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Semin Liver Dis 2022; 42(04): 423-433
DOI: 10.1055/a-1934-5588
Review Article

Three-Dimensional Organoids as a Model to Study Nonalcoholic Fatty Liver Disease

Yujin Park 
1   Department of Surgery, Division of Transplant Surgery, Indiana University School of Medicine, Indianapolis, Indiana
,
Deepthi Thadasina 
1   Department of Surgery, Division of Transplant Surgery, Indiana University School of Medicine, Indianapolis, Indiana
,
Ifeoluwa Bolujo 
1   Department of Surgery, Division of Transplant Surgery, Indiana University School of Medicine, Indianapolis, Indiana
,
Abdulkadir Isidan 
1   Department of Surgery, Division of Transplant Surgery, Indiana University School of Medicine, Indianapolis, Indiana
,
Arthur A. Cross-Najafi 
1   Department of Surgery, Division of Transplant Surgery, Indiana University School of Medicine, Indianapolis, Indiana
,
Kevin Lopez 
1   Department of Surgery, Division of Transplant Surgery, Indiana University School of Medicine, Indianapolis, Indiana
,
Ping Li 
1   Department of Surgery, Division of Transplant Surgery, Indiana University School of Medicine, Indianapolis, Indiana
,
Andrew M. Dahlem
2   Division of Clinical Pharmacology, Indiana University School of Medicine, Indianapolis, Indiana
,
Lindsey Kennedy 
3   Department of Medicine, Division of Gastroenterology and Hepatology, Indiana University School of Medicine, and Division of Research, Richard L. Roudebush VA Medical Center, Indianapolis, Indiana
,
Keisaku Sato 
3   Department of Medicine, Division of Gastroenterology and Hepatology, Indiana University School of Medicine, and Division of Research, Richard L. Roudebush VA Medical Center, Indianapolis, Indiana
,
Heather Francis 
3   Department of Medicine, Division of Gastroenterology and Hepatology, Indiana University School of Medicine, and Division of Research, Richard L. Roudebush VA Medical Center, Indianapolis, Indiana
,
Gianfranco Alpini 
3   Department of Medicine, Division of Gastroenterology and Hepatology, Indiana University School of Medicine, and Division of Research, Richard L. Roudebush VA Medical Center, Indianapolis, Indiana
,
Wenjun Zhang    *
1   Department of Surgery, Division of Transplant Surgery, Indiana University School of Medicine, Indianapolis, Indiana
,
Burcin Ekser    *
1   Department of Surgery, Division of Transplant Surgery, Indiana University School of Medicine, Indianapolis, Indiana
› Author Affiliations Funding This work was partially supported by ASTS Faculty Development Grant (BE), Indiana University Health Values Fund for Research Award (VFR-457-Ekser) (BE), and IU Health Foundation Jerome A. Josephs Fund for Transplant Innovation Grant (BE), the Hickam Endowed Chair, Gastroenterology, Medicine, Indiana University, the Indiana University Health – Indiana University School of Medicine Strategic Research Initiative, the Senior Research Career Scientist Award (IK6 BX004601) and the VA Merit award (5I01BX000574) to GA and the Research Career Scientist Award (IK6BX005226) and the VA Merit award (1I01BX003031) to HF, and Career Development Award (1IK2BX004306) to LK from the United States Department of Veteran's Affairs, Biomedical Laboratory Research and Development Service and NIH grants DK108959 and DK119421 (HF), DK054811, DK115184, DK076898, DK107310, DK110035, DK062975 and AA028711 (GA) and the PSC Partners Seeking a Cure (GA). Portions of these studies were supported by resources at the Richard L. Roudebush VA Medical Center, Indianapolis, IN. The views expressed in this article are those of the authors and do not necessarily represent the views of the Department of Veterans Affairs.
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Abstract

Despite the rising prevalence of nonalcoholic fatty liver disease (NAFLD), the underlying disease pathophysiology remains unclear. There is a great need for an efficient and reliable “human” in vitro model to study NAFLD and the progression to nonalcoholic steatohepatitis (NASH), which will soon become the leading indication for liver transplantation. Here, we review the recent developments in the use of three-dimensional (3D) liver organoids as a model to study NAFLD and NASH pathophysiology and possible treatments. Various techniques that are currently used to make liver organoids are discussed, such as the use of induced pluripotent stem cells versus primary cell lines and human versus murine cells. Moreover, methods for inducing lipid droplet accumulation and fibrosis to model NAFLD are explored. Finally, the limitations specific to the 3D organoid model for NAFLD/NASH are reviewed, highlighting the need for further development of multilineage models to include hepatic nonparenchymal cells and immune cells. The ultimate goal is to be able to accurately recapitulate the complex liver microenvironment in which NAFLD develops and progresses to NASH.

Keywords

NAFLD - NASH - human liver organoids - 3D cell culture

Authors' Contribution

Y.P. drafted the first manuscript, D.T. and I.B. helped with writing and reviewing the literature. B.E. and W.Z. initiated the concept and assisted in writing and critically reviewing the literature. All authors critically reviewed and participated in the writing of the paper. All authors approved the final version.


* These senior authors contributed equally to the study.




Publication History

Accepted Manuscript online:
31 August 2022

Article published online:
14 October 2022

© 2022. Thieme. All rights reserved.

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  • References

  • 1 Perumpail BJ, Khan MA, Yoo ER, Cholankeril G, Kim D, Ahmed A. Clinical epidemiology and disease burden of nonalcoholic fatty liver disease. World J Gastroenterol 2017; 23 (47) 8263-8276
    CrossrefPubMedGoogle Scholar
  • 2 Wong RJ, Aguilar M, Cheung R. et al. Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States. Gastroenterology 2015; 148 (03) 547-555
    CrossrefPubMedGoogle Scholar
  • 3 Musso G, Gambino R, Cassader M, Pagano G. Meta-analysis: natural history of non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-invasive tests for liver disease severity. Ann Med 2011; 43 (08) 617-649
    CrossrefPubMedGoogle Scholar
  • 4 Younossi ZM, Blissett D, Blissett R. et al. The economic and clinical burden of nonalcoholic fatty liver disease in the United States and Europe. Hepatology 2016; 64 (05) 1577-1586
    CrossrefPubMedGoogle Scholar
  • 5 Day CP. From fat to inflammation. Gastroenterology 2006; 130 (01) 207-210
    CrossrefPubMedGoogle Scholar
  • 6 Day CP, James OF. Steatohepatitis: a tale of two “hits”?. Gastroenterology 1998; 114 (04) 842-845
    CrossrefPubMedGoogle Scholar
  • 7 Buzzetti E, Pinzani M, Tsochatzis EA. The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metabolism 2016; 65 (08) 1038-1048
    CrossrefPubMedGoogle Scholar
  • 8 Malaguarnera M, Di Rosa M, Nicoletti F, Malaguarnera L. Molecular mechanisms involved in NAFLD progression. J Mol Med (Berl) 2009; 87 (07) 679-695
    CrossrefPubMedGoogle Scholar
  • 9 Friedman SL, Neuschwander-Tetri BA, Rinella M, Sanyal AJ. Mechanisms of NAFLD development and therapeutic strategies. Nat Med 2018; 24 (07) 908-922
    CrossrefPubMedGoogle Scholar
  • 10 Simian M, Bissell MJ. Organoids: A historical perspective of thinking in three dimensions. J Cell Biol 2017; 216 (01) 31-40
    CrossrefPubMedGoogle Scholar
  • 11 Shamir ER, Ewald AJ. Three-dimensional organotypic culture: experimental models of mammalian biology and disease. Nat Rev Mol Cell Biol 2014; 15 (10) 647-664
    CrossrefPubMedGoogle Scholar
  • 12 Marsee A, Roos FJM, Verstegen MMA. et al; HPB Organoid Consortium. Building consensus on definition and nomenclature of hepatic, pancreatic, and biliary organoids. Cell Stem Cell 2021; 28 (05) 816-832
    CrossrefPubMedGoogle Scholar
  • 13 Soto-Gutierrez A, Navarro-Alvarez N, Yagi H, Nahmias Y, Yarmush ML, Kobayashi N. Engineering of an hepatic organoid to develop liver assist devices. Cell Transplant 2010; 19 (06) 815-822
    CrossrefPubMedGoogle Scholar
  • 14 Takebe T, Sekine K, Enomura M. et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 2013; 499 (7459): 481-484
    CrossrefPubMedGoogle Scholar
  • 15 Freitas-Lopes MA, Mafra K, David BA, Carvalho-Gontijo R, Menezes GB. Differential location and distribution of hepatic immune cells. Cells 2017; 6 (04) 6
    PubMedGoogle Scholar
  • 16 Müller FA, Sturla SJ. Human in vitro models of nonalcoholic fatty liver disease. Curr Opin Toxicol 2019; 16: 9-16
    CrossrefPubMedGoogle Scholar
  • 17 Kunst RF, Niemeijer M, van der Laan LJW, Spee B, van de Graaf SFJ. From fatty hepatocytes to impaired bile flow: matching model systems for liver biology and disease. Biochem Pharmacol 2020; 180: 114173
    CrossrefPubMedGoogle Scholar
  • 18 Trefts E, Gannon M, Wasserman DH. The liver. Curr Biol 2017; 27 (21) R1147-R1151
    CrossrefPubMedGoogle Scholar
  • 19 Bissell DM, Tilles JG. Morphology and function of cells of human embryonic liver in monolayer culture. J Cell Biol 1971; 50 (01) 222-231
    CrossrefPubMedGoogle Scholar
  • 20 Bell CC, Hendriks DF, Moro SM. et al. Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease. Sci Rep 2016; 6: 25187
    CrossrefPubMedGoogle Scholar
  • 21 Hu H, Gehart H, Artegiani B. et al. Long-term expansion of functional mouse and human hepatocytes as 3D organoids. Cell 2018; 175 (06) 1591-1606.e19
    CrossrefPubMedGoogle Scholar
  • 22 Xiang C, Du Y, Meng G. et al. Long-term functional maintenance of primary human hepatocytes in vitro. Science 2019; 364 (6438): 399-402
    CrossrefPubMedGoogle Scholar
  • 23 Zhou Y, Shen JX, Lauschke VM. Comprehensive evaluation of organotypic and microphysiological liver models for prediction of drug-induced liver injury. Front Pharmacol 2019; 10: 1093
    CrossrefPubMedGoogle Scholar
  • 24 Shinozawa T, Kimura M, Cai Y. et al. High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids. Gastroenterology 2021; 160 (03) 831-846.e10
    CrossrefPubMedGoogle Scholar
  • 25 LiverTox: Clinical and Research Information on Drug-Induced Liver Injury [Internet]. Bethesda (MD): National Institute of Diabetes and Digestive and Kidney Diseases; 2012. Troglitazone. [Updated June 6, 2018]. Accessed on Sept 28, 2022, at: https://www.ncbi.nlm.nih.gov/books/NBK548142/
    PubMedGoogle Scholar
  • 26 Ramli MNB, Lim YS, Koe CT. et al. Human pluripotent stem cell-derived organoids as models of liver disease. Gastroenterology 2020; 159 (04) 1471-1486.e12
    CrossrefPubMedGoogle Scholar
  • 27 Wang F, Weaver VM, Petersen OW. et al. Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology. Proc Natl Acad Sci U S A 1998; 95 (25) 14821-14826
    CrossrefPubMedGoogle Scholar
  • 28 Weaver VM, Lelièvre S, Lakins JN. et al. beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. Cancer Cell 2002; 2 (03) 205-216
    CrossrefPubMedGoogle Scholar
  • 29 Reeves HL, Burt AD, Wood S, Day CP. Hepatic stellate cell activation occurs in the absence of hepatitis in alcoholic liver disease and correlates with the severity of steatosis. J Hepatol 1996; 25 (05) 677-683
    CrossrefPubMedGoogle Scholar
  • 30 Kisseleva T, Brenner DA. Role of hepatic stellate cells in fibrogenesis and the reversal of fibrosis. J Gastroenterol Hepatol 2007; 22 (Suppl. 01) S73-S78
    CrossrefPubMedGoogle Scholar
  • 31 Pingitore P, Sasidharan K, Ekstrand M, Prill S, Lindén D, Romeo S. Human multilineage 3D spheroids as a model of liver steatosis and fibrosis. Int J Mol Sci 2019; 20 (07) 20
    CrossrefPubMedGoogle Scholar
  • 32 Pingitore P, Romeo S. The role of PNPLA3 in health and disease. Biochim Biophys Acta Mol Cell Biol Lipids 2019; 1864 (06) 900-906
    CrossrefPubMedGoogle Scholar
  • 33 Xu JJ, Diaz D, O'Brien PJ. Applications of cytotoxicity assays and pre-lethal mechanistic assays for assessment of human hepatotoxicity potential. Chem Biol Interact 2004; 150 (01) 115-128
    CrossrefPubMedGoogle Scholar
  • 34 Gerets HH, Tilmant K, Gerin B. et al. Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biol Toxicol 2012; 28 (02) 69-87
    CrossrefPubMedGoogle Scholar
  • 35 McCarron S, Bathon B, Conlon DM. et al. Functional characterization of organoids derived from irreversibly damaged liver of patients with NASH. Hepatology 2021; 74 (04) 1825-1844
    CrossrefPubMedGoogle Scholar
  • 36 Broutier L, Andersson-Rolf A, Hindley CJ. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc 2016; 11 (09) 1724-1743
    CrossrefPubMedGoogle Scholar
  • 37 Prill S, Caddeo A, Baselli G. et al. The TM6SF2 E167K genetic variant induces lipid biosynthesis and reduces apolipoprotein B secretion in human hepatic 3D spheroids. Sci Rep 2019; 9 (01) 11585
    CrossrefPubMedGoogle Scholar
  • 38 Tostões RM, Leite SB, Serra M. et al. Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing. Hepatology 2012; 55 (04) 1227-1236
    CrossrefPubMedGoogle Scholar
  • 39 Park Y, Thadasina D, Isidan K. et al. Optimization of human liver organoid model for nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. [abstract] Am J Transplant 2022; 22 (s3): 413
    PubMedGoogle Scholar
  • 40 Zhang W, Isidan A, Sato K. et al. Fr366 advanced 3D human liver organoids created by multiple-hepatic lineage cells for the study of liver diseases. . [abstract] Gastroenterology 2021; 160: S-798-S-799
    CrossrefPubMedGoogle Scholar
  • 41 Akbari S, Sevinç GG, Ersoy N. et al. Robust, long-term culture of endoderm-derived hepatic organoids for disease modeling. Stem Cell Reports 2019; 13 (04) 627-641
    CrossrefPubMedGoogle Scholar
  • 42 Nuciforo S, Heim MH. Organoids to model liver disease. JHEP Rep 2020; 3 (01) 100198
    CrossrefPubMedGoogle Scholar
  • 43 Gurevich I, Burton SA, Munn C. et al. iPSC-derived hepatocytes generated from NASH donors provide a valuable platform for disease modeling and drug discovery. Biol Open 2020; 9 (12) 9
    PubMedGoogle Scholar
  • 44 Ouchi R, Togo S, Kimura M. et al. Modeling steatohepatitis in humans with pluripotent stem cell-derived organoids. Cell Metab 2019; 30 (02) 374-384.e6
    CrossrefPubMedGoogle Scholar
  • 45 Tsuchida T, Friedman SL. Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol 2017; 14 (07) 397-411
    CrossrefPubMedGoogle Scholar
  • 46 Chavez-Tapia NC, Rosso N, Tiribelli C. Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease. BMC Gastroenterol 2012; 12: 20
    CrossrefPubMedGoogle Scholar
  • 47 Giraudi PJ, Becerra VJ, Marin V, Chavez-Tapia NC, Tiribelli C, Rosso N. The importance of the interaction between hepatocyte and hepatic stellate cells in fibrogenesis induced by fatty accumulation. Exp Mol Pathol 2015; 98 (01) 85-92
    CrossrefPubMedGoogle Scholar
  • 48 Stephenson K, Kennedy L, Hargrove L. et al. Updates on dietary models of nonalcoholic fatty liver disease: current studies and insights. Gene Expr 2018; 18 (01) 5-17
    CrossrefPubMedGoogle Scholar
  • 49 Farrell G, Schattenberg JM, Leclercq I. et al. Mouse models of nonalcoholic steatohepatitis: toward optimization of their relevance to human nonalcoholic steatohepatitis. Hepatology 2019; 69 (05) 2241-2257
    CrossrefPubMedGoogle Scholar
  • 50 Hansen HH, Feigh M, Veidal SS, Rigbolt KT, Vrang N, Fosgerau K. Mouse models of nonalcoholic steatohepatitis in preclinical drug development. Drug Discov Today 2017; 22 (11) 1707-1718
    CrossrefPubMedGoogle Scholar
  • 51 Elbadawy M, Yamanaka M, Goto Y. et al. Efficacy of primary liver organoid culture from different stages of non-alcoholic steatohepatitis (NASH) mouse model. Biomaterials 2020; 237: 119823
    CrossrefPubMedGoogle Scholar
  • 52 Jiang M, Wu N, Chen X. et al. Pathogenesis of and major animal models used for nonalcoholic fatty liver disease. J Int Med Res 2019; 47 (04) 1453-1466
    CrossrefPubMedGoogle Scholar
  • 53 Prior N, Inacio P, Huch M. Liver organoids: from basic research to therapeutic applications. Gut 2019; 68 (12) 2228-2237
    CrossrefPubMedGoogle Scholar
  • 54 Liang W, Menke AL, Driessen A. et al. Establishment of a general NAFLD scoring system for rodent models and comparison to human liver pathology. PLoS One 2014; 9 (12) e115922
    CrossrefPubMedGoogle Scholar
  • 55 Van Norman GA. Limitations of animal studies for predicting toxicity in clinical trials: is it time to rethink our current approach?. JACC Basic Transl Sci 2019; 4 (07) 845-854
    CrossrefPubMedGoogle Scholar
  • 56 Lasli S, Kim HJ, Lee K. et al. A human liver-on-a-chip platform for modeling nonalcoholic fatty liver disease. Adv Biosyst 2019; 3 (08) e1900104
    CrossrefPubMedGoogle Scholar
  • 57 Soret PA, Magusto J, Housset C, Gautheron J. In vitro and in vivo models of non-alcoholic fatty liver disease: a critical appraisal. J Clin Med 2020; 10 (01) 36
    CrossrefPubMedGoogle Scholar
  • 58 Wang Y, Wang H, Deng P. et al. Modeling human nonalcoholic fatty liver disease (NAFLD) with an organoids-on-a-chip system. ACS Biomater Sci Eng 2020; 6 (10) 5734-5743
    CrossrefPubMedGoogle Scholar
  • 59 Hassan S, Sebastian S, Maharjan S. et al. Liver-on-a-chip models of fatty liver disease. Hepatology 2020; 71 (02) 733-740
    CrossrefPubMedGoogle Scholar
  • 60 Gori M, Simonelli MC, Giannitelli SM, Businaro L, Trombetta M, Rainer A. Investigating nonalcoholic fatty liver disease in a liver-on-a-chip microfluidic device. PLoS One 2016; 11 (07) e0159729
    CrossrefPubMedGoogle Scholar
  • 61 Nishimura M, Hagi M, Ejiri Y. et al. Secretion of albumin and induction of CYP1A2 and CYP3A4 in novel three-dimensional culture system for human hepatocytes using micro-space plate. Drug Metab Pharmacokinet 2010; 25 (03) 236-242
    CrossrefPubMedGoogle Scholar
  • 62 Goral VN, Hsieh YC, Petzold ON, Clark JS, Yuen PK, Faris RA. Perfusion-based microfluidic device for three-dimensional dynamic primary human hepatocyte cell culture in the absence of biological or synthetic matrices or coagulants. Lab Chip 2010; 10 (24) 3380-3386
    CrossrefPubMedGoogle Scholar
  • 63 Kaur G, Dufour JM. Cell lines: valuable tools or useless artifacts. Spermatogenesis 2012; 2 (01) 1-5
    CrossrefPubMedGoogle Scholar
  • 64 Nozaki K, Mochizuki W, Matsumoto Y. et al. Co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes. J Gastroenterol 2016; 51 (03) 206-213
    CrossrefPubMedGoogle Scholar
  • 65 Li X, Wang H. Multiple organs involved in the pathogenesis of non-alcoholic fatty liver disease. Cell Biosci 2020; 10 (01) 140
    CrossrefPubMedGoogle Scholar
  • 66 Rogoz A, Reis BS, Karssemeijer RA, Mucida D. A 3-D enteroid-based model to study T-cell and epithelial cell interaction. J Immunol Methods 2015; 421: 89-95
    CrossrefPubMedGoogle Scholar