Liver cell types derived from induced pluripotent stem cells (iPSCs) share the potential to investigate development, toxicity, as well as genetic and infectious disease in ways currently limited by the availability of primary tissue. With the added advantage of patient specificity, which can play a role in all of these areas. Many iPSC differentiation protocols focus on 3 dimensional (3D) or organotypic differentiation, as these offer the advantage of more closely mimicking in vivo systems including; the formation of tissue like architecture and interactions/crosstalk between different cell types. Ultimately such models have the potential to be used clinically and either with or more aptly, in place of animal models. Along with the development of organotypic and micro-tissue models, there will be a need to co-develop imaging technologies to enable their visualization. A variety of liver models termed "organoids" have been reported in the literature ranging from simple spheres or cysts of a single cell type, usually hepatocytes, to those containing multiple cell types combined during the differentiation process such as hepatic stellate cells, endothelial cells, and mesenchymal cells, often leading to an improved hepatic phenotype. These allow specific functions or readouts to be examined such as drug metabolism, protein secretion or an improved phenotype, but because of their relative simplicity they lack the flexibility and general applicability of ex vivo tissue culture. In the liver field these are more often constructed rather than developed together organotypically as seen in other organoid models such as brain, kidney, lung and intestine. Having access to organotypic liver like surrogates containing multiple cell types with in vivo like interactions/architecture, would provide vastly improved models for disease, toxicity and drug development, combining disciplines such as microfluidic chip technology with organoids and ultimately paving the way to new therapies.

Department of Pediatric Research Oslo University Hospital Oslo Norway

Hybrid Technology Hub Center of Excellence Institute of Basic Medical Sciences University of Oslo Oslo Norway

Institute of Immunology Oslo University Hospital Oslo Norway

Institute of Physics of the Czech Academy of Sciences Prague Czechia

Norwegian Center for Stem Cell Research Oslo University Hospital University of Oslo Oslo Norway

See more in PubMed

Huch M, Knoblich JA, Lutolf MP, Martinez-Arias A. The hope and the hype of organoid research. Development. (2017) 144:938–41. 10.1242/dev.150201 PubMed DOI

Xu H, Jiao Y, Qin S, Zhao W, Chu Q, Wu K. Organoid technology in disease modeling, drug development, personalized treatment and regeneration medicine. Exp Hematol Oncol. (2018) 7:30. 10.1186/s40164-018-0122-9 PubMed DOI PMC

Clevers H. Modeling development and disease with organoids. Cell. (2016) 165:1586–97. 10.1016/j.cell.2016.05.082 PubMed DOI

Qian X, Song H, Ming GL. Brain organoids: advances, applications and challenges. Development. (2019) 146:dev166074. 10.1242/dev.166074 PubMed DOI PMC

Patterson M, Chan DN, Ha I, Case D, Cui Y, Van Handel B, et al. . Defining the nature of human pluripotent stem cell progeny. Cell Res. (2012) 22:178–93. 10.1038/cr.2011.133 PubMed DOI PMC

Baxter M, Withey S, Harrison S, Segeritz CP, Zhang F, Atkinson-Dell R, et al. . Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes. J Hepatol. (2015) 62:581–9. 10.1016/j.jhep.2014.10.016 PubMed DOI PMC

Sato T, Vries RG, Snippert HJ, Van De Wetering M, Barker N, Stange DE, et al. . Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. (2009) 459:262–5. 10.1038/nature07935 PubMed DOI

Eiraku M, Sasai Y. Self-formation of layered neural structures in three-dimensional culture of ES cells. Curr Opin Neurobiol. (2012) 22:768–77. 10.1016/j.conb.2012.02.005 PubMed DOI

Workman MJ, Mahe MM, Trisno S, Poling HM, Watson CL, Sundaram N, et al. . Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system. Nat Med. (2017) 23:49–59. 10.1038/nm.4233 PubMed DOI PMC

Lee WM. Acute liver failure in the United States. Semin Liver Dis. (2003) 23:217–26. 10.1055/s-2003-42641 PubMed DOI

Lyubimov AVX, Das LB, Prakash C. CYP450 enzymes in drug discovery and development: an overview. In: Encyclopedia of Drug Metabolism and Interactions. John Wiley & Sons, Inc. (2012). p. 1–35.

Tomalik-Scharte D, Lazar A, Fuhr U, Kirchheiner J. The clinical role of genetic polymorphisms in drug-metabolizing enzymes. Pharmacogenomics J. (2008) 8:4–15. 10.1038/sj.tpj.6500462 PubMed DOI

Crews KR, Gaedigk A, Dunnenberger HM, Leeder JS, Klein TE, Caudle KE, et al. . Clinical pharmacogenetics implementation consortium guidelines for cytochrome P450 2D6 genotype and codeine therapy: 2014 update. Clin Pharmacol Ther. (2014) 95:376–82. 10.1038/clpt.2013.254 PubMed DOI PMC

Driehuis E, Clevers H. CRISPR/Cas 9 genome editing and its applications in organoids. Am J Physiol Gastrointest Liver Physiol. (2017) 312:G257–65. 10.1152/ajpgi.00410.2016 PubMed DOI

Artegiani B, Hendriks D, Beumer J, Kok R, Zheng X, Joore I, et al. . Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing. Nat Cell Biol. (2020) 22:321–31. 10.1038/s41556-020-0472-5 PubMed DOI

Nicolas CT, Hickey RD, Chen HS, Mao SA, Lopera Higuita M, Wang Y, et al. . Concise review: liver regenerative medicine: from hepatocyte transplantation to bioartificial livers and bioengineered grafts. Stem Cells. (2017) 35:42–50. 10.1002/stem.2500 PubMed DOI PMC

Takebe T, Sekine K, Kimura M, Yoshizawa E, Ayano S, Koido M, et al. . Massive and reproducible production of liver buds entirely from human pluripotent stem cells. Cell Rep. (2017) 21:2661–70. 10.1016/j.celrep.2017.11.005 PubMed DOI

Ishigami M, Masumoto H, Ikuno T, Aoki T, Kawatou M, Minakata K, et al. . Human iPS cell-derived cardiac tissue sheets for functional restoration of infarcted porcine hearts. PLoS ONE. (2018) 13:e0201650. 10.1371/journal.pone.0201650 PubMed DOI PMC

Achberger K, Probst C, Haderspeck J, Bolz S, Rogal J, Chuchuy J, et al. . Merging organoid and organ-on-a-chip technology to generate complex multi-layer tissue models in a human retina-on-a-chip platform. Elife. (2019) 8:e43533. 10.7554/eLife.46188.034 PubMed DOI PMC

Park SE, Georgescu A, Huh D. Organoids-on-a-chip. Science. (2019) 364:960–5. 10.1126/science.aaw7894 PubMed DOI PMC

Eicher AK, Berns HM, Wells JM. Translating developmental principles to generate human gastric organoids. Cell Mol Gastroenterol Hepatol. (2018) 5:353–63. 10.1016/j.jcmgh.2017.12.014 PubMed DOI PMC

Rossi G, Manfrin A, Lutolf MP. Progress and potential in organoid research. Nat Rev Genet. (2018) 19:671–87. 10.1038/s41576-018-0051-9 PubMed DOI

Takebe T, Wells JM, Helmrath MA, Zorn AM. Organoid center strategies for accelerating clinical translation. Cell Stem Cell. (2018) 22:806–9. 10.1016/j.stem.2018.05.008 PubMed DOI PMC

Gunther C, Brevini T, Sampaziotis F, Neurath MF. What gastroenterologists and hepatologists should know about organoids in 2019. Dig Liver Dis. (2019) 51:753–60. 10.1016/j.dld.2019.02.020 PubMed DOI

Kubo A, Shinozaki K, Shannon JM, Kouskoff V, Kennedy M, Woo S, et al. . Development of definitive endoderm from embryonic stem cells in culture. Development. (2004) 131:1651–62. 10.1242/dev.01044 PubMed DOI

Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. (2008) 132:661–80. 10.1016/j.cell.2008.02.008 PubMed DOI

Nakanishi M, Kurisaki A, Hayashi Y, Warashina M, Ishiura S, Kusuda-Furue M, et al. . Directed induction of anterior posterior primitive streak by Wnt from embryonic stem cells cultured in a chemically defined serum-free medium. FASEB J. (2009) 23:114–22. 10.1096/fj.08-111203 PubMed DOI

Wells JM, Melton DA. Vertebrate endoderm development. Annu Rev Cell Dev Biol. (1999) 15:393–410. 10.1146/annurev.cellbio.15.1.393 PubMed DOI

Kamiya A, Kinoshita T, Ito Y, Matsui T, Morikawa Y, Senba E, et al. . Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer. EMBO J. (1999) 18:2127–36. 10.1093/emboj/18.8.2127 PubMed DOI PMC

Deutsch G, Jung J, Zheng M, Lora J, Zaret KS. A bipotential precursor population for pancreas and liver within the embryonic endoderm. Development. (2001) 128:871–81. 10.1242/dev.128.6.871 PubMed DOI

Jung J, Zheng M, Goldfarb M, Zaret KS. Initiation of mammalian liver development from endoderm by fibroblast growth factors. Science. (1999) 284:1998–2003. 10.1126/science.284.5422.1998 PubMed DOI

Rossi JM, Dunn NR, Hogan BLM, Zaret KS. Distinct mesodermal signals, including BMPs from the septum transversum mesenchyme, are required in combination for hepatogenesis from the endoderm. Genes Dev. (2001) 15:1998–2009. 10.1101/gad.904601 PubMed DOI PMC

Chung WS, Shin CH, Stainier DY. Bmp2 signaling regulates the hepatic versus pancreatic fate decision. Dev Cell. (2008) 15:738–48. 10.1016/j.devcel.2008.08.019 PubMed DOI PMC

Rogler LE. Selective bipotential differentiation of mouse embryonic hepatoblasts in vitro. Am J Pathol. (1997) 150:591–602. PubMed PMC

Spagnoli FM, Amicone L, Tripodi M, Weiss MC. Identification of a bipotential precursor cell in hepatic cell lines derived from transgenic mice expressing cyto-met in the liver. J Cell Biol. (1998) 143:1101–12. 10.1083/jcb.143.4.1101 PubMed DOI PMC

Kinoshita T, Sekiguchi T, Xu MJ, Ito Y, Kamiya A, Tsuji K, et al. . Hepatic differentiation induced by oncostatin M attenuates fetal liver hematopoiesis. Proc Natl Acad Sci USA. (1999) 96:7265–70. 10.1073/pnas.96.13.7265 PubMed DOI PMC

Kamiya A, Kinoshita T, Miyajima A. Oncostatin M and hepatocyte growth factor induce hepatic maturation via distinct signaling pathways. FEBS Lett. (2001) 492:90–4. 10.1016/S0014-5793(01)02140-8 PubMed DOI

Russell DW, Setchell KD. Bile acid biosynthesis. Biochemistry. (1992) 31:4737–49. 10.1021/bi00135a001 PubMed DOI

Si-Tayeb K, Lemaigre FP, Duncan SA. Organogenesis and development of the liver. Dev Cell. (2010) 18:175–89. 10.1016/j.devcel.2010.01.011 PubMed DOI

Clotman F, Lannoy VJ, Reber M, Cereghini S, Cassiman D, Jacquemin P, et al. . The onecut transcription factor HNF6 is required for normal development of the biliary tract. Development. (2002) 129:1819–28. 10.1242/dev.129.8.1819 PubMed DOI

Shiojiri N, Katayama H. Secondary joining of the bile ducts during the hepatogenesis of the mouse embryo. Anat Embryol. (1987) 177:153–63. 10.1007/BF00572540 PubMed DOI

Lemaigre FP. Development of the biliary tract. Mech Dev. (2003) 120:81–7. 10.1016/S0925-4773(02)00334-9 PubMed DOI

Antoniou A, Raynaud P, Cordi S, Zong Y, Tronche F, Stanger B, et al. . Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9. Gastroenterology. (2009) 136:2325–33. 10.1053/j.gastro.2009.02.051 PubMed DOI PMC

Van Eyken P, Sciot R, Callea F, Van Der Steen K, Moerman P, Desmet V. The development of the intrahepatic bile ducts in man: a keratin-immunohistochemical study. Hepatology. (1988) 8:1586–95. 10.1002/hep.1840080619 PubMed DOI

Enzan H, Hara H, Yamashita Y, Ohkita T, Yamane T. Fine structure of hepatic sinusoids and their development in human embryos and fetuses. Acta Pathol Jpn. (1983) 33:447–66. 10.1111/j.1440-1827.1983.tb00352.x PubMed DOI

Enzan H, Himeno H, Hiroi M, Kiyoku H, Saibara T, Onishi S. Development of hepatic sinusoidal structure with special reference to the ito cells. Microsc Res Tech. (1997) 39:336–49. 10.1002/(SICI)1097-0029(19971115)39:4<336::AID-JEMT4>3.0.CO;2-F PubMed DOI

Couvelard A, Scoazec JY, Dauge MC, Bringuier AF, Potet F, Feldmann G. Structural and functional differentiation of sinusoidal endothelial cells during liver organogenesis in humans. Blood. (1996) 87:4568–80. 10.1182/blood.V87.11.4568.bloodjournal87114568 PubMed DOI

Collardeau-Frachon S, Scoazec JY. Vascular development and differentiation during human liver organogenesis. Anat Rec. (2008) 291:614–27. 10.1002/ar.20679 PubMed DOI

Burt AD. Pathobiology of hepatic stellate cells. J Gastroenterol. (1999) 34:299–304. 10.1007/s005350050264 PubMed DOI

Loo CK, Wu XJ. Origin of stellate cells from submesothelial cells in a developing human liver. Liver Int. (2008) 28:1437–45. 10.1111/j.1478-3231.2008.01788.x PubMed DOI

Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev. (2008) 88:125–72. 10.1152/physrev.00013.2007 PubMed DOI PMC

Asahina K, Tsai S, Li P, Ishii M, Maxson R, Sucov H, et al. . Mesenchymal origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liver development. Hepatology. (2009) 49:998–1011. 10.1002/hep.22721 PubMed DOI PMC

Matsumoto T, Kawakami M. The unit-concept of hepatic parenchyma–a re-examination based on angioarchitectural studies. Acta Pathol Jpn. (1982) 32 (Suppl. 2):285–314 PubMed

Lalor PF, Lai WK, Curbishley SM, Shetty S, Adams DH. Human hepatic sinusoidal endothelial cells can be distinguished by expression of phenotypic markers related to their specialised functions in vivo. World J Gastroenterol. (2006) 12:5429–39. 10.3748/wjg.v12.i34.5429 PubMed DOI PMC

Smedsrod B, Pertoft H, Gustafson S, Laurent TC. Scavenger functions of the liver endothelial cell. Biochem J. (1990) 266:313–27. 10.1042/bj2660313 PubMed DOI PMC

Matsumoto K, Yoshitomi H, Rossant J, Zaret KS. Liver organogenesis promoted by endothelial cells prior to vascular function. Science. (2001) 294:559–63. 10.1126/science.1063889 PubMed DOI

Lecouter J, Moritz DR, Li B, Phillips GL, Liang XH, Gerber HP, et al. . Angiogenesis-independent endothelial protection of liver: role of VEGFR-1. Science. (2003) 299:890–3. 10.1126/science.1079562 PubMed DOI

Deleve LD, Wang X, Hu L, Mccuskey MK, Mccuskey RS. Rat liver sinusoidal endothelial cell phenotype is maintained by paracrine and autocrine regulation. Am J Physiol Gastrointest Liver Physiol. (2004) 287:G757–63. 10.1152/ajpgi.00017.2004 PubMed DOI

Jungermann K, Katz N. Functional specialization of different hepatocyte populations. Physiol Rev. (1989) 69:708–64. 10.1152/physrev.1989.69.3.708 PubMed DOI

Giwa S, Lewis JK, Alvarez L, Langer R, Roth AE, Church GM, et al. . The promise of organ and tissue preservation to transform medicine. Nat Biotechnol. (2017) 35:530–42. 10.1038/nbt.3889 PubMed DOI PMC

Messner F, Guo Y, Etra JW, Brandacher G. Emerging technologies in organ preservation, tissue engineering and regenerative medicine: a blessing or curse for transplantation? Transpl Int. (2019) 32:673–85. 10.1111/tri.13432 PubMed DOI

Rowe C, Gerrard DT, Jenkins R, Berry A, Durkin K, Sundstrom L, et al. . Proteome-wide analyses of human hepatocytes during differentiation and dedifferentiation. Hepatology. (2013) 58:799–809. 10.1002/hep.26414 PubMed DOI PMC

Mederacke I, Dapito DH, Affo S, Uchinami H, Schwabe RF. High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers. Nat Protoc. (2015) 10:305–15. 10.1038/nprot.2015.017 PubMed DOI PMC

Perea L, Coll M, Sancho-Bru P. Assessment of liver fibrotic insults in vitro. Methods Mol Biol. (2015) 1250:391–401. 10.1007/978-1-4939-2074-7_30 PubMed DOI

Cassim S, Raymond V-A, Lapierre P, Bilodeau M. From in vivo to in vitro: major metabolic alterations take place in hepatocytes during and following isolation. PLoS ONE. (2017) 12:e0190366. 10.1371/journal.pone.0190366 PubMed DOI PMC

Duffy PA, Bonner F. Stem Cells for Safer Medicines (SC4SM): A public–private consortium to enhance medicines safety assessment. Toxicology. (2010) 278:370. 10.1016/j.tox.2010.08.128 DOI

Xu L, Hui AY, Albanis E, Arthur MJ, O'byrne SM, Blaner WS, et al. . Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut. (2005) 54:142–151. 10.1136/gut.2004.042127 PubMed DOI PMC

Herrmann J, Gressner AM, Weiskirchen R. Immortal hepatic stellate cell lines: useful tools to study hepatic stellate cell biology and function? J Cell Mol Med. (2007) 11:704–22. 10.1111/j.1582-4934.2007.00060.x PubMed DOI PMC

D'amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol. (2005) 23:1534–41. 10.1038/nbt1163 PubMed DOI

D'amour K, Bang A, Eliazer S, Kelly O, Agulnick A, Smart N, et al. . Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol. (2006) 24:1392–1401. 10.1038/nbt1259 PubMed DOI

Agarwal S, Holton KL, Lanza R. Efficient differentiation of functional hepatocytes from human embryonic stem cells. Stem Cells. (2008) 26:1117–27. 10.1634/stemcells.2007-1102 PubMed DOI

Hay DC, Fletcher J, Payne C, Terrace JD, Gallagher RC, Snoeys J, et al. . Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling. Proc Natl Acad Sci USA. (2008) 105:12301–6. 10.1073/pnas.0806522105 PubMed DOI PMC

Baxter M, Rowe C, Alder J, Harrison S, Hanley K, Park B, et al. . Generating hepatic cell lineages from pluripotent stem cells for drug toxicity screening. Stem Cell Res. (2010) 5:4–22. 10.1016/j.scr.2010.02.002 PubMed DOI PMC

Si-Tayeb K, Noto F, Nagaoka M, Li J, Battle M, Duris C, et al. . Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology. (2010) 51:297–305. 10.1002/hep.23354 PubMed DOI PMC

Sullivan GJ, Hay DC, Park IH, Fletcher J, Hannoun Z, Payne CM, et al. . Generation of functional human hepatic endoderm from human induced pluripotent stem cells. Hepatology. (2010) 51:329–35. 10.1002/hep.23335 PubMed DOI PMC

Siller R, Greenhough S, Naumovska E, Sullivan GJ. Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells. Stem Cell Reports. (2015) 4:939–52. 10.1016/j.stemcr.2015.04.001 PubMed DOI PMC

Mathapati S, Siller R, Impellizzeri AAR, Lycke M, Vegheim K, et al. . Small-molecule-directed hepatocyte-like cell differentiation of human pluripotent stem cells. Curr Protoc Stem Cell Biol. (2016) 38:1G.6.1–8. 10.1002/cpsc.13 PubMed DOI

Du C, Feng Y, Qiu D, Xu Y, Pang M, Cai N, et al. . Highly efficient and expedited hepatic differentiation from human pluripotent stem cells by pure small-molecule cocktails. Stem Cell Res Ther. (2018) 9:58. 10.1186/s13287-018-0794-4 PubMed DOI PMC

Ghodsizadeh A, Taei A, Totonchi M, Seifinejad A, Gourabi H, Pournasr B, et al. . Generation of liver disease-specific induced pluripotent stem cells along with efficient differentiation to functional hepatocyte-like cells. Stem Cell Rev. (2010) 6:622–32. 10.1007/s12015-010-9189-3 PubMed DOI

Rashid ST, Corbineau S, Hannan N, Marciniak SJ, Miranda E, Alexander G, et al. . Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest. (2010) 120:3127–36. 10.1172/JCI43122 PubMed DOI PMC

Cayo MA, Cai J, Delaforest A, Noto FK, Nagaoka M, Clark BS, et al. . JD induced pluripotent stem cell-derived hepatocytes faithfully recapitulate the pathophysiology of familial hypercholesterolemia. Hepatology. (2012) 56:2163–71. 10.1002/hep.25871 PubMed DOI PMC

Siller R, Greenhough S, Park I-H, Sullivan GJ. Modeling human disease with pluripotent stem cells. Curr Gene Ther. (2013) 13:99–110. 10.2174/1566523211313020004 PubMed DOI PMC

Yusa K, Rashid ST, Strick-Marchand H, Varela I, Liu PQ, Paschon DE, et al. . Targeted gene correction of alpha1-antitrypsin deficiency in induced pluripotent stem cells. Nature. (2011) 478:391–4. 10.1038/nature10424 PubMed DOI PMC

Fattahi F, Asgari S, Pournasr B, Seifinejad A, Totonchi M, Taei A, et al. . Disease-corrected hepatocyte-like cells from familial hypercholesterolemia-induced pluripotent stem cells. Mol Biotechnol. (2013) 54:863–73. 10.1007/s12033-012-9635-3 PubMed DOI

Sivaraman A, Leach JK, Townsend S, Iida T, Hogan BJ, Stolz DB, et al. . A microscale in vitro physiological model of the liver: predictive screens for drug metabolism and enzyme induction. Curr Drug Metab. (2005) 6:569–91. 10.2174/138920005774832632 PubMed DOI

Eiraku M, Watanabe K, Matsuo-Takasaki M, Kawada M, Yonemura S, Matsumura M, et al. . Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell. (2008) 3:519–32. 10.1016/j.stem.2008.09.002 PubMed DOI

Huch M, Dorrell C, Boj SF, Van Es JH, Li VS, Van De Wetering M, et al. . In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature. (2013) 494:247–50. 10.1038/nature11826 PubMed DOI PMC

Coll M, Perea L, Boon R, Leite SB, 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. (2018) 23:101–13.e107. 10.1016/j.stem.2018.05.027 PubMed DOI

Wu F, Wu D, Ren Y, Huang Y, Feng B, Zhao N, et al. . Generation of hepatobiliary organoids from human induced pluripotent stem cells. J Hepatol. (2019) 70:1145–58. 10.1016/j.jhep.2018.12.028 PubMed DOI

Guan Y, Xu D, Garfin PM, Ehmer U, Hurwitz M, Enns G, et al. . Human hepatic organoids for the analysis of human genetic diseases. JCI Insight. (2017) 2:e94954. 10.1172/jci.insight.94954 PubMed DOI PMC

Ouchi R, Togo S, Kimura M, Shinozawa T, Koido M, Koike H, et al. . Modeling steatohepatitis in humans with pluripotent stem cell-derived organoids. Cell Metabolism. (2019) 30:374–84.376. 10.1016/j.cmet.2019.05.007 PubMed DOI PMC

Huch M, Gehart H, Van Boxtel R, Hamer K, Blokzijl F, Verstegen MM, et al. . Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell. (2015) 160:299–312. 10.1016/j.cell.2014.11.050 PubMed DOI PMC

Den Braver-Sewradj SP, Den Braver MW, Vermeulen NPE, Commandeur JNM, Richert L, Vos JC. Inter-donor variability of phase I/phase II metabolism of three reference drugs in cryopreserved primary human hepatocytes in suspension and monolayer. Toxicology in Vitro. (2016) 33:71–9. 10.1016/j.tiv.2016.02.013 PubMed DOI

Bell CC, Hendriks DFG, Moro SML, Ellis E, Walsh J, Renblom A, et al. . Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease. Sci Rep. (2016) 6:25187–25187. 10.1038/srep25187 PubMed DOI PMC

Ogawa S, Surapisitchat J, Virtanen C, Ogawa M, Niapour M, Sugamori KS, et al. . Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes. Development. (2013) 140:3285–96. 10.1242/dev.090266 PubMed DOI PMC

Yamamoto J, Udono M, Miura S, Sekiya S, Suzuki A. Cell aggregation culture induces functional differentiation of induced hepatocyte-like cells through activation of hippo signaling. Cell Rep. (2018) 25:183–98. 10.1016/j.celrep.2018.09.010 PubMed DOI

Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I, et al. . Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad. Sci. (2002) 99:15655. 10.1073/pnas.232137699 PubMed DOI PMC

Guillouzo A, Corlu A, Aninat C, Glaise D, Morel F, Guguen-Guillouzo C. The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem Biol Interact. (2007) 168:66–73. 10.1016/j.cbi.2006.12.003 PubMed DOI

Yin C, Evason KJ, Asahina K, Stainier DYR. Hepatic stellate cells in liver development, regeneration, and cancer. J Clin Invest. (2013) 123:1902–10. 10.1172/JCI66369 PubMed DOI PMC

Bell CC, Lauschke VM, Vorrink SU, Palmgren H, Duffin R, Andersson TB, et al. . Transcriptional, functional, and mechanistic comparisons of stem cell–derived hepatocytes, heparg cells, and three-dimensional human hepatocyte spheroids as predictive in vitro systems for drug-induced liver injury. Drug Metab Disposit. (2017) 45:419. 10.1124/dmd.116.074369 PubMed DOI PMC

Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T, et al. . Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature. (2013) 499:481–4. 10.1038/nature12271 PubMed DOI

Takebe T, Enomura M, Yoshizawa E, Kimura M, Koike H, Ueno Y, et al. . Vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven condensation. Cell Stem Cell. (2015) 16:556–65. 10.1016/j.stem.2015.03.004 PubMed DOI

Flynn DM, Nijjar S, Hubscher SG, De Goyet Jde V, Kelly DA, Strain AJ, et al. . The role of notch receptor expression in bile duct development and disease. J Pathol. (2004) 204:55–64. 10.1002/path.1615 PubMed DOI

Tanimizu N, Miyajima A. Notch signaling controls hepatoblast differentiation by altering the expression of liver-enriched transcription factors. J Cell Sci. (2004) 117:3165–74. 10.1242/jcs.01169 PubMed DOI

Zong YW, Panikkar A, Xu J, Antoniou A, Raynaud P, Lemaigre F, et al. . Notch signaling controls liver development by regulating biliary differentiation. Development. (2009) 136:1727–39. 10.1242/dev.029140 PubMed DOI PMC

Landing BH, Wells TR. Considerations of some architectural properties of the biliary tree and liver in childhood. Perspect Pediatr Pathol. (1991) 14:122–42. PubMed

Furuyama K, Kawaguchi Y, Akiyama H, Horiguchi M, Kodama S, Kuhara T, et al. . Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine. Nat Genet. (2011) 43:34–41. 10.1038/ng.722 PubMed DOI

Hu H, Gehart H, Artegiani B, Löpez-Iglesias C, Dekkers F, Basak O, et al. . Long-Term expansion of functional mouse and human hepatocytes as 3d organoids. Cell. (2018) 175:1591–606.e1519. 10.1016/j.cell.2018.11.013 PubMed DOI

Campbell JS, Hughes SD, Gilbertson DG, Palmer TE, Holdren MS, Haran AC, et al. . Platelet-derived growth factor C induces liver fibrosis, steatosis, hepatocellular carcinoma. Proc Natl Acad Sci USA. (2005) 102:3389–94. 10.1073/pnas.0409722102 PubMed DOI PMC

Miyao M, Kotani H, Ishida T, Kawai C, Manabe S, Abiru H, et al. . Pivotal role of liver sinusoidal endothelial cells in NAFLD/NASH progression. Lab Invest. (2015) 95:1130–44. 10.1038/labinvest.2015.95 PubMed DOI

Cheng L, Hansen, Nancy F, Zhao L, Du Y, Zou C, et al. . Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression. Cell Stem Cell. (2012) 10:337–44. 10.1016/j.stem.2012.01.005 PubMed DOI PMC

Hindley CJ, Cordero-Espinoza L, Huch M. Organoids from adult liver and pancreas: stem cell biology and biomedical utility. Dev Biol. (2016) 420:251–61. 10.1016/j.ydbio.2016.06.039 PubMed DOI

Nuciforo S, Heim MH. Organoids to model liver disease. JHEP Rep. (2021) 3:100198. 10.1016/j.jhepr.2020.100198 PubMed DOI PMC

Moorman AF, Vermeulen JL, Charles R, Lamers WH. Localization of ammonia-metabolizing enzymes in human liver: ontogenesis of heterogeneity. Hepatology. (1989) 9:367–72. 10.1002/hep.1840090305 PubMed DOI

Pradere JP, Troeger JS, Dapito DH, Mencin AA, Schwabe RF. Toll-like receptor 4 and hepatic fibrogenesis. Semin Liver Dis. (2010) 30:232–44. 10.1055/s-0030-1255353 PubMed DOI PMC

Song IJ, Yang YM, Inokuchi-Shimizu S, Roh YS, Yang L, Seki E. The contribution of toll-like receptor signaling to the development of liver fibrosis and cancer in hepatocyte-specific TAK1-deleted mice. Int J Cancer. (2018) 142:81–91. 10.1002/ijc.31029 PubMed DOI PMC

Lau JKC, Zhang X, Yu J. Animal models of non-alcoholic fatty liver disease: current perspectives and recent advances. J Pathol. (2017) 241:36–44. 10.1002/path.4829 PubMed DOI PMC

Müller FA, Sturla SJ. Human in vitro models of nonalcoholic fatty liver disease. Curr Opin Toxicol. (2019) 16:9–16. 10.1016/j.cotox.2019.03.001 DOI

Graffmann N, Ring S, Kawala MA, Wruck W, Ncube A, Trompeter HI, 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. (2016) 25:1119–33. 10.1089/scd.2015.0383 PubMed DOI PMC

Parafati M, Kirby RJ, 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. Dis Model Mech. (2018) 11:dmm033530. 10.1242/dmm.033530 PubMed DOI PMC

Leite SB, Roosens T, El Taghdouini A, Mannaerts I, Smout AJ, Najimi M, et al. . Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitro. Biomaterials. (2016) 78:1–10. 10.1016/j.biomaterials.2015.11.026 PubMed DOI

Weltin A, Hammer S, Noor F, Kaminski Y, Kieninger J, Urban GA. Accessing 3D microtissue metabolism: lactate and oxygen monitoring in hepatocyte spheroids. Biosen Bioelectro. (2017) 87:941–8. 10.1016/j.bios.2016.07.094 PubMed DOI

Rubiano A, Indapurkar A, Yokosawa R, Miedzik A, Rosenzweig B, Arefin A, et al. . Characterizing the reproducibility in using a liver microphysiological system for assaying drug toxicity, metabolism, and accumulation. Clin Transl Sci. (2021). 10.1111/cts.12969. [Epub ahead of print]. PubMed DOI PMC

Bhise NS, Manoharan V, Massa S, Tamayol A, Ghaderi M, Miscuglio M, et al. . A liver-on-a-chip platform with bioprinted hepatic spheroids. Biofabrication. (2016) 8:014101. 10.1088/1758-5090/8/1/014101 PubMed DOI

Norona LM, Nguyen DG, Gerber DA, Presnell SC, Lecluyse EL. Editor's highlight: modeling compound-induced fibrogenesis in vitro using three-dimensional bioprinted human liver tissues. Toxicol Sci. (2016) 154:354–67. 10.1093/toxsci/kfw169 PubMed DOI PMC

Sasaki K, Akagi T, Asaoka T, Eguchi H, Fukuda Y, Iwagami Y, et al. . Construction of three-dimensional vascularized functional human liver tissue using a layer-by-layer cell coating technique. Biomaterials. (2017) 133:263–74. 10.1016/j.biomaterials.2017.02.034 PubMed DOI

Schermelleh L, Heintzmann R, Leonhardt H. A guide to super-resolution fluorescence microscopy. J Cell Biol. (2010) 190:165–75. 10.1083/jcb.201002018 PubMed DOI PMC

Rios AC, Fu NY, Lindeman GJ, Visvader JE. In situ identification of bipotent stem cells in the mammary gland. Nature. (2014) 506:322–7. 10.1038/nature12948 PubMed DOI

Richardson DS, Lichtman JW. Clarifying Tissue Clearing. Cell. (2015) 162:246–57. 10.1016/j.cell.2015.06.067 PubMed DOI PMC

Richardson DS, Lichtman JW. SnapShot: Tissue Clearing. Cell. (2017) 171:496–6.941. 10.1016/j.cell.2017.09.025 PubMed DOI

Sahl SJ, Hell SW, Jakobs S. Fluorescence nanoscopy in cell biology. Nat Rev Mol Cell Biol. (2017) 18:685–701. 10.1038/nrm.2017.71 PubMed DOI

Rios AC, Clevers H. Imaging organoids: a bright future ahead. Nat Methods. (2018) 15:24–6. 10.1038/nmeth.4537 PubMed DOI

Rakotoson I, Delhomme B, Djian P, Deeg A, Brunstein M, Seebacher C, et al. . Fast 3-D imaging of brain organoids with a new single-objective planar-illumination two-photon microscope. Front Neuroanat. (2019) 13:77. 10.3389/fnana.2019.00077 PubMed DOI PMC

Akkerman N, Defize LH. Dawn of the organoid era: 3D tissue and organ cultures revolutionize the study of development, disease, and regeneration. Bioessays. (2017) 39:1600244. 10.1002/bies.201600244 PubMed DOI

Thomas C, Devries P, Hardin J, White J. Four-dimensional imaging: Computer visualization of 3D movements in living specimens. Science. (1996) 273:603–7. 10.1126/science.273.5275.603 PubMed DOI

Conchello JA, Lichtman JW. Optical sectioning microscopy. Nat Methods. (2005) 2:920–31. 10.1038/nmeth815 PubMed DOI

Gustafsson MG. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci USA. (2005) 102:13081–6. 10.1073/pnas.0406877102 PubMed DOI PMC

Hayashi S, Okada Y. Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics. Mol Biol Cell. (2015) 26:1743–51. 10.1091/mbc.E14-08-1287 PubMed DOI PMC

Dekkers JF, Alieva M, Wellens LM, Ariese HCR, Jamieson PR, Vonk AM, et al. . High-resolution 3D imaging of fixed and cleared organoids. Nat Protoc. (2019) 14:1756–71. 10.1038/s41596-019-0160-8 PubMed DOI

Shimozawa T, Yamagata K, Kondo T, Hayashi S, Shitamukai A, Konno D, et al. . Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging. Proc Natl Acad Sci USA. (2013) 110:3399–404. 10.1073/pnas.1216696110 PubMed DOI PMC

Inoue S, Inoue T. Direct-view high-speed confocal scanner: the CSU-10. Methods Cell Biol. (2002) 70:87–127. 10.1016/S0091-679X(02)70003-4 PubMed DOI

Yamagata K, Suetsugu R, Wakayama T. Long-Term, six-dimensional live-cell imaging for the mouse preimplantation embryo that does not affect full-term development. J Reprod Dev. (2009) 55:343–50. 10.1262/jrd.20166 PubMed DOI

Ueda J, Maehara K, Mashiko D, Ichinose T, Yao T, Hori M, et al. . Heterochromatin dynamics during the differentiation process revealed by the DNA methylation reporter mouse, MethylRO. Stem Cell Rep. (2014) 2:910–24. 10.1016/j.stemcr.2014.05.008 PubMed DOI PMC

Denk W, Strickler JH, Webb WW. Two-photon laser scanning fluorescence microscopy. Science. (1990) 248:73–6. 10.1126/science.2321027 PubMed DOI

Miller DR, Jarrett JW, Hassan AM, Dunn AK. Deep tissue imaging with multiphoton fluorescence microscopy. Curr Opin Biomed Eng. (2017) 4:32–9. 10.1016/j.cobme.2017.09.004 PubMed DOI PMC

Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, et al. . Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci. (2011) 14:1481–8. 10.1038/nn.2928 PubMed DOI

Huisken J, Swoger J, Del Bene F, Wittbrodt J, Stelzer EH. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science. (2004) 305:1007–9. 10.1126/science.1100035 PubMed DOI

Santi PA. Light sheet fluorescence microscopy: a review. J Histochem Cytochem. (2011) 59:129–38. 10.1369/0022155410394857 PubMed DOI PMC

Glaser AK, Reder NP, Chen Y, Mccarty EF, Yin C, Wei L, et al. . Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat Biomed Eng. (2017) 1:0084. 10.1038/s41551-017-0084 PubMed DOI PMC

Wang F, Wan H, Ma Z, Zhong Y, Sun Q, Tian Y, et al. . Light-sheet microscopy in the near-infrared II window. Nat Methods. (2019) 16:545–52. 10.1038/s41592-019-0398-7 PubMed DOI PMC

Harrison SP, Siller R, Tanaka Y, Xiang Y, Patterson B, Kempf H, et al. . Scalable production of tissue-like vascularised liver organoids from human PSCs. bioRxiv. (2020) 2020.2012.2002.406835. 10.1101/2020.12.02.406835 PubMed DOI PMC

Avior Y, Levy G, Zimerman M, Kitsberg D, Schwartz R, Sadeh R, et al. . Microbial-derived lithocholic acid and vitamin K2 drive the metabolic maturation of pluripotent stem cells–derived and fetal hepatocytes. Hepatology. (2015) 62:265–78. 10.1002/hep.27803 PubMed DOI

Cahan P, Li H, Morris SA, Lummertz Da Rocha E, Daley GQ, Collins JJ. CellNet: network biology applied to stem cell engineering. Cell. (2014) 158:903–15. 10.1016/j.cell.2014.07.020 PubMed DOI PMC

Boon R, Kumar M, Tricot T, Elia I, Ordovas L, Jacobs F, et al. . Amino acid levels determine metabolism and CYP450 function of hepatocytes and hepatoma cell lines. Nat Commun. (2020) 11:1393. 10.1038/s41467-020-15058-6 PubMed DOI PMC

Kia R, Kelly L, Sison-Young RL, Zhang F, Pridgeon CS, Heslop JA, et al. . MicroRNA-122: a novel hepatocyte-enriched in vitro marker of drug-induced cellular toxicity. Toxicol Sci. (2014) 144:173–85. 10.1093/toxsci/kfu269 PubMed DOI PMC

Horslen SP, Fox IJ. Hepatocyte transplantation. Transplantation. (2004) 77:1481–6. 10.1097/01.TP.0000113809.53415.C2 PubMed DOI

Jensen KJ, Alpini G, Glaser S. Hepatic nervous system and neurobiology of the liver. Compr Physiol. (2013) 3:655–65. 10.1002/cphy.c120018 PubMed DOI PMC

Muntener M, Kagi U, Stevens LC, Walt Innervation H, maturation of muscular tissue in testicular teratomas in strain 129/Sv-ter mice . Virchows Arch B Cell Pathol. (1990) 59:223–9. 10.1007/BF02899408 PubMed DOI

Borden P, Houtz J, Leach, Steven D, Kuruvilla R. Sympathetic innervation during development is necessary for pancreatic islet architecture and functional maturation. Cell Rep. (2013) 4:287–301. 10.1016/j.celrep.2013.06.019 PubMed DOI PMC

Koike H, Iwasawa K, Ouchi R, Maezawa M, Giesbrecht K, Saiki N, et al. . Modeling human hepato-biliary-pancreatic organogenesis from the foregut–midgut boundary. Nature. (2019) 574:112–6. 10.1038/s41586-019-1598-0 PubMed DOI PMC

Lin A, Sved Skottvoll F, Rayner S, Pedersen-Bjergaard S, Sullivan G, Krauss S, et al. . 3D cell culture models and organ-on-a-chip: meet separation science and mass spectrometry. Electrophoresis. (2020) 41:56–64. 10.1002/elps.201900170 PubMed DOI

Eckel J. Adipose tissue. in the cellular secretome and organ crosstalk. In: Eckel J, editor. The Cellular Secretome and Organ Crosstalk. London; San Diego, CA: Academic Press; Elsevier Inc. (2018). p. 9–63. 10.1016/B978-0-12-809518-8.00002-7 DOI

Gwosch KC, Pape JK, Balzarotti F, Hoess P, Ellenberg J, Ries J, et al. . MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat Methods. (2020) 17:217–24. 10.1038/s41592-019-0688-0 PubMed DOI

Carlton PM, Boulanger J, Kervrann C, Sibarita JB, Salamero J, Gordon-Messer S, et al. . Fast live simultaneous multiwavelength four-dimensional optical microscopy. Proc Natl Acad Sci USA. (2010) 107:16016–22. 10.1073/pnas.1004037107 PubMed DOI PMC

Laissue PP, Alghamdi RA, Tomancak P, Reynaud EG, Shroff H. Assessing phototoxicity in live fluorescence imaging. Nat Methods. (2017) 14:657–61. 10.1038/nmeth.4344 PubMed DOI

Artifacts of light . Nat Methods. (2013) 10:1135. 10.1038/nmeth.2760 DOI

Van De Linde S, Sauer M. How to switch a fluorophore: from undesired blinking to controlled photoswitching. Chem Soc Rev. (2014) 43:1076–87. 10.1039/C3CS60195A PubMed DOI

Waldchen S, Lehmann J, Klein T, Van De Linde S, Sauer M. Light-induced cell damage in live-cell super-resolution microscopy. Sci Rep. (2015) 5:15348. 10.1038/srep15348 PubMed DOI PMC

Lynnyk A, Lunova M, Jirsa M, Egorova D, Kulikov A, Kubinova S, et al. . Manipulating the mitochondria activity in human hepatic cell line Huh7 by low-power laser irradiation. Biomed Opt Express. (2018) 9:1283–300. 10.1364/BOE.9.001283 PubMed DOI PMC

Lunova M, Smolkova B, Uzhytchak M, Janouskova KZ, Jirsa M, Egorova D, et al. . Light-induced modulation of the mitochondrial respiratory chain activity: possibilities and limitations. Cell Mol Life Sci. (2019) 77:2815–38. 10.1007/s00018-019-03321-z PubMed DOI PMC

Swoboda M, Henig J, Cheng HM, Brugger D, Haltrich D, Plumere N, et al. . Enzymatic oxygen scavenging for photostability without pH drop in single-molecule experiments. ACS Nano. (2012) 6:6364–9. 10.1021/nn301895c PubMed DOI PMC

Kilian N, Goryaynov A, Lessard MD, Hooker G, Toomre D, Rothman JE, et al. . Assessing photodamage in live-cell STED microscopy. Nat Methods. (2018) 15:755–6. 10.1038/s41592-018-0145-5 PubMed DOI PMC

Bottanelli F, Kromann EB, Allgeyer ES, Erdmann RS, Wood Baguley S, Sirinakis G, et al. . Two-colour live-cell nanoscale imaging of intracellular targets. Nat Commun. (2016) 7:10778. 10.1038/ncomms10778 PubMed DOI PMC

Hayashi S. Resolution doubling using confocal microscopy via analogy with structured illumination microscopy. Jpn J Appl Phys. (2016) 55:082501. 10.7567/JJAP.55.082501 DOI

The interactions between DNA nanostructures and cells: A critical overview from a cell biology perspective