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A humanized mouse model of liver fibrosis following expansion of transplanted hepatic stellate cells

Laboratory Investigationvolume 98pages525536 (2018) | Download Citation


Hepatic stellate cells (HSCs) are major contributors to liver fibrosis, as hepatic injuries may cause their transdifferentiation into myofibroblast-like cells capable of producing excessive extracellular matrix proteins. Also, HSCs can modulate engraftment of transplanted hepatocytes and contribute to liver regeneration. Therefore, understanding the biology of human HSCs (hHSCs) is important, but effective methods have not been available to address their fate in vivo. To investigate whether HSCs could engraft and repopulate the liver, we transplanted GFP-transduced immortalized hHSCs into immunodeficient NOD/SCID mice. Biodistribution analysis with radiolabeled hHSCs showed that after intrasplenic injection, the majority of transplanted cells rapidly translocated to the liver. GFP-immunohistochemistry demonstrated that transplanted hHSCs engrafted alongside hepatic sinusoids. Prior permeabilization of the sinusoidal endothelial layer with monocrotaline enhanced engraftment of hHSCs. Transplanted hHSCs remained engrafted without relevant proliferation in the healthy liver. However, after CCl4 or bile duct ligation-induced liver damage, transplanted hHSCs expanded and contributed to extracellular matrix production, formation of bridging cell-septae and cirrhosis-like hepatic pseudolobules. CCl4-induced injury recruited hHSCs mainly to zone 3, whereas after bile duct ligation, hHSCs were mainly in zone 1 of the liver lobule. Transplanted hHSCs neither transdifferentiated into other cell types nor formed tumors in these settings. In conclusion, a humanized mouse model was generated by transplanting hHSCs, which proliferated during hepatic injury and inflammation, and contributed to liver fibrosis. The ability to repopulate the liver with transplanted hHSCs will be particularly significant for mechanistic studies of cell-cell interactions and fibrogenesis within the liver.

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Additional information

Daniel Benten and Johannes Kluwe contributed equally to this work.


  1. 1.

    Follenzi A, Benten D, Novikoff P, et al. Transplanted endothelial cells repopulate the liver endothelium and correct the phenotype of hemophilia A mice. J Clin Invest. 2008;118:935–45.

  2. 2.

    Gupta S, Inada M, Joseph B, et al. Emerging insights into liver-directed cell therapy for genetic and acquired disorders. Transpl Immunol. 2004;12:289–302.

  3. 3.

    Ding BS, Nolan DJ, Butler JM, et al. Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature. 2010;468:310–5.

  4. 4.

    Omenetti A, Porrello A, Jung Y, et al. Hedgehog signaling regulates epithelial-mesenchymal transition during biliary fibrosis in rodents and humans. J Clin Invest. 2008;118:3331–42.

  5. 5.

    Gupta S, Bhargava KK, Novikoff PM. Mechanisms of cell engraftment during liver repopulation with hepatocyte transplantation. Semin Liver Dis. 1999;19:15–26.

  6. 6.

    Benten D, Kumaran V, Joseph B, et al. Hepatocyte transplantation activates hepatic stellate cells with beneficial modulation of cell engraftment in the rat. Hepatology. 2005;42:1072–81.

  7. 7.

    Brenner DA, Waterboer T, Choi SK, et al. New aspects of hepatic fibrosis. J Hepatol. 2000;32:32–8.

  8. 8.

    Murphy FR, Issa R, Zhou X, et al. Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition: implications for reversibility of liver fibrosis. J Biol Chem. 2002;277:11069–76.

  9. 9.

    Pinzani M, Marra F. Cytokine receptors and signaling in hepatic stellate cells. Semin Liver Dis. 2001;21:397–416.

  10. 10.

    Sanz S, Pucilowska JB, Liu S, et al. Expression of insulin-like growth factor I by activated hepatic stellate cells reduces fibrogenesis and enhances regeneration after liver injury. Gut. 2005;54:134–41.

  11. 11.

    Schaefer B, Rivas-Estilla AM, Meraz-Cruz N, et al. Reciprocal modulation of matrix metalloproteinase-13 and type I collagen genes in rat hepatic stellate cells. Am J Pathol. 2003;162:1771–80.

  12. 12.

    Benyon RC, Arthur MJ. Extracellular matrix degradation and the role of hepatic stellate cells. Semin Liver Dis. 2001;21:373–84.

  13. 13.

    Issa R, Zhou X, Constandinou CM, et al. Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking. Gastroenterology. 2004;126:1795–808.

  14. 14.

    Kluwe J, Pradere JP, Gwak GY, et al. Modulation of hepatic fibrosis by c-Jun-N-terminal kinase inhibition. Gastroenterology. 2010;138:347–59.

  15. 15.

    Maher JJ. Interactions between hepatic stellate cells and the immune system. Semin Liver Dis. 2001;21:417–26.

  16. 16.

    Mederacke I, Hsu CC, Troeger JS, et al. Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun. 2013;4:2823.

  17. 17.

    Schnabl B, Choi YH, Olsen JC, et al. Immortal activated human hepatic stellate cells generated by ectopic telomerase expression. Lab Invest. 2002;82:323–33.

  18. 18.

    Benten D, Follenzi A, Bhargava KK, et al. Hepatic targeting of transplanted liver sinusoidal endothelial cells in intact mice. Hepatology. 2005;42:140–8.

  19. 19.

    Benten D, Cheng K, Gupta S. Identification of transplanted human cells in animal tissues. Methods Mol Biol. 2006;326:189–201.

  20. 20.

    Volz T, Lutgehetmann M, Wachtler P, et al. Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients. Gastroenterology. 2007;133:843–52.

  21. 21.

    Berna MJ, Seiz O, Nast JF, et al. CCK1 and CCK2 receptors are expressed on pancreatic stellate cells and induce collagen production. J Biol Chem. 2010;285:38905–14.

  22. 22.

    Kluwe J, Wongsiriroj N, Troeger JS, et al. Absence of hepatic stellate cell retinoid lipid droplets does not enhance hepatic fibrosis but decreases hepatic carcinogenesis. Gut. 2011;60:1260–8.

  23. 23.

    De Minicis S, Seki E, Uchinami H, et al. Gene expression profiles during hepatic stellate cell activation in culture and in vivo. Gastroenterology. 2007;132:1937–46.

  24. 24.

    Pradere JP, Kluwe J, De Minicis S, et al. Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice. Hepatology. 2013;58:1461–73.

  25. 25.

    Sancho-Bru P, Bataller R, Gasull X, et al. Genomic and functional characterization of stellate cells isolated from human cirrhotic livers. J Hepatol. 2005;43:272–82.

  26. 26.

    Merlin S, Bhargava KK, Ranaldo G, et al. Kupffer Cell Transplantation in Mice for Elucidating Monocyte/Macrophage Biology and for Potential in Cell or Gene Therapy. Am J Pathol. 2016;186:539–51.

  27. 27.

    Gupta S, Lee CD, Vemuru RP, et al. 111Indium labeling of hepatocytes for analysis of short-term biodistribution of transplanted cells. Hepatology. 1994;19:750–7.

  28. 28.

    Joseph B, Kumaran V, Berishvili E, et al. Monocrotaline promotes transplanted cell engraftment and advances liver repopulation in rats via liver conditioning. Hepatology. 2006;44:1411–20.

  29. 29.

    Bonacchi A, Romagnani P, Romanelli RG, et al. Signal transduction by the chemokine receptor CXCR3: activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes. J Biol Chem. 2001;276:9945–54.

  30. 30.

    Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology. 2008;134:1655–69.

  31. 31.

    Marra F, Romanelli RG, Giannini C, et al. Monocyte chemotactic protein-1 as a chemoattractant for human hepatic stellate cells. Hepatology. 1999;29:140–8.

  32. 32.

    Seki E, De Minicis S, Gwak GY, et al. CCR1 and CCR5 promote hepatic fibrosis in mice. J Clin Invest. 2009;119:1858–70.

  33. 33.

    Kordes C, Sawitza I, Gotze S, et al. Hepatic stellate cells contribute to progenitor cells and liver regeneration. J Clin Invest. 2014;124:5503–15.

  34. 34.

    Choi SS, Omenetti A, Witek RP, et al. Hedgehog pathway activation and epithelial-to-mesenchymal transitions during myofibroblastic transformation of rat hepatic cells in culture and cirrhosis. Am J Physiol Gastrointest Liver Physiol. 2009;297:G1093–106.

  35. 35.

    Roskams T. Relationships among stellate cell activation, progenitor cells, and hepatic regeneration. Clin Liver Dis. 2008;12:853–60.

  36. 36.

    Zalzman M, Gupta S, Giri RK, et al. Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells. Proc Natl Acad Sci Usa. 2003;100:7253–8.

  37. 37.

    Chen CH, Kuo LM, Chang Y, et al. In vivo immune modulatory activity of hepatic stellate cells in mice. Hepatology. 2006;44:1171–81.

  38. 38.

    Chou HS, Hsieh CC, Yang HR, et al. Hepatic stellate cells regulate immune response by way of induction of myeloid suppressor cells in mice. Hepatology. 2011;53:1007–19.

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This work was supported in part by NIH grants R01 DK071111 and P30-DK41296. DB received support from Deutsche Forschungsgemeinschaft, grants BE 2559/2–1 and BE 2559/2–2 and SFB 841. JK received support from the Deutsche Forschungsgemeinschaft, grant KL2140/2–1 and SFB 841. JK and DB received support from Forschungsförderungsfonds Medizin, Hamburg University.

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Author notes


    1. Departments of Medicine and Pathology, Marion Bessin Liver Research Center, Diabetes Center, Cancer Center, Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Institute for Clinical and Translational Research, Albert Einstein College of Medicine, Bronx, NY, USA

      • Daniel Benten
      •  & Sanjeev Gupta
    2. Department of Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany

      • Daniel Benten
      • , Johannes Kluwe
      • , Jan W. Wirth
      • , Nina D. Thiele
      • , Michael Koepke
      • , Reni Tjandra
      • , Tassilo Volz
      •  & Marc Lutgehetmann
    3. Helios Klinikum Duisburg, Duisburg, Germany

      • Daniel Benten
    4. Department of HealthSciences, Università del Piemonte Orientale “A. Avogadro”, Novara, Italy

      • Antonia Follenzi
    5. Division of Nuclear Medicine and Molecular Imaging, Long Island Jewish Health Center, NorthWell Health, New Hyde Park, NY, USA

      • Kuldeep K. Bhargava
      •  & Christopher J. Palestro


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    The authors declare that they have no conflict of interest.

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    Correspondence to Daniel Benten or Sanjeev Gupta.

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