high-resolution reference map of the human lung in health and disease. Am J Respir Cell Mol Biol 61 (2019), 31–41.
Dobie, R., Wilson-Kanamori, J.R., Henderson, B.E.P., Smith, J.R., Matchett, K.P., Portman, J.R., et al. Single-cell transcriptomics uncovers zonation of function in the mesenchyme during liver fibrosis. Cell Rep 29 (2019), 1832–1847.
MacParland, S.A., Liu, J.C., Ma, X.Z., Innes, B.T., Bartczak, A.M., Gage, B.K., et al. Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations. Nat Commun, 9, 2018, 4383.
Segal, J.M., Kent, D., Wesche, D.J., Ng, S.S., Serra, M., Oules, B., et al. Single cell analysis of human foetal liver captures the transcriptional profile of hepatobiliary hybrid progenitors. Nat Commun, 10, 2019, 3350.
Aizarani, N., Saviano, A., Sagar, Mailly, L., Durand, S., Herman, J.S., et al. A human liver cell atlas reveals heterogeneity and epithelial progenitors. Nature 572 (2019), 199–204.
Ramachandran, P., Dobie, R., Wilson-Kanamori, J.R., Dora, E.F., Henderson, B.E.P., Luu, N.T., et al. Resolving the fibrotic niche of human liver cirrhosis at single-cell level. Nature 575 (2019), 512–518.
Saviano, A., Henderson, N.C., Baumert, T.F., Single-cell genomics and spatial transcriptomics: discovery of novel cell states and cellular interactions in liver physiology and disease biology. J Hepatol 73 (2020), 1219–1230.
Halpern, K.B., Shenhav, R., Matcovitch-Natan, O., Toth, B., Lemze, D., Golan, M., et al. Single-cell spatial reconstruction reveals global division of labour in the mammalian liver. Nature 542 (2017), 352–356.
Wiederkehr, J.C., Igreja, M.R., Nogara, M.S., Goncalves, N., Montemezzo, G.P., Wiederkehr, H.A., et al. Use of IGL-1 preservation solution in liver transplantation. Transpl Proc 46 (2014), 1809–1811.
Coppin, L., Sokal, E., Stephenne, X., Hepatocyte transplantation in children. Methods Mol Biol 1506 (2017), 295–315.
Najimi, M., Khuu, D.N., Lysy, P.A., Jazouli, N., Abarca, J., Sempoux, C., et al. Adult-derived human liver mesenchymal-like cells as a potential progenitor reservoir of hepatocytes?. Cell Transpl 16 (2007), 717–728.
Butler, A., Hoffman, P., Smibert, P., Papalexi, E., Satija, R., Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36 (2018), 411–420.
Stuart, T., Butler, A., Hoffman, P., Hafemeister, C., Papalexi, E., Mauck, W.M. 3rd, et al. Comprehensive integration of single-cell data. Cell 177 (2019), 1888–1902.
Kowalczyk, M.S., Tirosh, I., Heckl, D., Rao, T.N., Dixit, A., Haas, B.J., et al. Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells. Genome Res 25 (2015), 1860–1872.
Eden, E., Lipson, D., Yogev, S., Yakhini, Z., Discovering motifs in ranked lists of DNA sequences. PLoS Comput Biol, 3, 2007, e39.
Eden, E., Navon, R., Steinfeld, I., Lipson, D., Yakhini, Z., GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinformatics, 10, 2009, 48.
Uhlen, M., Fagerberg, L., Hallstrom, B.M., Lindskog, C., Oksvold, P., Mardinoglu, A., et al. Proteomics. Tissue-based map of the human proteome. Science, 347, 2015, 1260419.
Wang, F., Flanagan, J., Su, N., Wang, L.C., Bui, S., Nielson, A., et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 14 (2012), 22–29.
Becht, E., McInnes, L., Healy, J., Dutertre, C.A., Kwok, I.W.H., Ng, L.G., et al. Dimensionality reduction for visualizing single-cell data using UMAP. Nat Biotechnol 37 (2018), 38–44.
Kuscuoglu, D., Janciauskiene, S., Hamesch, K., Haybaeck, J., Trautwein, C., Strnad, P., Liver - master and servant of serum proteome. J Hepatol 69 (2018), 512–524.
Ku, N.O., Strnad, P., Bantel, H., Omary, M.B., Keratins: biomarkers and modulators of apoptotic and necrotic cell death in the liver. Hepatology 64 (2016), 966–976.
Endo, Y., Matsushita, M., Fujita, T., Role of ficolin in innate immunity and its molecular basis. Immunobiology 212 (2007), 371–379.
Meyer, D.H., Krull, N., Dreher, K.L., Gressner, A.M., Biglycan and decorin gene expression in normal and fibrotic rat liver: cellular localization and regulatory factors. Hepatology 16 (1992), 204–216.
Yang, S., Corbett, S.E., Koga, Y., Wang, Z., Johnson, W.E., Yajima, M., et al. Decontamination of ambient RNA in single-cell RNA-seq with DecontX. Genome Biol, 21, 2020, 57.
Strauss, O., Phillips, A., Ruggiero, K., Bartlett, A., Dunbar, P.R., Immunofluorescence identifies distinct subsets of endothelial cells in the human liver. Sci Rep, 7, 2017, 44356.
Haussinger, D., Lamers, W.H., Moorman, A.F., Hepatocyte heterogeneity in the metabolism of amino acids and ammonia. Enzyme 46 (1992), 72–93.
Oinonen, T., Lindros, K.O., Zonation of hepatic cytochrome P-450 expression and regulation. Biochem J 329 (1998), 17–35.
Braeuning, A., Ittrich, C., Kohle, C., Hailfinger, S., Bonin, M., Buchmann, A., et al. Differential gene expression in periportal and perivenous mouse hepatocytes. FEBS J 273 (2006), 5051–5061.
Schleicher, J., Tokarski, C., Marbach, E., Matz-Soja, M., Zellmer, S., Gebhardt, R., et al. Zonation of hepatic fatty acid metabolism - the diversity of its regulation and the benefit of modeling. Biochim Biophys Acta 1851 (2015), 641–656.
Mederacke, I., Hsu, C.C., Troeger, J.S., Huebener, P., Mu, X., Dapito, D.H., et al. Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun, 4, 2013, 2823.
Motoyama, H., Komiya, T., Thuy le, T.T., Tamori, A., Enomoto, M., Morikawa, H., et al. Cytoglobin is expressed in hepatic stellate cells, but not in myofibroblasts, in normal and fibrotic human liver. Lab Invest 94 (2014), 192–207.
Cassiman, D., Denef, C., Desmet, V.J., Roskams, T., Human and rat hepatic stellate cells express neurotrophins and neurotrophin receptors. Hepatology 33 (2001), 148–158.
Maher, J.J., Cell-specific expression of hepatocyte growth factor in liver. Upregulation in sinusoidal endothelial cells after carbon tetrachloride. J Clin Invest 91 (1993), 2244–2252.
Schirmacher, P., Geerts, A., Pietrangelo, A., Dienes, H.P., Rogler, C.E., Hepatocyte growth factor/hepatopoietin A is expressed in fat-storing cells from rat liver but not myofibroblast-like cells derived from fat-storing cells. Hepatology 15 (1992), 5–11.
Asahina, K., Sato, H., Yamasaki, C., Kataoka, M., Shiokawa, M., Katayama, S., et al. Pleiotrophin/heparin-binding growth-associated molecule as a mitogen of rat hepatocytes and its role in regeneration and development of liver. Am J Pathol 160 (2002), 2191–2205.
Azimifar, S.B., Nagaraj, N., Cox, J., Mann, M., Cell-type-resolved quantitative proteomics of murine liver. Cell Metab 20 (2014), 1076–1087.
Coppock, D., Kopman, C., Gudas, J., Cina-Poppe, D.A., Regulation of the quiescence-induced genes: quiescin Q6, decorin, and ribosomal protein S29. Biochem Biophys Res Commun 269 (2000), 604–610.
Ilani, T., Alon, A., Grossman, I., Horowitz, B., Kartvelishvily, E., Cohen, S.R., et al. A secreted disulfide catalyst controls extracellular matrix composition and function. Science 341 (2013), 74–76.
Scarl, R.T., Lawrence, C.M., Gordon, H.M., Nunemaker, C.S., STEAP4: its emerging role in metabolism and homeostasis of cellular iron and copper. J Endocrinol 234 (2017), R123–R134.
Xie, G., Karaca, G., Swiderska-Syn, M., Michelotti, G.A., Kruger, L., Chen, Y., et al. Cross-talk between Notch and Hedgehog regulates hepatic stellate cell fate in mice. Hepatology 58 (2013), 1801–1813.
Chen, Y., Zheng, S., Qi, D., Zheng, S., Guo, J., Zhang, S., et al. Inhibition of Notch signaling by a gamma-secretase inhibitor attenuates hepatic fibrosis in rats. PLoS One, 7, 2012, e46512.
Sawitza, I., Kordes, C., Reister, S., Haussinger, D., The niche of stellate cells within rat liver. Hepatology 50 (2009), 1617–1624.
Liu, X., Xu, J., Rosenthal, S., Zhang, L.J., McCubbin, R., Meshgin, N., et al. Identification of lineage-specific transcription factors that prevent activation of hepatic stellate cells and promote fibrosis resolution. Gastroenterology 158 (2020), 1728–1744.
Lee, T.F., Mak, K.M., Rackovsky, O., Lin, Y.L., Kwong, A.J., Loke, J.C., et al. Downregulation of hepatic stellate cell activation by retinol and palmitate mediated by adipose differentiation-related protein (ADRP). J Cell Physiol 223 (2010), 648–657.
Coll, M., El Taghdouini, A., Perea, L., Mannaerts, I., Vila-Casadesus, M., Blaya, D., et al. Integrative miRNA and gene expression profiling analysis of human quiescent hepatic stellate cells. Sci Rep, 5, 2015, 11549.
Coll, M., Perea, L., Boon, R., Leite, S.B., Vallverdu, J., Mannaerts, I., et al. Generation of hepatic stellate cells from human pluripotent stem cells enables in vitro modeling of liver fibrosis. Cell Stem Cell 23 (2018), 101–113.
Nakano, Y., Kamiya, A., Sumiyoshi, H., Tsuruya, K., Kagawa, T., Inagaki, Y., A Deactivation factor of fibrogenic hepatic stellate cells induces regression of liver fibrosis in mice. Hepatology 71 (2020), 1437–1452.
Wandzioch, E., Kolterud, A., Jacobsson, M., Friedman, S.L., Carlsson, L., Lhx2-/- mice develop liver fibrosis. Proc Natl Acad Sci U S A 101 (2004), 16549–16554.
Genz, B., Thomas, M., Putzer, B.M., Siatkowski, M., Fuellen, G., Vollmar, B., et al. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells. Exp Cell Res 328 (2014), 429–443.
Kisseleva, T., Cong, M., Paik, Y., Scholten, D., Jiang, C., Benner, C., et al. Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis. Proc Natl Acad Sci U S A 109 (2012), 9448–9453.
Troeger, J.S., Mederacke, I., Gwak, G.Y., Dapito, D.H., Mu, X., Hsu, C.C., et al. Deactivation of hepatic stellate cells during liver fibrosis resolution in mice. Gastroenterology 143 (2012), 1073–1083.
Breitkopf, K., Sawitza, I., Westhoff, J.H., Wickert, L., Dooley, S., Gressner, A.M., Thrombospondin 1 acts as a strong promoter of transforming growth factor beta effects via two distinct mechanisms in hepatic stellate cells. Gut 54 (2005), 673–681.
Xiong, X., Kuang, H., Ansari, S., Liu, T., Gong, J., Wang, S., et al. Landscape of intercellular crosstalk in healthy and NASH liver revealed by single-cell secretome gene analysis. Mol Cell 75 (2019), 644–660.
Dey, A., Chao, S.H., Lane, D.P., HEXIM1 and the control of transcription elongation: from cancer and inflammation to AIDS and cardiac hypertrophy. Cell Cycle 6 (2007), 1856–1863.
Rembold, C.M., Regulation of contraction and relaxation in arterial smooth muscle. Hypertension 20 (1992), 129–137.
Rensen, S.S., Doevendans, P.A., van Eys, G.J., Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Neth Heart J 15 (2007), 100–108.
van den Brink, S.C., Sage, F., Vertesy, A., Spanjaard, B., Peterson-Maduro, J., Baron, C.S., et al. Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations. Nat Methods 14 (2017), 935–936.
Friedman, S.L., Rockey, D.C., McGuire, R.F., Maher, J.J., Boyles, J.K., Yamasaki, G., Isolated hepatic lipocytes and Kupffer cells from normal human liver: morphological and functional characteristics in primary culture. Hepatology 15 (1992), 234–243.
Van Rossen, E., Vander Borght, S., van Grunsven, L.A., Reynaert, H., Bruggeman, V., Blomhoff, R., et al. Vinculin and cellular retinol-binding protein-1 are markers for quiescent and activated hepatic stellate cells in formalin-fixed paraffin embedded human liver. Histochem Cell Biol 131 (2009), 313–325.
Barbatis, C., Kelly, P., Greveson, J., Heryet, A., McGee, J.O., Immunocytochemical analysis of HLA class II (DR) antigens in liver disease in man. J Clin Pathol 40 (1987), 879–884.
Alevra Sarika, N., Payen, V.L., Fleron, M., Ravau, J., Brusa, D., Najimi, M., et al. Human liver-derived extracellular matrix for the culture of distinct human primary liver cells. Cells, 9, 2020, 1357.
Naba, A., Clauser, K.R., Ding, H., Whittaker, C.A., Carr, S.A., Hynes, R.O., The extracellular matrix: tools and insights for the “omics” era. Matrix Biol 49 (2016), 10–24.
Naba, A., Clauser, K.R., Whittaker, C.A., Carr, S.A., Tanabe, K.K., Hynes, R.O., Extracellular matrix signatures of human primary metastatic colon cancers and their metastases to liver. BMC Cancer, 14, 2014, 518.
Jungermann, K., Thurman, R.G., Hepatocyte heterogeneity in the metabolism of carbohydrates. Enzyme 46 (1992), 33–58.
Bishop-Bailey, D., Thomson, S., Askari, A., Faulkner, A., Wheeler-Jones, C., Lipid-metabolizing CYPs in the regulation and dysregulation of metabolism. Annu Rev Nutr 34 (2014), 261–279.
Shimada, S., Kawaguchi-Miyashita, M., Kushiro, A., Sato, T., Nanno, M., Sako, T., et al. Generation of polymeric immunoglobulin receptor-deficient mouse with marked reduction of secretory IgA. J Immunol 163 (1999), 5367–5373.
Gougelet, A., Torre, C., Veber, P., Sartor, C., Bachelot, L., Denechaud, P.D., et al. T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice. Hepatology 59 (2014), 2344–2357.
Riccalton-Banks, L., Bhandari, R., Fry, J., Shakesheff, K.M., A simple method for the simultaneous isolation of stellate cells and hepatocytes from rat liver tissue. Mol Cell Biochem 248 (2003), 97–102.
Meyer, J., Gonelle-Gispert, C., Morel, P., Buhler, L., Methods for isolation and purification of murine liver sinusoidal endothelial cells: a systematic review. PLoS One, 11, 2016, e0151945.
Wake, K., Sato, T., Intralobular heterogeneity of perisinusoidal stellate cells in porcine liver. Cell Tissue Res 273 (1993), 227–237.
Bomble, M., Tacke, F., Rink, L., Kovalenko, E., Weiskirchen, R., Analysis of antigen-presenting functionality of cultured rat hepatic stellate cells and transdifferentiated myofibroblasts. Biochem Biophys Res Commun 396 (2010), 342–347.
Winau, F., Hegasy, G., Weiskirchen, R., Weber, S., Cassan, C., Sieling, P.A., et al. Ito cells are liver-resident antigen-presenting cells for activating T cell responses. Immunity 26 (2007), 117–129.
Vinas, O., Bataller, R., Sancho-Bru, P., Gines, P., Berenguer, C., Enrich, C., et al. Human hepatic stellate cells show features of antigen-presenting cells and stimulate lymphocyte proliferation. Hepatology 38 (2003), 919–929.
Jiang, G., Yang, H.R., Wang, L., Wildey, G.M., Fung, J., Qian, S., et al. Hepatic stellate cells preferentially expand allogeneic CD4+ CD25+ FoxP3+ regulatory T cells in an IL-2-dependent manner. Transplantation 86 (2008), 1492–1502.
Raicevic, G., Najar, M., Najimi, M., El Taghdouini, A., van Grunsven, L.A., Sokal, E., et al. Influence of inflammation on the immunological profile of adult-derived human liver mesenchymal stromal cells and stellate cells. Cytotherapy 17 (2015), 174–185.
Mehrfeld, C., Zenner, S., Kornek, M., Lukacs-Kornek, V., The contribution of non-professional antigen-presenting cells to immunity and tolerance in the liver. Front Immunol, 9, 2018, 635.
