Abstract
Metabolic dysfunction-associated steatotic liver disease (MASLD) is one of the most pervasive liver disorders. It can progress to metabolic dysfunction-associated steatohepatitis (MASH), hallmarked by increased inflammation and inclination to permanent liver damage. Given the limited treatment options available to patients, models that recapitulate critical features of disease pathogenesis are needed to improve drug development. Here, a MASLD model was developed by differentiating hepatocyte-like cells (HLCs) from human induced pluripotent stem cells (hiPSCs) in 2D and 3D, in which hepatic aggregates exhibited enhanced functionality. Induced HLCs exposed to free fatty acids led to a steatotic phenotype, partially reducing hepatic function. Biochemical cues released from induced HLCs promoted activation of fibroblasts, resulting in increased inflammatory cytokine secretion and extracellular matrix (ECM) deposition. These findings suggest paracrine signaling from hepatocytes to the surrounding stroma can trigger the progression of MASH and fibrosis. Collectively, our results demonstrate the utility of hiPSC derivatives for modeling liver diseases and reveal how heterotypic interactions can drive disease progression.
Data availability
All data generated in this study to demonstrate the key findings are provided in the manuscript and the Supplementary Information. Any other data supporting this study are available from the corresponding author upon reasonable request.
References
Bhatia, S. N., Underhill, G. H., Zaret, K. S. & Fox, I. J. Cell and tissue engineering for liver disease. Sci. Transl Med. (2014). https://doi.org/10.1126/SCITRANSLMED.3005975
Eslam, M. et al. A new definition for metabolic dysfunction-associated fatty liver disease: an international expert consensus statement. J. Hepatol. 73, 202–209. https://doi.org/10.1016/J.JHEP.2020.03.039 (2020).
Lim, G. E. H. et al. An observational data Meta-analysis on the differences in prevalence and risk factors between MAFLD vs NAFLD. Clin. Gastroenterol. Hepatol. 21, 619–629e7. https://doi.org/10.1016/J.CGH.2021.11.038 (2023).
Nassir, F. NAFLD: Mechanisms, treatments, and biomarkers. Biomolecules (2022). https://doi.org/10.3390/BIOM12060824
Anstee, Q. M. & Day, C. P. The genetics of NAFLD. Nat. Rev. Gastroenterol. Hepatol. 10, 645–655. https://doi.org/10.1038/NRGASTRO.2013.182 (2013).
Feldstein, A. E. et al. Diet associated hepatic steatosis sensitizes to Fas mediated liver injury in mice. J. Hepatol. 39, 978–983. https://doi.org/10.1016/S0168-8278(03)00460-4 (2003).
Malhi, H., Bronk, S. F., Werneburg, N. W. & Gores, G. J. Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis. J. Biol. Chem. 281, 12093–12101. https://doi.org/10.1074/JBC.M510660200 (2006).
De Gottardi, A. et al. Microarray analyses and molecular profiling of steatosis induction in immortalized human hepatocytes. Lab. Invest. 87, 792–806. https://doi.org/10.1038/LABINVEST.3700590 (2007).
Gómez-Lechón, M. J. et al. A human hepatocellular in vitro model to investigate steatosis. Chem. Biol. Interact. 165, 106–116. https://doi.org/10.1016/J.CBI.2006.11.004 (2007).
Srivastava, S. & Chan, C. Application of metabolic flux analysis to identify the mechanisms of free fatty acid toxicity to human hepatoma cell line. Biotechnol. Bioeng. 99, 399–410. https://doi.org/10.1002/BIT.21568 (2008).
Flynn, T. J. & Ferguson, M. S. Multiendpoint mechanistic profiling of hepatotoxicants in HepG2/C3A human hepatoma cells and novel statistical approaches for development of a prediction model for acute hepatotoxicity. Toxicol. In Vitro. 22, 1618–1631. https://doi.org/10.1016/J.TIV.2008.04.016 (2008).
Ricchi, M. et al. Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes. J. Gastroenterol. Hepatol. (Australia). 24, 830–840. https://doi.org/10.1111/J.1440-1746.2008.05733.X (2009).
Garcia, M. C., Amankwa-Sakyi, M. & Flynn, T. J. Cellular glutathione in fatty liver in vitro models. Toxicol. In Vitro. 25, 1501–1506. https://doi.org/10.1016/J.TIV.2011.05.011 (2011).
Anthérieu, S., Rogue, A., Fromenty, B., Guillouzo, A. & Robin, M. A. Induction of vesicular steatosis by Amiodarone and Tetracycline is associated with up-regulation of lipogenic genes in Heparg cells. Hepatology 53, 1895–1905. https://doi.org/10.1002/HEP.24290 (2011).
Barbero-Becerra, V. J. et al. The interplay between hepatic stellate cells and hepatocytes in an in vitro model of NASH. Toxicol. In Vitro. 29, 1753–1758. https://doi.org/10.1016/J.TIV.2015.07.010 (2015).
Green, C. J. et al. Characterization of lipid metabolism in a novel immortalized human hepatocyte cell line. Am. J. Physiol. Endocrinol. Metab. 309, E511–E522. https://doi.org/10.1152/AJPENDO.00594.2014 (2015).
Leite, S. B. et al. Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitro. Biomaterials 78, 1–10. https://doi.org/10.1016/J.BIOMATERIALS.2015.11.026 (2016).
Tolosa, L. et al. Advantageous use of HepaRG cells for the screening and mechanistic study of drug-induced steatosis. Toxicol. Appl. Pharmacol. 302, 1–9. https://doi.org/10.1016/J.TAAP.2016.04.007 (2016).
Michaut, A. et al. A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: application to acetaminophen. Toxicol. Appl. Pharmacol. 292, 40–55. https://doi.org/10.1016/J.TAAP.2015.12.020 (2016).
Gunn,P. J., Green, C. J., Pramfalk, C. & Hodson, L. In vitro cellular models of human hepatic fatty acid metabolism: Differences between Huh7 and HepG2 cell lines in human and fetal bovine culturing serum. Physiol. Rep. https://doi.org/10.14814/PHY2.13532 (2017).
Bai, X. et al. Valproate induced hepatic steatosis by enhanced fatty acid uptake and triglyceride synthesis. Toxicol. Appl. Pharmacol. 324, 12–25. https://doi.org/10.1016/J.TAAP.2017.03.022 (2017).
Luckert, C. et al. Adverse outcome Pathway-Driven analysis of liver steatosis in vitro: A case study with cyproconazole. Chem. Res. Toxicol. 31, 784–798. https://doi.org/10.1021/ACS.CHEMRESTOX.8B00112 (2018).
Cui, W., Chen, S. L. & Hu, K. Q. Quantification and mechanisms of oleic acid-induced steatosis in HepG2 cells. Am. J. Transl Res. 2, 95–104 (2010).
Donato, M. T. et al. Potential impact of steatosis on cytochrome P450 enzymes of human hepatocytes isolated from fatty liver grafts. Drug Metab. Dispos. 34, 1556–1562. https://doi.org/10.1124/DMD.106.009670 (2006).
Wobser, H. et al. Lipid accumulation in hepatocytes induces fibrogenic activation of hepatic stellate cells. Cell. Res. 19, 996–1005. https://doi.org/10.1038/CR.2009.73 (2009).
Wanninger, J. et al. Lipid accumulation impairs adiponectin-mediated induction of activin A by increasing TGFbeta in primary human hepatocytes. Biochim. Biophys. Acta Mol. Cell. Biol. Lipids. 1811, 626–633. https://doi.org/10.1016/J.BBALIP.2010.11.001 (2011).
Sharma, S., Mells, J. E., Fu, P. P., Saxena, N. K. & Anania, F. A. GLP-1 analogs reduce hepatocyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy. PLoS One, https://doi.org/10.1371/JOURNAL.PONE.0025269 (2011).
Feaver, R. E. et al. Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis. JCI Insight. https://doi.org/10.1172/JCI.INSIGHT.90954 (2016).
Bell, C. C. et al. Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease. Sci. Rep. https://doi.org/10.1038/SREP25187 (2016).
Kirovski, G. et al. Hepatic steatosis causes induction of the chemokine RANTES in the absence of significant hepatic inflammation. Int. J. Clin. Exp. Pathol. 3, 675–680 (2010).
Flessa, C. M. et al. Genetic and diet-induced animal models for non-alcoholic fatty liver disease (NAFLD) Research. Int. J. Mol. Sci. https://doi.org/10.3390/IJMS232415791 (2023).
Graffmann, N. et al. Modeling nonalcoholic fatty liver disease with human pluripotent stem Cell-Derived immature Hepatocyte-Like cells reveals activation of PLIN2 and confirms regulatory functions of peroxisome Proliferator-Activated receptor alpha. Stem Cells Dev. 25, 1119–1133. https://doi.org/10.1089/SCD.2015.0383 (2016).
Parafati, M., Kirby, R. J., Khorasanizadeh, S., Rastinejad, F. & Malany, S. A nonalcoholic fatty liver disease model in human induced pluripotent stem cell-derived hepatocytes, created by endoplasmic reticulum stress-induced steatosis. DMM Disease Models Mech, https://doi.org/10.1242/DMM.033530 (2018).
Ouchi, R. et al. Modeling steatohepatitis in humans with pluripotent stem Cell-Derived organoids. Cell. Metab. 30, 374–384e6. https://doi.org/10.1016/J.CMET.2019.05.007 (2019).
Sinton, M. C. et al. A human pluripotent stem cell model for the analysis of metabolic dysfunction in hepatic steatosis. iScience https://doi.org/10.1016/J.ISCI.2020.101931 (2021).
Holmgren, G. et al. Characterization of human induced pluripotent stem cell-derived hepatocytes with mature features and potential for modeling metabolic diseases. Int. J. Mol. Sci. https://doi.org/10.3390/IJMS21020469 (2020).
Gurevich, I. et al. iPSC-derived hepatocytes generated from NASH donors provide a valuable platform for disease modeling and drug discovery. Biol Open, https://doi.org/10.1242/BIO.055087 (2020).
Tilson, S. G. et al. Modeling PNPLA3-Associated NAFLD using Human-Induced pluripotent stem cells. Hepatology 74, 2998–3017. https://doi.org/10.1002/HEP.32063 (2021).
Kimura, M. et al. En masse organoid phenotyping informs metabolic-associated genetic susceptibility to NASH. Cell 185, 4216–4232e16. https://doi.org/10.1016/J.CELL.2022.09.031 (2022).
Kumar, M. et al. A fully defined matrix to support a pluripotent stem cell derived multi-cell-liver steatohepatitis and fibrosis model. Biomaterials. https://doi.org/10.1016/J.BIOMATERIALS.2021.121006 (2021).
Park, J. et al. IL-6/STAT3 axis dictates the PNPLA3-mediated susceptibility to non-alcoholic fatty liver disease. J. Hepatol. 78, 45–56. https://doi.org/10.1016/j.jhep.2022.08.022 (2023).
Rezvani, M., Vallier, L. & Guillot, A. Modeling nonalcoholic fatty liver disease in the dish using Human-Specific platforms: strategies and limitations. Cell. Mol. Gastroenterol. Hepatol. 15, 1135–1145. https://doi.org/10.1016/j.jcmgh.2023.01.014 (2023).
Graffmann, N., Scherer, B. & Adjaye, J. In vitro differentiation of pluripotent stem cells into hepatocyte like cells – Basic principles and current progress. Stem Cell Res, https://doi.org/10.1016/J.SCR.2022.102763 (2022).
Dao Thi, V. L. et al. Stem cell-derived polarized hepatocytes. Nat. Commun. https://doi.org/10.1038/S41467-020-15337-2 (2020).
Dollé, L. et al. EpCAM and the biology of hepatic stem/progenitor cells. Am. J. Physiology-Gastrointestinal Liver Physiol. 308, G233–G250. https://doi.org/10.1152/ajpgi.00069.2014 (2015).
March, S. et al. Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens. Nat. Protoc. 10, 2027–2053. https://doi.org/10.1038/nprot.2015.128 (2015).
Caldwell, S. et al. Hepatocellular ballooning in NASH. J. Hepatol. 53, 719–723. https://doi.org/10.1016/j.jhep.2010.04.031 (2010).
Tomaz, R. A. et al. Generation of functional hepatocytes by forward programming with nuclear receptors. Elife, https://doi.org/10.7554/eLife.71591 (2022).
Nass, K. J. et al. High prevalence of Apolipoprotein B dyslipoproteinemias in non-alcoholic fatty liver disease: the lifelines cohort study. Metabolism 72, 37–46. https://doi.org/10.1016/J.METABOL.2017.04.004 (2017).
Wang, J. et al. Serum ApoB levels independently predict the development of non-alcoholic fatty liver disease: A 7-year prospective study. Liver Int. 37, 1202–1208. https://doi.org/10.1111/LIV.13363 (2017).
Dutta, A. K. Adiponutrin (PNPLA3) in liver fibrogenesis: is unaltered HepG2 cell line a better model system compared to murine models? Med. Hypotheses. 85, 736–739. https://doi.org/10.1016/J.MEHY.2015.10.019 (2015).
Xu, Q., Norman, J. T., Shrivastav, S., Lucio-Cazana, J. & Kopp, J. B. In vitro models of TGF-β-induced fibrosis suitable for high-throughput screening of antifibrotic agents. Am. J. Physiol. Ren. Physiol., https://doi.org/10.1152/AJPRENAL.00379.2006 (2007).
Caliari, S. R. et al. Stiffening hydrogels for investigating the dynamics of hepatic stellate cell mechanotransduction during myofibroblast activation. Sci. Rep. 6, 21387. https://doi.org/10.1038/srep21387 (2016).
Zhong, L. et al. Runx2 activates hepatic stellate cells to promote liver fibrosis via transcriptionally regulating Itgav expression. Clin. Transl Med. 13, e1316. https://doi.org/10.1002/CTM2.1316 (2023).
Wang, Z. Y. et al. Single-cell and bulk transcriptomics of the liver reveals potential targets of NASH with fibrosis. Sci. Rep. 11, 1–15 https://doi.org/10.1038/s41598-021-98806-y (2021).
She, S. et al. Functional roles of chemokine receptor CCR2 and its ligands in liver. Disease Front. Immunol. https://doi.org/10.3389/fimmu.2022.812431 (2022).
Li, L. et al. CCL13 and human diseases. Front. Immunol. https://doi.org/10.3389/fimmu.2023.1176639 (2023).
Poulsen, K. L., Ross, C. K. C. D., Chaney, J. K. & Nagy, L. E. Role of the chemokine system in liver fibrosis: a narrative review. Dig. Med. Res. 5, 30–30. https://doi.org/10.21037/dmr-21-87 (2022).
Liang, Y. et al. Integrated single-cell transcriptomics reveals the hypoxia‐induced inflammation‐cancer transformation in NASH‐derived hepatocellular carcinoma. Cell. Prolif. https://doi.org/10.1111/cpr.13576 (2024).
Zhang, Y. et al. GCSF deficiency attenuates nonalcoholic fatty liver disease through regulating GCSFR-SOCS3-JAK-STAT3 pathway and immune cells infiltration. Am. J. Physiology-Gastrointestinal Liver Physiol. 320, G531–G542. https://doi.org/10.1152/ajpgi.00342.2020 (2021).
Gieseck, R. L. et al. Interleukin-13 Activates distinct cellular pathways leading to ductular reaction, steatosis, and fibrosis. Immunity 45, 145–158 https://doi.org/10.1016/j.immuni.2016.06.009 (2016).
Lin, T. Y. et al. IL-29 promoted obesity-induced inflammation and insulin resistance. Cell. Mol. Immunol. 17, 369–379. https://doi.org/10.1038/s41423-019-0262-9 (2020).
Shoji, H. et al. Interleukin-34 as a fibroblast-derived marker of liver fibrosis in patients with non-alcoholic fatty liver disease. Sci. Rep. 6, 28814. https://doi.org/10.1038/srep28814 (2016).
Coulon, S. et al. Evaluation of inflammatory and angiogenic factors in patients with non-alcoholic fatty liver disease. Cytokine 59, 442–449. https://doi.org/10.1016/j.cyto.2012.05.001 (2012).
Shen, H. et al. Hepatocyte-derived VEGFA accelerates the progression of non-alcoholic fatty liver disease to hepatocellular carcinoma via activating hepatic stellate cells. Acta Pharmacol. Sin. 43, 2917–2928. https://doi.org/10.1038/s41401-022-00907-5 (2022).
Younesi, F. S., Miller, A. E., Barker, T. H., Rossi, F. M. V. & Hinz, B. Fibroblast and myofibroblast activation in normal tissue repair and fibrosis. Nat. Rev. Mol. Cell. Biol. 25, 617–638. https://doi.org/10.1038/s41580-024-00716-0 (2024).
Alisafaei, F. et al. Vimentin is a key regulator of cell mechanosensing through opposite actions on actomyosin and microtubule networks. Commun. Biol. 7, 658. https://doi.org/10.1038/s42003-024-06366-4 (2024).
Wang, Y., Keshavarz, M., Barhouse, P. & Smith, Q. Strategies for regenerative vascular tissue engineering. Adv. Biol. https://doi.org/10.1002/adbi.202200050 (2023).
Shinozawa, T. et al. High-Fidelity Drug-Induced liver injury screen using human pluripotent stem Cell–Derived organoids. Gastroenterology 160, 831–846e10. https://doi.org/10.1053/j.gastro.2020.10.002 (2021).
Wiering, L., Subramanian, P. & Hammerich, L. Hepatic stellate cells: dictating outcome in nonalcoholic fatty liver disease. Cell. Mol. Gastroenterol. Hepatol. 15, 1277–1292. https://doi.org/10.1016/j.jcmgh.2023.02.010 (2023).
Cheng, Z., Chu, H., Seki, E., Lin, R. & Yang, L. Hepatocyte programmed cell death: The trigger for inflammation and fibrosis in metabolic dysfunction-associated steatohepatitis. Front. Cell. Dev. Biol. https://doi.org/10.3389/fcell.2024.1431921 (2024).
Song, H. H. G. et al. Transient support from fibroblasts is sufficient to drive functional vascularization in engineered tissues. Adv. Funct. Mater. https://doi.org/10.1002/adfm.202003777 (2020).
Chhabra, A. et al. A vascularized model of the human liver mimics regenerative responses. Proc. Natl. Acad. Sci. U S A. 119, e2115867119. https://doi.org/10.1073/PNAS.2115867119/SUPPL_FILE/PNAS.2115867119.SM01.AVI (2022).
Wang, E., Andrade, M. J. & Smith, Q. Vascularized liver-on-a-chip model to investigate nicotine-induced dysfunction. Biomicrofluidics https://doi.org/10.1063/5.0172677 (2023).
Grigoryan, B. et al. Multivascular networks and functional intravascular topologies within biocompatible hydrogels. Science 364, 458–464. https://doi.org/10.1126/science.aav9750 (2019).
Geng, Y., Faber, K. N., de Meijer, V. E., Blokzijl, H. & Moshage, H. How does hepatic lipid accumulation lead to lipotoxicity in non-alcoholic fatty liver disease? Hepatol. Int. 15, 21–35. https://doi.org/10.1007/s12072-020-10121-2 (2021).
Clark, C. T., Wang, Y., Johnson, D. C., Lee, S. C. & Smith, Q. Effects of PDMS culture on stem cell differentiation towards definitive endoderm and hepatocytes. Acta Biomater. 200, 508–519. https://doi.org/10.1016/j.actbio.2025.05.017 (2025).
Li, Y., Townsend, K. M., Dorn, R. S., Prescher, J. A. & Potma, E. O. Enhancing Alkyne-Based Raman tags with a sulfur linker. J. Phys. Chem. B. 127, 1976–1982. https://doi.org/10.1021/acs.jpcb.2c09093 (2023).
Acknowledgements
We thank Dr. Edwin Monuki for kindly providing the KOLF iPSC line.
Funding
The California Institute for Regenerative Medicine supported research reported in this publication under Award Number EDUC4-12822 to Y.W. and M.A. The content is solely the responsibility of the authors and does not necessarily represent the official views of the California Institute for Regenerative Medicine. This study was made possible in part through access to the Optical Biology Core Facility of the Developmental Biology Center, a shared resource supported by the Cancer Center Support Grant (CA-62203) and NIH-S10OD032327-01. Q.S. acknowledges the support from the National Institutes of Health (NIH) (Grant No. R35GM151099) and the Hanna Gray Fellowship Program from the Howard Hughes Medical Institute (Grant No. GT15187).
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Y.W. designed the study, performed most of the experiments, analyzed data, and wrote the manuscript. Y.L. performed the experiments, analyzed the data, and wrote the manuscript on Raman spectroscopy and imaging. D.B., L.M.K, Z.Q., J.F., C.T.C., D.C., and M.A. supported the conduct of select experiments. D.B. partook in the processing of confocal images. M.A. supported the study design. E.P. designed and supervised the study regarding Raman spectroscopy and imaging. Q.S. led the design and execution of the study and wrote the manuscript.
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Wang, Y., Berlin, D., Li, Y. et al. Modeling fatty liver disease and progression with stem cell derived hepatocytes. Sci Rep (2026). https://doi.org/10.1038/s41598-025-34762-1
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DOI: https://doi.org/10.1038/s41598-025-34762-1