Pluripotent Stem Cell-Derived Human Liver Organoids Enter the Realm of High-Throughput Drug Screening

See "High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids," by Shinozawa T, Kimura M, Cai Y, et al, on page 831. See "High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids," by Shinozawa T, Kimura M, Cai Y, et al, on page 831. Drug-induced liver injury (DILI) is a leading cause of drug attrition because animals are not fully predictive of human DILI owing to species-specific differences in drug metabolism pathways and the lack of genetic diversity in used rodent strains. Therefore, human liver models are essential for compound screening.1Kaplowitz N. Idiosyncratic drug hepatotoxicity.Nat Rev Drug Discov. 2005; 4: 489-499Crossref PubMed Scopus (820) Google Scholar Although 2-dimensional cultures of primary human hepatocytes (PHH) can be generated on collagen-coated plastic/glass with or without a gelled extracellular matrix overlay, hepatic functions rapidly decline over 24–72 hours, which restricts the sensitivity (detection of true positives) of DILI detection to approximately 50%.2Godoy P. Hewitt N.J. Albrecht U. et al.Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling, and ADME.Arch Toxicol. 2013; 87: 1315-1530Crossref PubMed Scopus (798) Google Scholar,3Xu J.J. Henstock P.V. Dunn M.C. et al.Cellular imaging predictions of clinical drug-induced liver injury.Toxicol Sci. 2008; 105: 97-105Crossref PubMed Scopus (380) Google Scholar To mitigate such issues, several advanced culture techniques have been developed, including micropatterned co-cultures, 3-dimensional self-assembled spheroids, bioprinted tissues, microfluidic cultures, and lumen-containing organoids embedded within gelled extracellular matrix.4Underhill G.H. Khetani S.R. Bioengineered liver models for drug testing and cell differentiation studies.Cell Mol Gastroenterol Hepatol. 2018; 5: 426-439 e1Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar In this issue of Gastroenterology, Shinozawa et al5Shinozawa T. Kimura M. Cai Y. et al.High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids.Gastroenterology. 2021; 160: 831-846Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar bring induced pluripotent stem cell (iPSC)-derived human liver organoids (HLO) into the realm of high-throughput compound screening.5Shinozawa T. Kimura M. Cai Y. et al.High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids.Gastroenterology. 2021; 160: 831-846Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Although PHHs are the gold standard for fabricating in vitro human liver models, they are in limited supply for routine high-throughput screening of large compound libraries during early drug development when structure–activity relationship-based compound optimizations are feasible. Additionally, PHH lots suffer from considerable differences in viability and quality for in vitro use because they are often isolated from livers rejected for transplantation owing to health issues. Most important, limited PHH availability makes it difficult to determine the genetic basis of DILI.6Khetani S.R. Berger D.R. Ballinger K.R. et al.Microengineered liver tissues for drug testing.J Lab Autom. 2015; 20: 216-250Crossref PubMed Scopus (73) Google Scholar In contrast, human iPSC-derived hepatocyte-like cells (iHeps) could help to address these limitations with PHHs; however, in 2-dimensional monocultures, iHeps display a very immature phenotype, as the typically possess of <10% of adult PHH functional levels.7Schwartz R.E. Fleming H.E. Khetani S.R. et al.Pluripotent stem cell-derived hepatocyte-like cells.Biotech Adv. 2014; 32: 504-513Crossref PubMed Scopus (181) Google Scholar To address the limitations associated with 2-dimensional iHep culture, Shinozawa et al5Shinozawa T. Kimura M. Cai Y. et al.High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids.Gastroenterology. 2021; 160: 831-846Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar developed scalable HLOs in 384-well plates (Figure 1). Human iPSC monolayers were first differentiated into foregut cells over 6 days, which were detached and frozen at –80°C. After thawing, the foregut cells were placed within Matrigel to initiate organoid formation and liver specification for 4 days via the use of 5 factors (fibroblast growth factor 2, vascular endothelial growth factor, epidermal growth factor, glycogen synthase kinase 3 inhibitor, transforming growth factor beta inhibitor) that improved HLO yield; retinoic acid further enhanced bile canaliculi formation. Then, the organoids were harvested and re-embedded in Matrigel within 384-well plates and matured into hepatocyte-like cells over 10 days. Overall, HLOs upregulated drug metabolism/transporter genes, secreted similar albumin levels as PHHs, displayed bile canaliculi and transporter proteins, and secreted bile acids into their lumens. When the bile salt export protein was mutated using CRISPR gene editing, transport of a fluorescent bile analog into the lumens was inhibited. Single cell RNA sequencing revealed approximately 75% hepatocyte-like cells and approximately 25% non–parenchymal-like cells within HLOs, and some emergence of zonated gene expression across 2 hepatic clusters. To determine usefulness for DILI screening, Shinozawa et al5Shinozawa T. Kimura M. Cai Y. et al.High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids.Gastroenterology. 2021; 160: 831-846Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar incubated HLOs with 238 drugs at 4 concentrations and assessed cholestasis (inhibition of fluorescent bile analog into lumen) after 1 day and viability (ATP levels) after 3 days. The sensitivity and specificity from these 2 end points were found to be 88.7% and 88.9%, respectively (69%/100% by changing the decision thresholds). HLOs could detect mitochondrial dysfunction following drug incubation and when preloaded with oleic acid, were more susceptible to the toxicity of some drugs owing to preexisting lipotoxicity via generation of reactive oxygen species. Finally, the investigators screened HLOs generated from 5 iPSC lines with the CYP2C9∗2 gene (reduction in CYP2C9 activity by approximately 30% versus wild type) and 3 iPSC lines with wild-type CYP2C9 for inhibition of fluorescent bile analog excretion into lumens after bosentan incubation; excretion was impaired in the CYP2C9∗2 HLOs versus wild-type HLOs, potentially owing to impaired CYP2C9-mediated bosentan detoxification.8Markova S.M. De Marco T. Bendjilali N. et al.Association of CYP2C9∗2 with bosentan-induced liver injury.Clin Pharmacol Ther. 2013; 94: 678-686Crossref PubMed Scopus (39) Google Scholar Shinozawa et al are the first to show that HLOs (a) can be reproducibly generated using cryopreserved foregut cells (reduces approximately 6 days from the overall differentiation timeline and allows banking of large numbers of foregut cells for on-demand HLO generation), (b) can be scaled down to a 384-well plate format using a defined liver specification/maturation protocol containing 5 defined factors, (c) have sensitivity and specificity for DILI detection comparable with PHHs when evaluating both ATP levels and inhibition of canalicular transporters, and (d) can be used for DILI detection across different genetic backgrounds, both endogenous and generated via gene editing. HLOs achieved a sensitivity and specificity similar to PHHs even while not achieving complete maturity, a result consistent with micropatterned cocultures containing either PHHs or iHeps and supportive fibroblasts.9Berger D.R. Ware B.R. Davidson M.D. et al.Enhancing the functional maturity of induced pluripotent stem cell-derived human hepatocytes by controlled presentation of cell-cell interactions in vitro.Hepatology. 2015; 61: 1370-1381Crossref PubMed Scopus (126) Google Scholar, 10Ware B.R. Berger D.R. Khetani S.R. Prediction of drug-induced liver injury in micropatterned co-cultures containing iPSC-derived human hepatocytes.Toxicol Sci. 2015; 145: 252-262Crossref PubMed Scopus (119) Google Scholar, 11Khetani S.R. Kanchagar C. Ukairo O. et al.Use of micropatterned cocultures to detect compounds that cause drug-induced liver injury in humans.Toxicol Sci. 2013; 132: 107-117Crossref PubMed Scopus (140) Google Scholar Moving forward, further advances may help improve DILI sensitivity of HLOs (and other iHep-based models) closer to 100% and enable widespread adoption by the pharmaceutical/biotech industry for routine compound screening over other hepatic cell sources (ie, primary and transformed). First, HLO differentiation cost needs to be decreased considerably to justify use in high-throughput screening; current prices of commercially available iHeps (and likely those generated in-house) are approximately 30%–50% higher than PHHs and approximately 50%–70% higher than differentiated but transformed lines in our experience (eg, HepaRG12Hantz O. Parent R. Durantel D. et al.Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells.J Gen Virol. 2009; 90: 127-135Crossref PubMed Scopus (109) Google Scholar). Second, HLOs need to be compared with other liver models (eg, micropatterned co-cultures, liver-on-a-chip, spheroids, bioprinted tissues4Underhill G.H. Khetani S.R. Bioengineered liver models for drug testing and cell differentiation studies.Cell Mol Gastroenterol Hepatol. 2018; 5: 426-439 e1Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) using the same compounds and concentrations, as determined by a consortia of industrial partners and the US Food and Drug Administration, for sensitivity, specificity, and cost. Third, HLOs will need to include a functional cholangiocyte-lined biliary network to enable the investigation of drug and metabolite transport to blood and bile compartments. Fourth, although some non–parenchymal-like cells were found in HLOs, dedicated protocols to drive iPSCs into different liver cell types13Tasnim F. Xing J. Huang X. et al.Generation of mature Kupffer cells from human induced pluripotent stem cells.Biomaterials. 2019; 192: 377-391Crossref PubMed Scopus (25) Google Scholar, 14Sampaziotis F. de Brito M.C. Geti I. et al.Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells.Nat Protoc. 2017; 12: 814-827Crossref PubMed Scopus (55) Google Scholar, 15Danoy M. Poulain S. Koui Y. et al.Transcriptome profiling of hiPSC-derived LSECs with nanoCAGE.Mol Omics. 2020; 16: 138-146Crossref PubMed Google Scholar, 16Coll M. Perea L. Boon R. et al.Generation of hepatic stellate cells from human pluripotent stem cells enables in vitro modeling of liver fibrosis.Cell Stem Cell. 2018; 23: 101-113 e7Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar need to be further developed to allow fabrication of HLOs that better mimic liver sinusoid architecture and cellular composition, toward expanding DILI assessment to all liver cell types, preferably under perfusion to induce functional zonation and subject endothelia to physiological shear stress.17Li X. George S.M. Vernetti L. et al.A glass-based, continuously zonated and vascularized human liver acinus microphysiological system (vLAMPS) designed for experimental modeling of diseases and ADME/TOX.Lab Chip. 2018; 18: 2614-2631Crossref PubMed Google Scholar,18Kang Y.B.A. Eo J. Mert S. et al.Metabolic patterning on a chip: towards in vitro liver zonation of primary rat and human hepatocytes.Sci Rep. 2018; 8: 8951Crossref PubMed Scopus (40) Google Scholar Finally, adaptive immune cells will need to be included into HLOs with sufficient numbers of genetically diverse iPSC lines to help detect some forms of idiosyncratic DILI that manifests itself in 1:10,000 to 1:100,000 patients.1Kaplowitz N. Idiosyncratic drug hepatotoxicity.Nat Rev Drug Discov. 2005; 4: 489-499Crossref PubMed Scopus (820) Google Scholar With these continued advances, HLOs and other iHep-based models are well poised to revolutionize the detection of different types of human DILI during high-throughput preclinical screening toward significantly decreasing animal use and preventing harm in the clinic. High-Fidelity Drug-Induced Liver Injury Screen Using Human Pluripotent Stem Cell–Derived OrganoidsGastroenterologyVol. 160Issue 3PreviewPreclinical identification of compounds at risk of causing drug induced liver injury (DILI) remains a significant challenge in drug development, highlighting a need for a predictive human system to study complicated DILI mechanism and susceptibility to individual drug. Here, we established a human liver organoid (HLO)–based screening model for analyzing DILI pathology at organoid resolution. Full-Text PDF Open Access