From a Single Cell to a Whole Human Liver: Disease Modeling and Transplantation

Abstract

Although the underlying cause may vary across countries and demographic groups, liver disease is a major cause of morbidity and mortality globally. Orthotopic liver transplantation is the only definitive treatment for liver failure but is limited by the lack of donor livers. The development of drugs that prevent the progression of liver disease and the generation of alternative liver constructs for transplantation could help alleviate the burden of liver disease. Bioengineered livers containing human induced pluripotent stem cell (iPSC)–derived liver cells are being utilized to study liver disease and to identify and test potential therapeutics. Moreover, bioengineered livers containing pig hepatocytes and endothelial cells have been shown to function and survive after transplantation into pig models of liver failure, providing preclinical evidence toward future clinical applications. Finally, bioengineered livers containing human iPSC-derived liver cells have been shown to function and survive after transplantation in rodents but require considerable optimization and testing prior to clinical use. In conclusion, bioengineered livers have emerged as a suitable tool for modeling liver diseases and as a promising alternative graft for clinical transplantation. The integration of novel technologies and techniques for the assembly and analysis of bioengineered livers will undoubtedly expand future applications in basic research and clinical transplantation.

Keywords

bioengineered liver - disease modeling - transplantation - liver disease - induced pluripotent stem cells

Financial Support

This work was supported by NIH grants DK099257, DK119973, TR003289, TR002383, DK120531 and DK096990 to A.S.-G.. Funding received from U.S. Department of Health and Human Services, National Institutes of Health, National Center for Advancing Translational Sciences (002383, 003289); U.S. Department of Health and Human Services, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (096990, 099257, 119973, 120531).

^* These authors contributed equally to this work and share first authorship.

^# These authors contributed equally to this work and share the last authorship.

Publication History

Accepted Manuscript online:
31 August 2022

Article published online:
19 October 2022

• References

• 1 Villarroel MA, Blackwell DL, Jen A. Tables of Summary Health Statistics for U.S. Adults: 2018 National Health Interview Survey. National Center for Health Statistics; 2019. . Accessed on Sept 28, 2022, at: http://www.cdc.gov/nchs/nhis/SHS/tables.htm
• 2 Centers for Disease Control and Prevention, National Center for Health Statistics. National Vital Statistics System, Mortality 1999–2020 on CDC WONDER Online Database, released in 2021. Data are from the Multiple Cause of Death Files, 1999–2020, as compiled from data provided by the 57 vital statistics jurisdictions through the Vital Statistics Cooperative Program. Accessed on Sept 28, 2022, at: http://wonder.cdc.gov/ucd-icd10.html
• 3 Sharma A, Nagalli S. Chronic Liver Disease. Treasure Island (FL): StatPearls; 2020
• 4 Malarkey DE, Johnson K, Ryan L, Boorman G, Maronpot RR. New insights into functional aspects of liver morphology. Toxicol Pathol 2005; 33 (01) 27-34
• 5 Farkas S, Hackl C, Schlitt HJ. Overview of the indications and contraindications for liver transplantation. Cold Spring Harb Perspect Med 2014; 4 (05) 4
• 6 Kwong A, Kim WR, Lake JR. et al. OPTN/SRTR 2018 Annual Data Report: Liver. Am J Transplant 2020; 20 (Suppl s1): 193-299
• 7 Kwong AJ, Kim WR, Lake JR. et al. OPTN/SRTR 2019 Annual Data Report: Liver. Am J Transplant 2021; 21 (Suppl. 02) 208-315
• 8 Guo L, Dial S, Shi L. et al. Similarities and differences in the expression of drug-metabolizing enzymes between human hepatic cell lines and primary human hepatocytes. Drug Metab Dispos 2011; 39 (03) 528-538
• 9 Heslop JA, Rowe C, Walsh J. et al. Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile. Arch Toxicol 2017; 91 (01) 439-452
• 10 LeCluyse EL, Witek RP, Andersen ME, Powers MJ. Organotypic liver culture models: meeting current challenges in toxicity testing. Crit Rev Toxicol 2012; 42 (06) 501-548
• 11 Richert L, Liguori MJ, Abadie C. et al. Gene expression in human hepatocytes in suspension after isolation is similar to the liver of origin, is not affected by hepatocyte cold storage and cryopreservation, but is strongly changed after hepatocyte plating. Drug Metab Dispos 2006; 34 (05) 870-879
• 12 Takahashi K, Tanabe K, Ohnuki M. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131 (05) 861-872
• 13 Yu J, Vodyanik MA, Smuga-Otto K. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318 (5858): 1917-1920
• 14 Tapia N, Schöler HR. Molecular obstacles to clinical translation of iPSCs. Cell Stem Cell 2016; 19 (03) 298-309
• 15 Cayo MA, Cai J, DeLaForest A. et al. JD induced pluripotent stem cell-derived hepatocytes faithfully recapitulate the pathophysiology of familial hypercholesterolemia. Hepatology 2012; 56 (06) 2163-2171
• 16 Cayo MA, Mallanna SK, Di Furio F. et al. A drug screen using human iPSC-derived hepatocyte-like cells reveals cardiac glycosides as a potential treatment for hypercholesterolemia. Cell Stem Cell 2017; 20 (04) 478-489.e5
• 17 Chen YF, Tseng CY, Wang HW, Kuo HC, Yang VW, Lee OK. Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an efficient three-step protocol. Hepatology 2012; 55 (04) 1193-1203
• 18 Choi SM, Kim Y, Shim JS. et al. Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells. Hepatology 2013; 57 (06) 2458-2468
• 19 Fattahi F, Asgari S, Pournasr B. et al. Disease-corrected hepatocyte-like cells from familial hypercholesterolemia-induced pluripotent stem cells. Mol Biotechnol 2013; 54 (03) 863-873
• 20 Gough A, Soto-Gutierrez A, Vernetti L, Ebrahimkhani MR, Stern AM, Taylor DL. Human biomimetic liver microphysiology systems in drug development and precision medicine. Nat Rev Gastroenterol Hepatol 2021; 18 (04) 252-268
• 21 Overeem AW, Klappe K, Parisi S. et al. Pluripotent stem cell-derived bile canaliculi-forming hepatocytes to study genetic liver diseases involving hepatocyte polarity. J Hepatol 2019; 71 (02) 344-356
• 22 Parisi S, Polishchuk EV, Allocca S. et al. Characterization of the most frequent ATP7B mutation causing Wilson disease in hepatocytes from patient induced pluripotent stem cells. Sci Rep 2018; 8 (01) 6247
• 23 Rashid ST, Corbineau S, Hannan N. et al. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest 2010; 120 (09) 3127-3136
• 24 Si-Tayeb K, Noto FK, Nagaoka M. et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 2010; 51 (01) 297-305
• 25 Song Z, Cai J, Liu Y. et al. Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Res 2009; 19 (11) 1233-1242
• 26 Tafaleng EN, Chakraborty S, Han B. et al. Induced pluripotent stem cells model personalized variations in liver disease resulting from α1-antitrypsin deficiency. Hepatology 2015; 62 (01) 147-157
• 27 Takeishi K, Collin de l'Hortet A, Wang Y. et al. Assembly and function of a bioengineered human liver for transplantation generated solely from induced pluripotent stem cells. Cell Rep 2020; 31 (09) 107711
• 28 Vosough M, Omidinia E, Kadivar M. et al. Generation of functional hepatocyte-like cells from human pluripotent stem cells in a scalable suspension culture. Stem Cells Dev 2013; 22 (20) 2693-2705
• 29 Wilson AA, Ying L, Liesa M. et al. Emergence of a stage-dependent human liver disease signature with directed differentiation of alpha-1 antitrypsin-deficient iPS cells. Stem Cell Reports 2015; 4 (05) 873-885
• 30 Yusa K, Rashid ST, Strick-Marchand H. et al. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells. Nature 2011; 478 (7369): 391-394
• 31 Zhang S, Chen S, Li W. et al. Rescue of ATP7B function in hepatocyte-like cells from Wilson's disease induced pluripotent stem cells using gene therapy or the chaperone drug curcumin. Hum Mol Genet 2011; 20 (16) 3176-3187
• 32 Fukuhara T, Matsuura Y. Role of miR-122 and lipid metabolism in HCV infection. J Gastroenterol 2013; 48 (02) 169-176
• 33 Okuyama-Dobashi K, Kasai H, Tanaka T. et al. Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide. Sci Rep 2015; 5: 17047
• 34 Yoshida T, Takayama K, Kondoh M. et al. Use of human hepatocyte-like cells derived from induced pluripotent stem cells as a model for hepatocytes in hepatitis C virus infection. Biochem Biophys Res Commun 2011; 416 (1-2): 119-124
• 35 Sakurai F, Mitani S, Yamamoto T. et al. Human induced-pluripotent stem cell-derived hepatocyte-like cells as an in vitro model of human hepatitis B virus infection. Sci Rep 2017; 7: 45698
• 36 Nelson DR, Teckman J, Di Bisceglie AM, Brenner DA. Diagnosis and management of patients with α1-antitrypsin (A1AT) deficiency. Clin Gastroenterol Hepatol 2012; 10 (06) 575-580
• 37 D'Agostino M, Lemma V, Chesi G. et al. The cytosolic chaperone α-crystallin B rescues folding and compartmentalization of misfolded multispan transmembrane proteins. J Cell Sci 2013; 126 (Pt 18): 4160-4172
• 38 Ayonrinde OT. Historical narrative from fatty liver in the nineteenth century to contemporary NAFLD - reconciling the present with the past. JHEP Rep 2021; 3 (03) 100261
• 39 Duwaerts CC, Le Guillou D, Her CL. et al. Induced pluripotent stem cell-derived hepatocytes from patients with nonalcoholic fatty liver disease display a disease-specific gene expression profile. Gastroenterology 2021; 160 (07) 2591-2594.e6
• 40 Eslam M, Sanyal AJ, George J. International Consensus Panel. MAFLD: a consensus-driven proposed nomenclature for metabolic associated fatty liver disease. Gastroenterology 2020; 158 (07) 1999-2014.e1
• 41 Méndez-Sánchez N, Díaz-Orozco LE. Editorial: International Consensus Recommendations to Replace the Terminology of Non-Alcoholic Fatty Liver Disease (NAFLD) with Metabolic-Associated Fatty Liver Disease (MAFLD). Med Sci Monit 2021; 27: e933860
• 42 Mendez-Sanchez N, Arrese M, Gadano A. et al. The Latin American Association for the Study of the Liver (ALEH) position statement on the redefinition of fatty liver disease. Lancet Gastroenterol Hepatol 2021; 6 (01) 65-72
• 43 Zhang HX, Zhang Y, Yin H. Genome editing with mRNA encoding ZFN, TALEN, and Cas9. Mol Ther 2019; 27 (04) 735-746
• 44 Gupta RM, Musunuru K. Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9. J Clin Invest 2014; 124 (10) 4154-4161
• 45 Cong L, Ran FA, Cox D. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339 (6121): 819-823
• 46 Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012; 337 (6096): 816-821
• 47 Mali P, Yang L, Esvelt KM. et al. RNA-guided human genome engineering via Cas9. Science 2013; 339 (6121): 823-826
• 48 Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in human cells. eLife 2013; 2: e00471
• 49 Omer L, Hudson EA, Zheng S, Hoying JB, Shan Y, Boyd NL. CRISPR correction of a homozygous low-density lipoprotein receptor mutation in familial hypercholesterolemia induced pluripotent stem cells. Hepatol Commun 2017; 1 (09) 886-898
• 50 Okada H, Nakanishi C, Yoshida S. et al. Function and immunogenicity of gene-corrected iPSC-derived hepatocyte-like cells in restoring low density lipoprotein uptake in homozygous familial hypercholesterolemia. Sci Rep 2019; 9 (01) 4695
• 51 Brahimi N, Jambou M, Sarzi E. et al. The first founder DGUOK mutation associated with hepatocerebral mitochondrial DNA depletion syndrome. Mol Genet Metab 2009; 97 (03) 221-226
• 52 Johansson K, Ramaswamy S, Ljungcrantz C. et al. Structural basis for substrate specificities of cellular deoxyribonucleoside kinases. Nat Struct Biol 2001; 8 (07) 616-620
• 53 Dimmock DP, Zhang Q, Dionisi-Vici C. et al. Clinical and molecular features of mitochondrial DNA depletion due to mutations in deoxyguanosine kinase. Hum Mutat 2008; 29 (02) 330-331
• 54 Jing R, Corbett JL, Cai J. et al. A screen using iPSC-derived hepatocytes reveals NAD⁺ as a potential treatment for mtDNA depletion syndrome. Cell Rep 2018; 25 (06) 1469-1484.e5
• 55 Chai S, Wan X, Ramirez-Navarro A. et al. Physiological genomics identifies genetic modifiers of long QT syndrome type 2 severity. J Clin Invest 2018; 128 (03) 1043-1056
• 56 Bianchi L, Priori SG, Napolitano C. et al. Mechanisms of I(Ks) suppression in LQT1 mutants. Am J Physiol Heart Circ Physiol 2000; 279 (06) H3003-H3011
• 57 Wang W, Zheng Y, Sun S. et al. A genome-wide CRISPR-based screen identifies KAT7 as a driver of cellular senescence. Sci Transl Med 2021; 13 (575) 13
• 58 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
• 59 Collin de l'Hortet A, Takeishi K, Guzman-Lepe J. et al. Generation of human fatty livers using custom-engineered induced pluripotent stem cells with modifiable SIRT1 metabolism. Cell Metab 2019; 30 (02) 385-401.e9
• 60 Nakazawa K, Ijima H, Fukuda J. et al. Development of a hybrid artificial liver using polyurethane foam/hepatocyte spheroid culture in a preclinical pig experiment. Int J Artif Organs 2002; 25 (01) 51-60
• 61 Riordan SM, Williams R. Extracorporeal support and hepatocyte transplantation in acute liver failure and cirrhosis. J Gastroenterol Hepatol 1999; 14 (08) 757-770
• 62 Vishwakarma SK, Lakkireddy C, Bardia A. et al. Bioengineered functional humanized livers: an emerging supportive modality to bridge the gap of organ transplantation for management of end-stage liver diseases. World J Hepatol 2018; 10 (11) 822-836
• 63 Pullen LC. Bioengineered organs: not a matter of “If”. Am J Transplant 2022; 22 (01) 1-2
• 64 Dai Q, Jiang W, Huang F, Song F, Zhang J, Zhao H. Recent advances in liver engineering with decellularized scaffold. Front Bioeng Biotechnol 2022; 10: 831477
• 65 Anderson BD, Nelson ED, Joo D. et al. Functional characterization of a bioengineered liver after heterotopic implantation in pigs. Commun Biol 2021; 4 (01) 1157
• 66 Higashi H, Yagi H, Kuroda K. et al. Transplantation of bioengineered liver capable of extended function in a preclinical liver failure model. Am J Transplant 2022; 22 (03) 731-744
• 67 Shaheen MF, Joo DJ, Ross JJ. et al. Sustained perfusion of revascularized bioengineered livers heterotopically transplanted into immunosuppressed pigs. Nat Biomed Eng 2020; 4 (04) 437-445
• 68 Watt FM, Huck WT. Role of the extracellular matrix in regulating stem cell fate. Nat Rev Mol Cell Biol 2013; 14 (08) 467-473
• 69 Yagi H, Fukumitsu K, Fukuda K. et al. Human-scale whole-organ bioengineering for liver transplantation: a regenerative medicine approach. Cell Transplant 2013; 22 (02) 231-242
• 70 Porrett PM, Orandi BJ, Kumar V. et al. First clinical-grade porcine kidney xenotransplant using a human decedent model. Am J Transplant 2022; 22 (04) 1037-1053
• 71 Wang W, He W, Ruan Y, Geng Q. First pig-to-human heart transplantation. Innovation (Camb) 2022; 3 (02) 100223
• 72 Chih S, Chruscinski A, Ross HJ, Tinckam K, Butany J, Rao V. Antibody-mediated rejection: an evolving entity in heart transplantation. J Transplant 2012; 2012: 210210
• 73 Demetris AJ, Bellamy CO, Gandhi CR, Prost S, Nakanuma Y, Stolz DB. Functional immune anatomy of the liver-as an allograft. Am J Transplant 2016; 16 (06) 1653-1680
• 74 Gloor J, Stegall MD. Sensitized renal transplant recipients: current protocols and future directions. Nat Rev Nephrol 2010; 6 (05) 297-306
• 75 Lee BT, Fiel MI, Schiano TD. Antibody-mediated rejection of the liver allograft: An update and a clinico-pathological perspective. J Hepatol 2021; 75 (05) 1203-1216
• 76 Chen Y, Zhang W, Bao H, He W, Chen L. High mobility group box 1 contributes to the acute rejection of liver allografts by activating dendritic cells. Front Immunol 2021; 12: 679398
• 77 Mao JX, Guo WY, Guo M, Liu C, Teng F, Ding GS. Acute rejection after liver transplantation is less common, but predicts better prognosis in HBV-related hepatocellular carcinoma patients. Hepatol Int 2020; 14 (03) 347-361
• 78 Deuse T, Hu X, Gravina A. et al. Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nat Biotechnol 2019; 37 (03) 252-258
• 79 Han X, Wang M, Duan S. et al. Generation of hypoimmunogenic human pluripotent stem cells. Proc Natl Acad Sci U S A 2019; 116 (21) 10441-10446
• 80 Xu H, Wang B, Ono M. et al. Targeted disruption of HLA genes via CRISPR-Cas9 generates iPSCs with enhanced immune compatibility. Cell Stem Cell 2019; 24 (04) 566-578.e7
• 81 Minami T, Ishii T, Yasuchika K. et al. Novel hybrid three-dimensional artificial liver using human induced pluripotent stem cells and a rat decellularized liver scaffold. Regen Ther 2019; 10: 127-133
• 82 Acun A, Oganesyan R, Jaramillo M, Yarmush ML, Uygun BE. Human-origin iPSC-based recellularization of decellularized whole rat livers. Bioengineering (Basel) 2022; 9 (05) 9
• 83 Kuroda T, Yasuda S, Sato Y. In vitro detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human induced pluripotent stem cells. Methods Mol Biol 2014; 1210: 183-192
• 84 Tano K, Yasuda S, Kuroda T, Saito H, Umezawa A, Sato Y. A novel in vitro method for detecting undifferentiated human pluripotent stem cells as impurities in cell therapy products using a highly efficient culture system. PLoS One 2014; 9 (10) e110496
• 85 Tateno H, Onuma Y, Ito Y. et al. Elimination of tumorigenic human pluripotent stem cells by a recombinant lectin-toxin fusion protein. Stem Cell Reports 2015; 4 (05) 811-820
• 86 Sekine K, Tsuzuki S, Yasui R. et al. Robust detection of undifferentiated iPSC among differentiated cells. Sci Rep 2020; 10 (01) 10293
• 87 Jang S, Collin de l'Hortet A, Soto-Gutierrez A. Induced pluripotent stem cell-derived endothelial cells: overview, current advances, applications, and future directions. Am J Pathol 2019; 189 (03) 502-512
• 88 Kobayashi T, Yamaguchi T, Hamanaka S. et al. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell 2010; 142 (05) 787-799
• 89 Matsunari H, Nagashima H, Watanabe M. et al. Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs. Proc Natl Acad Sci U S A 2013; 110 (12) 4557-4562
• 90 Goto T, Hara H, Sanbo M. et al. Generation of pluripotent stem cell-derived mouse kidneys in Sall1-targeted anephric rats. Nat Commun 2019; 10 (01) 451
• 91 Hamanaka S, Umino A, Sato H. et al. Generation of vascular endothelial cells and hematopoietic cells by blastocyst complementation. Stem Cell Reports 2018; 11 (04) 988-997
• 92 Mori M, Furuhashi K, Danielsson JA. et al. Generation of functional lungs via conditional blastocyst complementation using pluripotent stem cells. Nat Med 2019; 25 (11) 1691-1698
• 93 Fu R, Yu D, Ren J. et al. Domesticated cynomolgus monkey embryonic stem cells allow the generation of neonatal interspecies chimeric pigs. Protein Cell 2020; 11 (02) 97-107
• 94 Das S, Koyano-Nakagawa N, Gafni O. et al. Generation of human endothelium in pig embryos deficient in ETV2. Nat Biotechnol 2020; 38 (03) 297-302
• 95 Ruiz-Estevez M, Crane AT, Rodriguez-Villamil P. et al. Liver development is restored by blastocyst complementation of HHEX knockout in mice and pigs. Stem Cell Res Ther 2021; 12 (01) 292
• 96 Tarazi S, Aguilera-Castrejon A, Joubran C. et al. Post-gastrulation synthetic embryos generated ex utero from mouse naive ESCs. Cell 2022; 185 (18) 3290-3306.e25
• 97 Longo SK, Guo MG, Ji AL, Khavari PA. Integrating single-cell and spatial transcriptomics to elucidate intercellular tissue dynamics. Nat Rev Genet 2021; 22 (10) 627-644
• 98 Taylor MJ, Liyu A, Vertes A, Anderton CR. Ambient single-cell analysis and native tissue imaging using laser-ablation electrospray ionization mass spectrometry with increased spatial resolution. J Am Soc Mass Spectrom 2021; 32 (09) 2490-2494
• 99 Taylor MJ, Lukowski JK, Anderton CR. Spatially resolved mass spectrometry at the single cell: recent innovations in proteomics and metabolomics. J Am Soc Mass Spectrom 2021; 32 (04) 872-894