Functional Analysis of Induced Human Ballooned Hepatocytes in a Cell Sheet-Based Three Dimensional Model



Ballooned hepatocytes (BH) are a key histological hallmark of nonalcoholic steatohepatitis (NASH), yet their consequences for liver-specific functions are unknown.


In our previous study, an experimental model of human induced-BHs (iBH) has been successfully developed based on cell sheet technology. This study aimed to determine the functions of iBHs in the primary human hepatocyte/normal human dermal fibroblast (PHH/NHDF) co-culture cell sheets. Normal hepatocytes in the PHH/3T3-J2 co-culture cell sheets were set as a control, since 3T3-J2 murine embryonic fibroblasts have exhibited previously long term maintenance of PHH functions.


It was found that, albumin secretion was not affected in iBHs, but urea synthesis as well as cytochrome P450 enzyme (CYP) activities including CYP1A2 and CYP3A4, were significantly reduced in iBHs. Besides, loss of bile canaliculi was observed in iBHs. These findings are consistent with clinical studies of human NASH. In addition, PHH/NHDF cell sheets demonstrated two fold higher TGF-β1 secretion compared with PHH/3T3-J2 cell sheets. Furthermore, treatment with a TGF-β inhibitor and a semi-synthetic bile acid analogue (obeticholic acid, phase 3 trial of NASH therapy) ameliorated the histological appearance of established iBHs.


In summary, this study demonstrates the priority of iBHs in recapitulating not only histology but also clinically relevant hepatic dysfunctions in human NASH and suggests TGF-β and bile acid related signal pathway may play important roles in the formation of iBHs.

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  1. 1.

    Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016;64:73–84.

    Article  Google Scholar 

  2. 2.

    Williams CD, Stengel J, Asike MI, Torres DM, Shaw J, Contreras M, et al. Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and liver biopsy: a prospective study. Gastroenterology. 2011;140:124–31.

    Article  Google Scholar 

  3. 3.

    Farrell GC, Wong VW, Chitturi S. NAFLD in Asia-as common and important as in the West. Nat Rev Gastroenterol Hepatol. 2013;10:307–18.

    CAS  Article  Google Scholar 

  4. 4.

    Thomsen KL, Grønbæk H, Glavind E, Hebbard L, Jessen N, Clouston A, et al. Experimental nonalcoholic steatohepatitis compromises ureagenesis, an essential hepatic metabolic function. Am J Physiol Gastrointest Liver Physiol. 2014;307:G295–301.

    CAS  Article  Google Scholar 

  5. 5.

    Appleby RN, Moghul I, Khan S, Yee M, Manousou P, Neal TD, et al. Non-alcoholic fatty liver disease is associated with dysregulated bile acid synthesis and diarrhea: a prospective observational study. PLoS One. 2019;14:e0211348.

    CAS  Article  Google Scholar 

  6. 6.

    Mouzaki M, Wang AY, Bandsma R, Comelli EM, Arendt BM, Zhang L, et al. Bile acids and dysbiosis in non-alcoholic fatty liver disease. PLoS One. 2016;11:e0151829.

    Article  Google Scholar 

  7. 7.

    Woolsey SJ, Mansell SE, Kim RB, Tirona RG, Beaton MD. CYP3A activity and expression in nonalcoholic fatty liver disease. Drug Metab Dispos. 2015;43:1484–90.

    CAS  Article  Google Scholar 

  8. 8.

    Yu Y, Ananthanarayanan A, Singh NH, Hong X, Sakban RB, Mittal N, et al. TGFβ1-mediated suppression of cytochrome P450(CYP) induction responses in rat hepatocyte-fibroblast co-cultures. Toxicol In Vitro. 2018;50:47–53.

    CAS  Article  Google Scholar 

  9. 9.

    Schuster N, Krieglstein K. Mechanisms of TGF-beta-mediated apoptosis. Cell Tissue Res. 2002;307:1–14.

    CAS  Article  Google Scholar 

  10. 10.

    Yang L, Roh YS, Song J, Zhang B, Liu C, Loomba R, et al. Transforming growth factor beta signaling in hepatocytes participates in steatohepatitis through regulation of cell death and lipid metabolism in mice. Hepatology. 2014;59:483–95.

    CAS  Article  Google Scholar 

  11. 11.

    Charlton M, Sreekumar R, Rasmussen D, Lindor K, Nair KS. Apolipoprotein synthesis in nonalcoholic steatohepatitis. Hepatology. 2002;35:898–904.

    CAS  Article  Google Scholar 

  12. 12.

    Guy CD, Suzuki A, Zdanowicz M, Abdelmalek MF, Burchette J, Unalp A, et al. Hedgehog pathway activation parallels histologic severity of injury and fibrosis in human non-alcoholic fatty liver disease. Hepatology. 2012;55:1711–21.

    CAS  Article  Google Scholar 

  13. 13.

    Syn WK, Choi SS, Liaskou E, Karaca GF, Agboola KM, Oo YH, et al. Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis. Hepatology. 2011;53:106–15.

    CAS  Article  Google Scholar 

  14. 14.

    Caldwell S, Ikura Y, Dias D, Isomoto K, Yabu A, Moskaluk C, et al. Hepatocellular ballooning in NASH. J Hepatol. 2010;53:719–23.

    Article  Google Scholar 

  15. 15.

    Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology. 1999;116:1413–9.

    CAS  Article  Google Scholar 

  16. 16.

    Guy CD, Suzuki A, Abdelmalek MF, Burchette JL, Diehl AM. Treatment response in the PIVENS trial is associated with decreased Hedgehog pathway activity. Hepatology. 2015;61:98–107.

    CAS  Article  Google Scholar 

  17. 17.

    Brunt EM, Kleiner DE, Wilson LA, Belt P, Neuschwander-Tetri BA. NASH Clinical Research Network (CRN). Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings. Hepatology. 2011;53:810–20.

    CAS  Article  Google Scholar 

  18. 18.

    Matsuda N, Shimizu T, Yamato M, Okano T. Tissue engineering based on cell sheet technology. Adv Mater. 2007;19:3089–99.

    CAS  Article  Google Scholar 

  19. 19.

    Haraguchi Y, Shimizu T, Sasagawa T, Sekine H, Sakaguchi K, Kikuchi T, et al. Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro. Nat Protoc. 2012;7:850–8.

    CAS  Article  Google Scholar 

  20. 20.

    Gao B, Sakaguchi K, Matsuura K, Ogawa T, Kagawa Y, Kubo H, et al. In vitro production of human ballooned hepatocytes in a cell sheet-based three-dimensional model. Tissue Eng Part A. 2020;26:93–101.

    CAS  Article  Google Scholar 

  21. 21.

    Khetani SR, Szulgit G, Del Rio JA, Barlow C, Bhatia SN. Exploring interactions between rat hepatocytes and nonparenchymal cells using gene expression profiling. Hepatology. 2004;40:545–54.

    CAS  Article  Google Scholar 

  22. 22.

    Khetani SR, Bhatia SN. Microscale culture of human liver cells for drug development. Nat Biotechnol. 2008;26:120–6.

    CAS  Article  Google Scholar 

  23. 23.

    Davidson MD, Kukla DA, Khetani SR. Microengineered cultures containing human hepatic stellate cells and hepatocytes for drug development. Integr Biol (Camb). 2017;9:662–77.

    CAS  Article  Google Scholar 

  24. 24.

    Khetani SR, Chen AA, Ranscht B, Bhatia SN. T-cadherin modulates hepatocyte functions in vitro. FASEB J. 2008;22:3768–75.

    CAS  Article  Google Scholar 

  25. 25.

    Shaheen A. Effect of the unfolded protein response on ER protein export: a potential new mechanism to relieve ER stress. Cell Stress Chaperones. 2018;23:797–806.

    CAS  Article  Google Scholar 

  26. 26.

    Bartalena L, Robbins J. Effect of tunicamycin and monensin on secretion of thyroxine-binding globulin by cultured human hepatoma (Hep G2) cells. J Biol Chem. 1984;259:13610–4.

    CAS  Article  Google Scholar 

  27. 27.

    Ota T, Gayet C, Ginsberg HN. Inhibition of apolipoprotein B100 secretion by lipid-induced hepatic endoplasmic reticulum stress in rodents. J Clin Invest. 2008;118:316–32.

    CAS  Article  Google Scholar 

  28. 28.

    De Chiara F, Heebøll S, Marrone G, Montoliu C, Hamilton-Dutoit S, Ferrandez A, et al. Urea cycle dysregulation in non-alcoholic fatty liver disease. J Hepatol. 2018;69:905–15.

    Article  Google Scholar 

  29. 29.

    Fisher CD, Lickteig AJ, Augustine LM, Ranger-Moore J, Jackson JP, Ferguson SS, et al. Hepatic cytochrome P450 enzyme alterations in humans with progressive stages of nonalcoholic fatty liver disease. Drug Metab Dispos. 2009;37:2087–94.

    CAS  Article  Google Scholar 

  30. 30.

    Abdel-Razzak Z, Corcos L, Fautrel A, Campion JP, Guillouzo A. Transforming growth factor-beta 1 down-regulates basal and polycyclic aromatic hydrocarbon-induced cytochromes P-450 1A1 and 1A2 in adult human hepatocytes in primary culture. Mol Pharmacol. 1994;46:1100–10.

    CAS  PubMed  Google Scholar 

  31. 31.

    Segovia-Miranda F, Morales-Navarrete H, Kücken M, Moser V, Seifert S, Repnik U, et al. Three-dimensional spatially resolved geometrical and functional models of human liver tissue reveal new aspects of NAFLD progression. Nat Med. 2019;25:1885–93.

    CAS  Article  Google Scholar 

  32. 32.

    Younossi ZM, Ratziu V, Loomba R, Rinella M, Anstee QM, Goodman Z, et al. Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial. Lancet. 2019;394:2184–96.

    CAS  Article  Google Scholar 

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This work is supported by Adaptable and Seamless Technology Transfer Program through Target-Driven Research and Development (Grant No. JP18im0302706) from Japan Agency for Medical Research and Development (AMED), Hundred Talents Program of Guangdong Academy of Sciences (Grant No. 2020GDASYL-20200102005) and Innovation Ability Construction program of Guangdong Medical Device Research Institute (Grant No. 2017GDASCX-0103).

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Correspondence to Tatsuya Shimizu.

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Tatsuya Shimizu is a member of the scientific advisory board and a stakeholder of CellSeed Inc. Tetsuya Ogawa, Yuki Kagawa and Hirotsugu Kubo are employees of Nihon Kohden Corporation. Tokyo Women’s Medical University was receiving research funds from CellSeed Inc. and Nihon Kohden Corporation.

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Gao, B., Sakaguchi, K., Ogawa, T. et al. Functional Analysis of Induced Human Ballooned Hepatocytes in a Cell Sheet-Based Three Dimensional Model. Tissue Eng Regen Med (2021).

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  • Cell sheet
  • Ballooned hepatocytes
  • Liver function
  • NASH