Liver organoid compositions and methods of making and using same

Abstract

A method for inducing the formation of liver organoids from progenitor cells such as iPSC cells is provided. [Solution] A method for inducing the formation of liver organoids from iPSC cells, comprising: a) contacting the iPSC cell-derived definitive endoderm (DE) with an FGF pathway activator and a Wnt signaling pathway activator for a period sufficient to form posterior foregut spheroids, preferably about 1 to about 3 days; and b) incubating the posterior foregut spheroids of step a in the presence of retinoic acid (RA) for a period sufficient to form the liver organoids, preferably about 1 to about 5 days, preferably about 4 days.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 517,414, filed June 9, 2017, the contents of each of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] The liver is a vital organ that provides many metabolic functions essential for life, such as detoxification and coagulation of exogenous compounds, as well as the production of lipids, proteins, ammonium, and bile. In vitro reconstruction of a patient's liver may offer applications including regenerative therapy, drug discovery, and drug toxicity studies. Existing methodologies using hepatocytes exhibit very poor functionality, primarily due to the lack of essential anatomical structures, which limits their practical use in the pharmaceutical industry.

[0003] In the pharmaceutical industry, billions of dollars are lost annually from drug development due to the failure of drug candidates identified in early screening, with nearly one-third of drugs being withdrawn from the market due to such failures (Takebe and Taniguchi, 2014). Drug candidate failure results in a significant loss of therapeutic opportunity for patients. Preclinical testing typically consists of in vitro evaluation as the primary efficacy screen to identify "hit" compounds, followed by in vitro and in vivo safety testing to assess metabolic and toxicity mechanisms. This inefficiency can be explained by the virtual lack of high-throughput, physiologically relevant preclinical models for assessing drug-induced liver injury (DILI) in humans. Therefore, there is an urgent need to develop in vitro human screening models to evaluate the vast numbers of ever-growing compound libraries.

[0004] Primary hepatocytes are a highly polarized metabolic cell type, forming bile canaliculi with microvilli channels that separate the peripheral circulation from the bile acid secretory pathway. The most upstream aspect of DILI involves hepatocyte detoxification of drugs (or their reactive metabolites) and their excretion into the bile canaliculi via transporters such as multidrug resistance-associated protein (MRP) transporters. This suggests the need to reconstitute these uniquely organized structures as a key in vivo property of hepatocytes to predict DILI pathology. However, as in the cases of troglitazone, nefazodone, and tolcapone (https: / / livertox.nlm.nih.gov / index.html), there are considerable differences in drug toxicity profiles between current simplified culture models involving the use of isolated primary human hepatocytes or hepatic cell lines and in vivo physiology. Therefore, while toxicological characterization primarily relies on animals as an essential step for drug development, fidelity to human results is severely lacking due to significant differences in human and animal physiology (Leslie et al., 2007; Yang et al., 2014). Furthermore, while idiosyncratic DILI (IDILI) is extremely rare, it is nonetheless responsible for approximately 10–15% of acute liver failure cases in the United States (Reuben et al., 2010) and is nearly impossible to predict (Kullak-Ublick et al., 2017). Collectively, effective human cell models are needed to screen compounds for proposed drug detoxification and excretion testing.

[0005] Despite innovative advances in methods for differentiating human hepatocytes from pluripotent stem cells (PSCs), clinical trials in a dish using human stem cells remain a "hype." There is a need for hepatocyte models for use in bioartificial liver devices, for example, as a bridge to transplantation and for precision (personalized) medicine, as well as for drug screening for efficacy and / or toxicity. The present disclosure seeks to address one or more of the aforementioned needs in the art. Summary of the Invention

[0006] Disclosed is a method for inducing the formation of liver organoid from progenitor cells such as iPSC cells.Disclosed liver organoid can be used for screening for serious adverse events (SAE), such as liver failure and / or drug-induced liver injury (DILI), and / or drug toxicity.Disclosed liver organoid can also be used to treat individuals with liver damage or to identify preferred therapeutic agents. [Brief explanation of the drawings]

[0007] Those skilled in the art will understand that the drawings, described below, are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way.

[0008] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the fee.

[0009] [Figure 1A] Generation of human liver organoids from iPSCs with luminal structures. A. Overview of differentiation methods for liver organoids. [Figure 1B] B. Phase contrast image of human liver organoids. [Figure 1C] C. Immunostaining of albumin (ALB), type IV collagen (collagen IV), and ZO-1 in organoids. Nuclei were stained with hematoxylin (blue). Bar, 50 μm. [Figure 1D]D. Quantitative RT-PCR of alpha-fetoprotein (AFP), albumin (ALB), retinol-binding protein 4 (RBP4), cytokeratin 19 (CK19), hepatocyte nuclear factor 6 (HNF6), and cytochrome P450 3A4 (CYP3A4) in undifferentiated iPSCs, organoids at days 7, 11, 20, and 30 of differentiation, and primary hepatocytes (PH). Relative expression values ​​were compared between undifferentiated iPSCs (AFP, ALB, RBP4, and CK19), day 7 organoids (HNF6), and day 11 organoids (CYP3A4). Bars represent mean ± SD, n=3. [Figure 1E] E. Principal component analysis based on RNA-seq data in undifferentiated iPS cells (iPSCs), definitive endoderm (DE), hepatic-specified cells (HS), hepatic progenitor cells (HP), iPSC-derived cholangiocytes (iDCs), normal human cholangiocytes (NHCs), iPSC-derived posterior foregut (pFG), iPSC-derived human liver organoids, primary hepatocytes, fetal liver tissue, hepatic tissue and right lobe of human liver. [Figure 1F] F. Albumin (ALB, n = 10) and fibrinogen (FBG, n = 4) secretion levels from organoids on days 25–30. Bars represent mean ± SEM. [Figure 1G] G. Complement factor secretion levels from organoids on days 25–30. [Figure 1H] FH: factor H, FB: factor B. Bars represent mean ± SEM, n=5. [Figure 2A] Bile acid synthesis, uptake, and excretion in human iPSC liver organoids. A. Immunostaining of multidrug resistance-associated protein 2 (MRP2) and bile salt export pump (BSEP) in a single organoid. Bar, 50 μm. [Figure 2B] B. Transilluminal electron microscopy of an organoid showing the microvilli (V) luminal surface; N: nuclei. Bar, 10 μm. [Figure 2C]C. Quantitative RT-PCR of ATP-binding cassette, subfamily B member 11 (ABCB11), and sodium taurocholate cotransporting polypeptide (NTCP) in undifferentiated iPSC cells, organoids at day 20 (NTCP) and day 30 (ABCB11) of differentiation, and primary hepatocytes (PH). Relative expression values ​​were compared in undifferentiated iPSCs (ABCB11) or organoids at day 11 (NTCP). Bars represent mean ± SD, n = 3. [Figure 2D] D. Total bile acid secretion levels inside organoids at day 27. Bars represent mean ± SEM, n=4. [Figure 2E] E. Bile acid uptake by organoids after 30 min of incubation in the presence of fluorescent bile acid (CGamF). [Figure 2F] F. CLF transport activity to organoids derived from four iPSC lines. T, W, 1, and F indicate the clonal names of the iPS cell lines. Green: CLF. [Figure 3A] Bosentan-induced cholestasis is specific to CYP2C9*2 iPSC-liver organoids. A. Representative allele images of rs1799853 in CYP2C9*2 and rs4148323 in UGT1A1*6 show the risk SNPs for bosentan- and irinotecan-induced DILI, respectively. The table shows the prevalence of the risk allele in each iPS cell line. [Figure 3B] B. Images of CLF transport activity and inhibition by bosentan. [Figure 3C] C. CLF intensity levels in individual organoids derived from four different iPS cell lines. *: p<0.01, **: p<1E-4, ****: p<1E-8, Wilcoxon-Mann-Whitney test. NS: not significant. In box plots, the top and bottom of the box represent the 75th and 25th percentiles, and the center line represents the median. Dots represent data from each organoid. [Figure 3D]D. CLF intensity levels in individual organoids derived from four different iPS cell lines. *: p<0.01, **: p<1E-4, ****: p<1E-8, Wilcoxon-Mann-Whitney test. NS: not significant. In box plots, the top and bottom of the box represent the 75th and 25th percentiles, and the center line represents the median. Dots represent data from each organoid. [Figure 4A] A high-fidelity organoid model of drug-induced cholestasis. A. Sequential imaging of fluorescein diacetate excretion from the outside to the inside of an organoid. [Figure 4B] B. Comparison of fluorescein diacetate efflux transport. [Figure 4C] C. Quantification of fluorescein diacetate efflux transport into organoids. The illustrative left figure shows the quantified ratio of fluorescein intensity between the inside and outside of the organoid. The right figure shows the results of a validation study using control (DMSO), cyclosporine A (CSA), and streptomycin (STP) as a negative control. Bars represent mean ± SD; **: p<0.01, n=4. [Figure 4D] D. Images of fluorescein diacetate transport inhibition after 24 h treatment with nine training compounds. [Figure 4E] E. Quantification of transport inhibition after treatment with training compounds. Bars represent mean ± SD, *: p<0.05, **: p<0.01, n=4-6. Quantification of MMP changes after treatment with training compounds. Bars represent mean ± SD, *: p<0.05, **: p<0.01, n=4-5. CON: control sample, STP: streptomycin, TOL: tolcapone, DICLO: diclofenac, BOS: bosentan, CSA: cyclosporine A. [Figure 5A] High-fidelity drug-induced mitochondrial toxicity screening using organoids. A. Images of mitochondrial membrane potential (MMP) on TMRM after treatment with nine training compounds. [Figure 5B]Bottom: Quantification of transport inhibition after treatment with training compounds. Bars represent mean ± SD, *: p<0.05, **: p<0.01, n=4-6. [Figure 5C] C. Quantification of MMP changes after treatment with training compounds. Bars represent mean ± SD. *: p<0.05, **: p<0.01, n=4–5. CON: control sample; STP: streptomycin; TOL: tolcapone; DICLO: diclofenac; BOS: bosentan; CSA: cyclosporine A; TRO: troglitazone; NEFA: nefazodone; ENTA: entacapone; PIO: pioglitazone. B. Classification of the set of nine training compounds (TCs) mentioned in Oorts et al., 2016 (Oorts et al., 2016). Class A represents TCs with known in vivo reports of DILI, while Class B represents TCs with in vivo reports of drug-induced cholestasis. The mechanism of toxicity based on literature data is also provided. Class C compounds are generally considered safe with respect to DILI. C. Analysis between survival rate 72 hours after drug treatment and dual risk parameters, potential drug-induced cholestasis and potential mitochondrial toxicity. [Figure 5D] Cholestatic and mitochondrial toxicity (Mito-tox) indices were derived from the data in Figure 3. The size of the circle indicated the magnitude of the loss of viability. [Figure 6A] Modeling drug-induced liver injury in a vulnerable state rescued by NAC exposure. A. Overview of evaluation of drug-induced cytotoxicity in a vulnerable organoid model. [Figure 6B] B. Profiling of the fragile model for lipid accumulation (blue: nuclei, green: lipids, red: F-actin). [Figure 6C] C ROS production (blue: nuclei, green: ROS) [Figure 6D] and D. Mitochondrial health (blue: nuclei, red: mitochondria). [Figure 6E] E. Images of organoids 24 hours after drug treatment. [Figure 6F]F. Viability assessment of lipid-induced fragile organoid models. Bars represent mean ± SD. *: p<0.05, n=5-6. CON: control, STP: streptomycin, TRO: troglitazone, NAC: N-acetylcysteine. [Figure 7] Multiplexed liver organoid-based screening for toxicity prediction. [Figure 8A] Optimization of retinoic acid treatment protocol A. Plan the timing and duration of retinoic acid treatment. RA: retinoic acid, HCM: hepatocyte culture medium. [Figure 8B] B. Albumin secretion levels in organoids at day 25 under different durations of RA treatment. [Figure 9] Organoid morphology at D20. The total number of organoids at D20 was 305: 216 organoids with a lumen and 89 organoids without a lumen. [Figure 10A] Conversion formula for determining cell number in organoids. A. Phase contrast image of a single organoid. [Figure 10B] B. Diameter and cell number of each single organoid. [Figure 10C] C. Correlation between diameter and cell number in single organoids. [Figure 11] Supplemental Figure 4. Generation of organoids from multiple PSC lines. Phase contrast images and albumin secretion levels of organoids derived from different iPS cell lines (317D6 and 1383D6). [Figure 12] Cell viability after 24 hours of treatment with 10 compounds. Viability assessment of a lipid-induced fragile organoid model. CON: control sample, STP: streptomycin, TOL: tolcapone, DICLO: diclofenac, AMIO: amiodarone, BOS: bosentan, CSA: cyclosporine A, TRO: troglitazone, NEFA: nefazodone, ENTA: entacapone, PIO: pioglitazone. Bars represent mean ± SD, n=4-6. [Figure 13A]Mitochondrial ROS generation and morphological changes in lipotoxic liver organoids. A. Ratio of ROS-producing cells to total cells in a lipid accumulation-induced fragile organoid model treated with 800 μM oleic acid (OA). [Figure 13B] B. Image of mitochondria in organoids for the fragile organoid model. Red: mitochondria, purple: F-actin, blue: nuclei. [Figure 13C] C. Mitochondrial number and size for the fragile organoid model. Bars represent mean ± SD. *: p<0.05, n=5-6. [Figure 14] Schematic of the cell-free Matrigel method. A schematic of the liver organoid generation method that does not use Matrigel to generate organoids is shown. DETAILED DESCRIPTION OF THE INVENTION

[0010] Unless otherwise specified, terms are to be understood according to conventional usage by those of ordinary skill in the art.

[0011] The term "about" or "approximately" means within an acceptable error range for a particular value, depending on how the value is measured or determined, e.g., the limitations of the measurement system, as determined by one of ordinary skill in the art. For example, "about" can mean within one or more standard deviations, in accordance with practice in the art. Alternatively, "about" can mean within a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within 10-fold, preferably within 5-fold, and more preferably within 2-fold of a value. Where specific values ​​are described in this application and claims, unless otherwise specified, the term "about" should be construed to mean within an acceptable error range for the particular value.

[0012] As used herein, the term "totipotent stem cells" (also known as omnipotent stem cells) are stem cells that can differentiate into embryonic and extraembryonic cell types. Such cells are capable of building complete, viable organisms. These cells are produced from the fusion of an egg cell and a sperm cell. Cells produced by the first few divisions of a fertilized egg are also totipotent.

[0013] As used herein, the term "pluripotent stem cells (PSCs)" encompasses any cell that can differentiate into almost any cell type in the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (inner gastric wall, digestive tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), and ectoderm (epidermal tissue and nervous system). PSCs can be the descendants of inner cell mass cells of preimplantation blastocysts or can be derived by the induction of non-pluripotent cells, such as adult somatic cells, by forcing the expression of specific genes. Pluripotent stem cells can be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including humans, rodents, pigs, and cattle.

[0014] As used herein, the term "induced pluripotent stem cells (iPSCs)," commonly abbreviated as iPS cells, refers to a type of pluripotent stem cell that is artificially induced from a normally non-pluripotent cell, such as an adult somatic cell, by inducing the "forced" expression of a specific gene. hiPSCs refer to human iPSCs.

[0015] As used herein, the term "embryonic stem cells (ESCs)," also commonly abbreviated as ES cells, refers to cells that are pluripotent and derived from the inner cell mass of an early embryo, the blastocyst. For purposes of the present invention, the term "ESCs" is used broadly to optionally include embryonic germ cells.

[0016] As used herein, the term "progenitor cell" encompasses any cell that can be used in the methods described herein, in which one or more progenitor cells acquire the ability to regenerate themselves or differentiate into one or more specialized cell types. In some embodiments, progenitor cells are pluripotent or have the ability to become pluripotent. In some embodiments, progenitor cells are subjected to treatment with external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, progenitor cells can be totipotent (totipotent or omnipotent) stem cells; pluripotent stem cells (induced or non-induced); multipotent stem cells; oligopotent stem cells, and unipotent stem cells. In some embodiments, progenitor cells can be derived from embryos, infants, children, or adults. In some embodiments, progenitor cells can be somatic cells that have undergone treatment such that pluripotency is conferred via genetic manipulation or protein / peptide treatment.

[0017] In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. As used herein, the term "directed differentiation" refers to the process by which less specialized cells become specific specialized target cell types. The specificity of the specialized target cell type can be determined by any applicable method that can be used to define or change the fate of initial cells. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0018] Embryonic cell-derived pluripotent stem cells In some embodiments, one step is to obtain stem cells that are pluripotent or can be induced to become pluripotent. In some embodiments, the pluripotent stem cells are derived from embryonic stem cells, which are derived from totipotent cells of early mammalian embryos and are capable of indefinite undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of an early-stage embryo, the blastocyst. Methods for deriving embryonic stem cells from blastocysts are well known in the art. Human embryonic stem cells H9 (H9-hESC) are used in the exemplary embodiments described herein, but those skilled in the art will understand that the methods and systems described herein are applicable to any stem cells.

[0019] Additional stem cells that may be used in embodiments according to the present invention include, but are not limited to, those provided by or described in databases maintained by the National Stem Cell Bank (NSCB), the Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF); the WISC cell Bank at the Wi Cell Research Institute; the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW-SCRMC); Novocell, Inc. (San Diego, Calif.); Cellartis AB (Göteborg, Sweden); ES Cell International Pte Ltd (Singapore); the Technion at the Israel Institute of Technology (Haifa, Israel); and the Stem Cell Database maintained by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that may be used in embodiments according to the present invention include, but are not limited to, SA01 (SA001); SA02 (SA002); ES01 (HES-1); ES02 (HES-2); ES03 (HES-3); ES04 (HES-4); ES05 (HES-5); ES06 (HES-6); BG01 (BGN-01); BG02 (BGN-02); BG03 (BGN-03); TE03 (13); TE04 (14); TE06 (16); UC01 (HSF1); UC06 (HSF6); WA01 (H1); WA07 (H7); WA09 (H9); WA13 (H13); WA14 (H14).

[0020] For further details on embryonic stem cells, see, for example, Thomson et al., 1998, "Embryonic Stem Cell Lines Derived from Human Blastocysts," Science 282(5391):1145-1147; Andrews et al., 2005, "Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin," Biochem Soc Trans 33:1526-1530; Martin 1980, "Teratocarcinomas and mammalian embryogenesis," Science 209(4458):768-776; Evans and Kaufman, 1981, "Establishment in culture of pluripotent cells from mouse embryos," Nature 292(5819):154-156; Klimanskaya et al., 2005, "Human embryonic stem cells derived without feeder "Cells," Lancet 365(9471):1636-1641, each of which is incorporated herein by reference in its entirety.

[0021] Induced pluripotent stem cells (iPSC) In some embodiments, iPSCs are derived by transfecting specific stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection is typically achieved through viral vectors such as retroviruses. Transfected genes include the master transcriptional regulators Oct-3 / 4 (Pouf51) and Sox2, although other genes have been suggested to increase the efficiency of induction. After 3–4 weeks, a small number of transfected cells become morphologically and biochemically similar to pluripotent stem cells and are typically isolated through morphological selection, doubling time, or reporter gene and antibiotic selection. As used herein, iPSCs include, but are not limited to, first-generation iPSCs, second-generation iPSCs in mice, and human induced pluripotent stem cells. In some embodiments, a retroviral system is used to transform human fibroblasts into pluripotent stem cells using four central genes: Oct-3 / 4, Sox2, Klf4, and c-Myc. In another embodiment, somatic cells are transformed with OCT4, SOX2, NANOG, and LIN28 using lentivirus system. The genes whose expression is induced in iPSCs include, but are not limited to, Oct-3 / 4 (for example, Pou5f1); certain members of the Sox gene family (for example, Sox1, Sox2, Sox3, and Sox15); certain members of the Klf family (for example, Klf1, Klf2, Klf4, and Klf5), certain members of the Myc family (for example, C-myc, L-myc, and N-myc), Nanog, and LIN28.

[0022] In some embodiments, non-viral techniques are used to generate iPSCs. In some embodiments, adenoviruses are used to deliver the four necessary genes into the DNA of mouse skin and liver cells, resulting in cells identical to embryonic stem cells. Because adenoviruses do not integrate any of their own genes into the target host, the risk of tumorigenesis is eliminated. In some embodiments, reprogramming can be achieved via plasmids without the use of viral transfection systems at all, albeit with very low efficiency. In other embodiments, direct protein delivery is used to generate iPSCs, thus eliminating the need for viral or genetic modification. In some embodiments, mouse iPSCs can be generated using similar methodologies: repeated treatment of cells with specific proteins delivered via polyarginine anchors was sufficient to induce pluripotency. In some embodiments, expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions.

[0023] For further details regarding embryonic stem cells, see Kaji et al., 2009, "Virus-free induction of pluripotency and subsequent excision of reprogramming factors," Nature 458:771-775; Woltjen et al., 2009, "piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells," Nature 458:766-770; Okita et al., 2008, "Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors," Science 322(5903):949-953; Stadtfeld et al., 2008, "Induced Pluripotent Stem Cells Generated without Viral Integration," Science 322(5903):945-949; and Zhou et al., 2009, "Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins," Cell Stem Cell 4(5):381-384; each of which is incorporated herein by reference in its entirety.

[0024] In some embodiments, exemplary iPS cell lines include, but are not limited to, iPS-DF19-9; iPS-DF19-9; iPS-DF4-3; iPS-DF6-9; iPS(foreskin); iPS(IMR90); and iPS(IMR90).

[0025] Further details regarding the function of signaling pathways involved in the development of DE can be found, for example, in Zorn and Wells, 2009, "Vertebrate endoderm development and organ formation," Annu Rev Cell Dev Biol 25:221-251; Dessimoz et al., 2006, "FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo," Mech Dev 123:42-55; McLin et al., 2007, "Repression of Wnt / β-catenin signaling in the anterior endoderm is essential for liver and pancreas development." Development 134:2207-2217; Wells and Melton, 2000, Development 127:1563-1572; de Santa Barbara et al., 2003, "Development and differentiation of the intestinal epithelium," Cell Mol Life Sci 60(7):1322-1332; each of which is incorporated herein by reference in its entirety.

[0026] Any method for generating definitive endoderm from pluripotent cells (e.g., iPSCs or ESCs) is applicable to the methods described herein. Any method for generating definitive endoderm from pluripotent cells (e.g., iPSCs or ESCs) is applicable to the methods described herein. Exemplary methods are described, for example, in US97 / 19068B2 (Wells et al.), "Methods and systems for converting precursor cells into intestinal tissues through directed differentiation," and US2017 / 0240866A1 (Wells et al.), "Methods and systems for converting precursor cells into gastric tissues through directed differentiation." In some embodiments, the pluripotent cells are derived from a morula. In some embodiments, the pluripotent stem cells are stem cells. Stem cells used in these methods can include, but are not limited to, embryonic stem cells. Embryonic stem cells can be derived from the inner cell mass of an embryo or the gonadal ridge of an embryo. Embryonic stem cells or germ cells can be derived from various animal species, including, but not limited to, various mammalian species, including humans. In some embodiments, human embryonic stem cells are used to produce definitive endoderm. In some embodiments, human embryonic germ cells are used to produce definitive endoderm. In some embodiments, iPSCs are used to produce definitive endoderm.Additional methods for obtaining or generating DE cells that can be used in the present invention include, but are not limited to, those described in U.S. Pat. No. 7,510,876 (D'Amour et al.); U.S. Pat. No. 7,326,572 (Fisk et al.); Kubol et al., 2004, "Development of definitive endoderm from embryonic stem cells in culture," Development 131:1651-1662; D'Amour et al., 2005, "Efficient differentiation of human embryonic stem cells to definitive endoderm," Nature Biotechnology 23:1534-1541; and Ang et al., 1993, "The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3 / forkhead proteins," Development 119:1301-1315.

[0027] The applicant has discovered a method for creating a 3D liver structure using human iPSCs. The structure contains microhepatic structures, including polarized hepatic epithelium, stellate cells, and canalicular structures. The disclosed composition exhibits improvements in liver function, bile transport activity, and durability compared to existing models. The 3D structure model can be used as a novel and robust model for drug screening and / or drug toxicity screening, transplantation, production of serum protein products, and development of personalized therapies. In one specific application, the composition and method can be used to screen drug compounds for liver toxicity.

[0028] While 3D aggregated hepatocytes have been reported, the disclosed compositions possess significantly higher functional activity, including albumin production (up to 10-fold increase compared to previous gold-standard models using iPSC-derived hepatocytes), and their internal luminal structure allows for improved oxygen and / or nutrient supply, thereby enabling much longer culture times (at least 60 days or more) and a long-term testing platform useful for drug testing. The disclosed compositions may also be useful for producing plasma products, such as albumin, a clotting factor product for the treatment of hypoalbuminemia, and for therapeutic transplantation, where miniature livers derived from human iPSCs can be transplanted to treat disorders in vivo. Finally, the disclosed compositions can be used for personalized medicine (individualized treatment).

[0029] In one embodiment, a method for inducing the formation of liver organoids from iPSC cells is disclosed.

[0030] The method may include the steps of: a) contacting iPSC cell-derived definitive endoderm (DE) with an FGF pathway activator and an activator of the Wnt signaling pathway (which can be activated by a GSK3 inhibitor) for a period sufficient to form posterior foregut spheroids, preferably about 1 to about 3 days; and b) incubating the posterior foregut spheroids obtained in step a in the presence of retinoic acid (RA) for a period sufficient to form liver organoids, preferably about 1 to about 5 days, preferably about 4 days.

[0031] Fibroblast growth factors (FGFs) are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. FGFs are heparin-binding proteins, and interaction with cell surface-associated heparan sulfate proteoglycans has been shown to be essential for FGF signal transduction. Suitable FGF pathway activators will be readily apparent to those skilled in the art. Exemplary FGF pathway activators include, but are not limited to, one or more molecules selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. In some embodiments, siRNA and / or shRNA targeting cellular components related to the FGF signaling pathway may be used to activate these pathways.

[0032] In some embodiments, DE cultures are treated with one or more molecules of the FGF signaling pathway described herein at a concentration of 10 ng / ml or more; 20 ng / ml or more; 50 ng / ml or more; 75 ng / ml or more; 100 ng / ml or more; 120 ng / ml or more; 150 ng / ml or more; 200 ng / ml or more; 500 ng / ml or more; 1,000 ng / ml or more; 1,200 ng / ml or more; 1,500 ng / ml or more; 2,000 ng / ml or more; 5,000 ng / ml or more; 7,000 ng / ml or more; 10,000 ng / ml or more; or 15,000 ng / ml or more. In some embodiments, the concentration of the signaling molecule is maintained constant during treatment. In other embodiments, the concentration of the signaling pathway molecule changes over the course of treatment. In some embodiments, the signaling molecules according to the present invention are suspended in a medium containing DMEM and fetal bovine serum (FBS). The FBS can be at a concentration of 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, or 50% or more. Those skilled in the art will understand that the regimens described herein can be applied alone or in combination to any known molecule in the signaling pathways described herein, including but not limited to, any molecule in the FGF signaling pathway.

[0033] Suitable FGF pathway activators will be readily apparent to those skilled in the art. In one embodiment, the FGF signaling pathway activator may be selected from a small molecule or protein FGF signaling pathway activator, FGF1, FGF2, FGF3, FGF4, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, or a combination thereof. The WNT signaling pathway activator may be selected from small molecule or protein Wnt signaling pathway activators, such as lithium chloride; 2-amino-4,6-disubstituted pyrimidine (hetero)arylpyrimidine; IQ1; QS11; NSC668036; DCA beta-catenin; 2-amino-4-[3,4-(methylenedioxy)-benzyl-amino]-6-(3-methoxyphenyl)pyrimidine, Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, GSK3 inhibitors, preferably CHIRON, R-spondin, or combinations thereof. In one embodiment, the BMP activator may be selected from BMP2, BMP4, BMP7, BMP9, small molecules that activate the BMP pathway, proteins that activate the BMP pathway, and may include noggin, dorsomorphin, LDN189, DMH-1, ventromophin, and combinations thereof. Suitable GSK3 inhibitors will be readily apparent to those skilled in the art. Exemplary GSK3 inhibitors include, but are not limited to, Chiron / CHIR99021, which inhibits GSK3β. Those skilled in the art will recognize suitable GSK3 inhibitors for practicing the disclosed methods. GSK3 inhibitors may be administered in amounts of about 1 μM to about 100 μM, or about 2 μM to about 50 μM, or about 3 μM to about 25 μM. Those skilled in the art will readily recognize appropriate amounts and durations. In some embodiments, siRNA and / or shRNA targeting cellular components associated with the FGF signaling pathway may be used to activate these pathways.

[0034] In one embodiment, the stem cells can be mammalian or human iPSCs.

[0035] In one embodiment, foregut spheroids can be embedded in a basement membrane matrix, such as the commercially available basement membrane matrix sold under the trademark Matrigel.

[0036] In one embodiment, liver organoid can be characterized by expressing alpha-fetoprotein (AFP), albumin (ALB), retinol-binding protein (RBP4), cytokeratin 19 (CK19), hepatocyte nuclear factor 6 (HNF6), cytochrome P450 3A4 (CYP3A4), HNF4a, E-cadherin, DAPI and Epcam.This expression can occur, for example, from day 40 to day 50.Expression level can be similar to that observed in human hepatocyte, for example, that of adult hepatocyte.

[0037] In one aspect, the liver organoids can be characterized as having bile transport activity.

[0038] In one aspect, liver organoid can be derived from stem cells, and can comprise the luminal structure that further comprises internalized microvilli cells and mesenchymal cells.Luminous structure can be surrounded by polarized hepatocytes and basement membrane.Liver organoid can comprise functional stellate cells and functional Kupffer cells.

[0039] In one embodiment, the liver organoids have the following characteristics: bile production capacity, bile transport activity, and a bile saturation of at least 50 ng / mL / 1xe 6 Complement factor H expression at least 40 ng / mL / 1xe per cell / 24 h 6 Complement factor B, at least 1000ng / mL / 1xe per cell / 24 hours 6 C3 expression at least 1000ng / mL / 1xe per cell / 24 hours 6 C4 expression at least 1000ng / mL / 1xe per cell / 24 hours 6fibrinogen production of at least 1000ng / mL / 1xe 6 In one embodiment, the liver organoids have at least one of the following characteristics: albumin production in cells / 24 hours; ... 6 Liver organoid can be characterized by having cell / 24 hours total liver protein expression.Liver organoid can be characterized by being able to express one or more genes selected from PROX1, RBP4, CYP2C9, CYP3A4, ABCC11, CFH, C3, C5, ALB, FBG, MRP2, ALCAM, CD68, CD34, CD31.In one embodiment, liver organoid can comprise the cell that comprises drug metabolism cytochrome mutant, such as CY2C9*2 mutant.Liver organoid can comprise vasculature, such as that described in US 2016 / 0177270.

[0040] In one embodiment, liver organoids can be characterized in that they do not contain inflammatory cells, such as T cells or other inflammatory secretory proteins.

[0041] In one embodiment, a method for screening for serious adverse events (SAE) is disclosed.SAE can be liver failure and / or drug-induced liver injury (DILI).This method can include contacting the target drug whose toxicity is being investigated with the liver organoid described herein.In one embodiment, the method can include measuring the uptake and / or excretion of fluorescein diacetate (FD), where impaired excretion indicates that the drug may induce serious adverse events.The toxicity of the target drug can be determined by measuring parameters selected from mitochondrial membrane potential, ROS measurement, liver mitochondrial swelling, and combinations thereof, where damage to mitochondria indicates that the drug may induce serious adverse events.In one embodiment, the method includes analyzing organoid viability, where impaired or reduced organoid viability indicates that the target drug may induce serious adverse events.

[0042] In one aspect, disclosed is the method for treating the individual with liver damage, and this method can comprise transplanting the liver organoid described herein into the individual who needs it.Liver damage can include, for example, metabolic liver disease, end-stage liver disease, or combinations thereof.

[0043] In one embodiment, disclosed is the method for identifying the preferred therapeutic agent for individual.In this embodiment, method can include contacting the liver organoid derived from target iPSC with candidate compound, wherein, for example, target iPSC comprises one or more mutations that are found in this individual, or for example, target iPSC is derived from the same ethical background as this individual, or further, target iPSC is derived from this individual. [Example]

[0044] In this study, we tested bile transport activity using fluorescein diacetate, which crosses the bile canalicular membrane and is excreted into the bile canalicular network by MRP2 (Tian et al., 2004). Troglitazone and cyclosporine have previously been reported to inhibit MRP2 (Chang et al., 2013; Lechner et al., 2010). Furthermore, the efflux transporter MRP2 mediates the transport of bosentan (Fahrmayr et al., 2013). Although inhibition of MRP2 by nefazodone has not been reported, because MRP2 is an ATP-dependent bile salt transporter for canalicular excretion of bile acids in hepatocytes, mitochondrial stress induced by nefazodone may be related to the decreased excretion of fluorescein diacetate in bile canalicular bile.

[0045] Preclinical detection of risk compounds for drug-induced liver injury (DILI) remains a key challenge in drug development, highlighting the need for predictive human systems. Here, we developed a human liver organoid (HLO) model for analyzing clinical DILI pathology at organoid resolution. Differentiated HLOs from human iPSCs contain polarized hepatocytes with a lumen lined by bile canaliculi-like structures, establishing a unidirectional bile acid transport pathway. We leveraged the structural features of organoids by modeling DILI using liver organoid imaging, termed LoT (Liver Organoid-Based Toxicity Screening). LoT is functionally validated with 10 marketed drugs and five different donors based on cholestatic and / or mitochondrial toxicity. Bosentan-induced cholestasis is specific to HLOs derived from CYP2C9 poor metabolizer donors. Interestingly, steatotic organoids were vulnerable to rosiglitazone toxicity, as suggested in the clinic, followed by chemical rescue from mass organoid death. Thus, LoT is a high-fidelity organoid model that can be used to analyze drug safety, yet is a cost-effective platform, facilitating compound optimization, providing mechanistic studies, and leading to personalized medicine and anti-DILI therapeutic screening applications.

[0046] In the pharmaceutical industry, billions of dollars are lost annually from drug development due to the failure of drug candidates identified in early screening, leading to the withdrawal of many drugs (one-third) from the market due to such failures (Takebe and Taniguchi, 2014). Despite promising efficacy, failed drug candidates result in significant lost treatment opportunities for patients. Preclinical testing typically consists of in vitro evaluation as the primary efficacy screen to identify "hit" compounds, followed by in vitro and in vivo safety testing to assess metabolic and toxicity mechanisms. This inefficiency can be explained by the virtual lack of physiologically relevant preclinical models for assessing drug-induced liver injury (DILI) in humans. Therefore, there is an urgent need to develop in vitro human screening models to evaluate vast numbers from ever-growing compound libraries.

[0047] Primary hepatocytes are a highly polarized metabolic cell type, forming bile canaliculi with microvilli channels that separate the peripheral circulation from the bile acid secretory pathway. The most upstream aspect of DILI involves hepatocyte detoxification of drugs (or their reactive metabolites) and their excretion into the bile canaliculi via transporters such as multidrug resistance-associated protein (MRP) transporters. This suggests the need to reconstitute these uniquely organized structures as a key in vivo property of hepatocytes to predict DILI pathology. However, as in the cases of troglitazone, nefazodone, and tolcapone (https: / / livertox.nlm.nih.gov / index.html), there are considerable differences in drug toxicity profiles between current simplified culture models involving the use of isolated primary human hepatocytes or hepatic cell lines and in vivo physiology. Therefore, toxicological characterization primarily relies on animals as an essential step for drug development, but fidelity to human results is severely lacking due to significant differences in human and animal physiology (Leslie et al., 2007; Yang et al., 2014). Furthermore, while idiosyncratic DILI (IDILI) is extremely rare, it is nonetheless responsible for approximately 10–15% of acute liver failure cases in the United States (Reuben et al., 2010) and is nearly impossible to predict (Kullak-Ublick et al., 2017). In summary, effective human cell models are highly anticipated for screening compounds to test proposed drug detoxification and excretion.

[0048] Despite innovative advances in differentiation methods for human hepatocytes from pluripotent stem cells (PSCs), clinical trials in a dish using human stem cells remain a "hype." To some extent, this can be explained by challenges with previous cell-based approaches, including (1) overcoming lot-to-lot variations, (2) minimizing experimental batch variations, (3) increasing assay throughput, and (4) improving correlation with clinical trial data. Applicant addresses these issues by developing a relatively simple and robust organoid-based testing platform using stably expandable human stem cells, i.e., iPSCs. Applicant first directed human PSCs into posterior foregut organoids and continued progressive hepatocyte differentiation through polarized culture with defined factors and matrices. The generated human liver organoids possess a luminal structure surrounded by polarized hepatocytes and have been shown to be capable of performing essential human hepatocyte functions, including protein and bile acid production and transport. Interestingly, we found that live-image-based dynamic detection of fluorescent diacetate uptake and excretion accurately modeled cholestasis induced by a series of DILI drugs characterized as inhibitors of biliary excretion with a high level of reproducibility. Separately, mitochondrial membrane potential assessment enabled independent risk assessment for each compound, reflecting the traditional classification of DILI drugs established by clinical trials. Furthermore, we extended our approach to model conditions induced by lipotoxic stress and confirmed the enhancement of DILI potential due to reactive oxygen species (ROS) production. Organoid-based viability assessment confirmed the reversal of DILI by N-acetylcysteine, highlighting the potential of our approach for anti-DILI drug screening. Taken together, this robust assay, termed the liver organoid-based toxicity screen (LoT), is considered the first functional readout developed in human liver organoids and will facilitate diagnostics, functional studies, drug development, and personalized medicine.

[0049] result Generation and Characterization of Polarized Liver Organoids from Multiple Human iPSCs We first established a novel liver organoid differentiation method using human iPSC-derived foregut spheroids (Spence et al., 2011) (Figure 1A). As a first step, we used BMP and activin A to promote differentiation into definitive endoderm, as previously described (D'Amour et al., 2005). Furthermore, we induced foregut spheroids using FGF4 and a GSK3 inhibitor (CHIR99021) and observed budding spheroids. After gently pipetting to detach mesenchymal cells seeded on the dish, the organoids were embedded in Matrigel. Retinoic acid (RA) has been reported to enhance cell polarity, as indicated by increased size and complexity of bile canaliculi and pericanalicular sheaths (Falasca et al., 1998). To generate polarized organoids suitable for bile transport modeling, we treated the organoids with RA. To optimize the organoid generation method, we first varied the duration of RA treatment. The albumin secretion levels of organoids were 1160, 1054, 3092, 4709, and 3865 ng / mL at D25 for 1, 2, 3, 4, and 5 days of RA treatment, respectively, with the 4-day RA treatment protocol tending to reach the highest level (Figure 8). Therefore, based on the level of albumin secretion, we set the RA duration to 4 days. Morphologically, approximately 10 days after RA treatment, over 300 organoids covered with epithelial cells were successfully generated, and the proportion of organoids with luminal structures was 71% (216 / 305) (Figure 1, panel B and Figure 9). Immunohistochemical analysis revealed that albumin was positive in the epithelial cells of the organoids, and interestingly, type IV collagen was localized to the outer surface, while ZO-1 (zona adherens occludens) stained the luminal inner layer, suggesting that these organoids have polarized properties (Figure 1, panel C).

[0050] Quantitative polymerase chain reaction (qPCR) analysis revealed that cells in organoids showed significantly increased expression of hepatic marker genes, such as alpha-fetoprotein (AFP), albumin (ALB), retinol-binding protein 4 (RBP4), cytokeratin 19 (CK19), hepatocyte nuclear factor 6 (HNF6), which controls cholangiocyte differentiation, and cytochrome P450 3A4 (CYP3A4) during differentiation (Figure 1, panel D). However, the expression levels of most hepatic genes extracted from bulk organoid-derived RNA were lower in organoids than in primary hepatocytes. While not intending to be limited by theory, these distinct mRNA profiles may be due in part to the presence of stromal lineages, as approximately 30% of cells identified by stromal cell markers are non-parenchymal cells, making organoids more similar to in vivo liver tissue than primary hepatocytes. The applicant further profiled the organoids by comprehensive gene expression analysis using RNA sequencing (RNA-seq). Principal component analysis (PCA) demonstrated that gene expression in organoids was not similar to that in iPSC-derived cholangiocytes and normal human cholangiocytes (Figure 1, Panel E). Furthermore, hepatocyte-specific proteins such as ALB, fibrinogen (Fbg), and complement factors were confirmed in the culture supernatant by ELISA (Figure 1, Panels F-G). To quantify the liver functionality of organoids, the applicant examined albumin secretion levels normalized by cell number (Figure 10). The albumin secretion level was 2133 ng / day / 10. 6 cells (Figure 1, panel F), and other experiments (150–1000 ng / day / 10 ) in 2D and 3D differentiation of hPSCs into HLCs related to published iPSC-derived hepatocytes (Miki et al., 2011 ; Song et al., 2015 ; Song et al., 2009 ; Vosough et al., 2013 ). 6 cells), whereas primary hepatocytes showed a mean of 30-40 μg / day / 10 in 3D scaffolds. 6These results demonstrate that liver organoids contain hepatocytes with albumin-secreting activity comparable to that of stem cell-derived hepatocytes (Davidson et al., 2016; Dvir-Ginzberg et al., 2003). Importantly, this organoid generation method is reproducible and therefore applicable to other PSC lines, as intraluminal organoids were generated from both the 317D6 and 1383D6 iPS cell lines with albumin-secreting activity (Figure 11). In summary, the applicant has established a protocol for generating large numbers of polarized liver organoids with hepatocyte characteristics.

[0051] Microanatomical characterization of bile acid-producing human iPSC-liver organoids Next, to test whether liver organoids possess bile transport activity, we first characterized the organoids by staining for key proteins involved in bile synthesis and excretory functions. Immunofluorescent staining of BSEP and MRP2 demonstrated that these proteins were preferentially localized in the intraluminal region (Figure 2, Panel A). Bile canaliculi are the smallest hepatic endocrine channels, and the bile canalicular lumen consists of a space formed by the modified apical regions of the opposing plasma membranes of adjacent hepatocytes (Cutrin et al., 1996; Tsukada et al., 1995). Furthermore, it is delimited by tight junction complexes, and microvilli are located inside the bile canalicular lumen (Tsukada et al., 1995). ZO-1 staining is known to stain the bile canalicular region of the liver, and Figure 1, Panel C, suggested that tight junctions are located inside the bile canalicular lumen. Transmission electron microscopy revealed that organoids contained microvilli oriented toward the lumen (Figure 2, panel B). Consistent with these anatomical features, qRT-PCR analysis revealed that organoids had gene expression of ABCB11 and Na+-taurocholate cotransporting polypeptide (NTCP), although the levels were lower in organoids than in primary hepatocytes (Figure 2, panel C). Thus, organoids contain polarized human hepatocytes separated from the lumen by adherens junctions, reflecting a unique microanatomical structure that mimics in vivo hepatic bile canaliculi.

[0052] Next, to determine bile acid (BA) production capacity, Applicant performed ELISA on the luminal fluid collected from organoid cultures. The level of the total BA pool in the luminal fluid was 26.7 μg / day / 10 6 cells (approximately 125 μmol / L in 200 μm diameter organoids) (Figure 2, panel D), and surprisingly, the BA concentration was comparable to that of primary hepatocytes derived from sandwich culture in a previous report (Ni et al., 2016) (approximately 40 μg / day / 10 6The organoids not only possessed bile canaliculi-like morphology but also possessed bile acid production and secretion activity, suggesting that the bile acid transport pathway was correctly established.

[0053] Dynamic visualization of bile acid uptake and excretion in human liver organoids Bile acid excretion is a major determinant of bile flow; therefore, defects in this system can result in impaired bile secretion (cholestasis), which is associated with various liver disease pathologies (Nishida et al., 1991). Efflux transport proteins located in the apical (bile canalicular) membrane of hepatocytes play a critical role in the hepatic elimination of many endogenous and exogenous compounds, including drugs and metabolites (Kock and Brouwer, 2012). BSEP and MRP2 mediate canalicular bile salt transport in humans. After demonstrating positive expression of key proteins for bile transport, we next wondered whether organoids could actively transport bile acids into their lumen. First, to examine bile acid uptake into organoids, we challenged them with cholylglycylamide-fluorescein (CGamF), a bile salt analog (Mork et al., 2012). After exogenous CGamF treatment, CGamF accumulation within the organoid lumen was successfully confirmed (Figure 2, Panel E). Similarly, the fluorescent bile acid cholyl-lysyl-fluorescein (CLF) was found to be reproducibly excreted and accumulated within organoids from multiple human iPSC lines (Figure 2, Panel F). To determine the specificity of this assay, we developed iPSC lines carrying a nonfunctionalized BSEP allele using a CRISPR-Cas9-based gene editing approach. BSEP is involved in bile transport, and consistent with this, BSEP-KO iPSC-organoids failed to accumulate fluorescent bile acids compared to parental control organoids. Collectively, these data suggest that organoids have the ability to ingest bile acids from the outside and excrete them into the organoid interior.

[0054] Bosentan-induced cholestasis specific to CYP2C9*2 iPSC-liver organoids To test the clinical relevance of our organoid-based cholestatic phenotyping method, we employed pharmacogenomic insights into our system to address the issue of fidelity. Specifically, multiple iPSC lines harboring known susceptibility gene variants (e.g., CYP2C9*2, as described in Clin Pharmacol Ther. 2013 Dec;94(6):678-86. doi:10.1038 / clpt.2013.143. Epub 2013 Jul 17. Association of CYP2C9*2 with bosentan-induced liver injury) were collected (Figure 3, Panel A) and their cholestatic potential was compared in the presence of bosentan (Figure 3, Panel B). Interestingly, CLF excretion into organoids was significantly impaired in CY2C9*2-harboring organoids but not in non-harboring organoids. This is consistent with the clinical trend toward bosentan-induced cholestasis shown in three different iPSC-derived organoids in the absence of CYP2C9*2, as shown in Figure 3, Panel C. In contrast, irinotecan-induced cholestasis was not specific to the CYP2C9*2 iPSC line. These results demonstrated that organoid-based cholestasis assays predict some aspects of human heterogeneity.

[0055] High-throughput drug-induced cholestasis assessment in organoids Given the important role of cholestasis in drug-induced DILI, we next considered whether this organoid model could reflect the pathology of DILI in the presence of specific compounds. Before testing a large number of compounds, we first attempted to develop a high-throughput fluorescence-based assay, as both CLF and CGamF have several issues that make them inapplicable to high-speed imaging: 1. strong background, requiring manual washing processes; 2. weak signal intensity, requiring careful acquisition settings. Alternatively, the use of fluorescein diacetate (FD), which has been reported to be a useful marker of efflux transport in hepatocytes (Barth and Schwarz, 1982; Bravo et al., 1998), has been proposed. The polar fluorescent metabolite fluorescein is trapped intracellularly until actively transported from the cell into the bile canalicular lumen (Malinen et al., 2014). To determine whether FD could be used for live assessment of transport capacity without medium changes or exposure dose adjustments, we further examined time-lapse hepatobiliary transport activity over time using time-lapse imaging. Organoids were incubated with fluorescein diacetate for 45 minutes, and intraluminal accumulation was observed inside the organoids 20 minutes after treatment (Figure 4, panels A and B). The opposite direction of this transport flow was determined by microinjection of FD into organoids. After microinjection of diacetate into the lumen, fluorescein remained inside and was not observed outside the organoids (Figure 4, panel C). In summary, this FD-based evaluation model has the potential for high-throughput assessment of unidirectional excretory bile transport in liver organoids by simple fluorescence live imaging analysis.

[0056] Next, we validated the fidelity of the FD-based assay by evaluating the feasible doses of 10 FDA-approved drugs to measure secondary cell damage. We successfully identified optimal doses for nine compounds with acceptable viability. In contrast, amiodarone (AMIO) was significantly toxic to organoids within the range tested; therefore, AMIO was excluded from further potential DILI evaluation studies (Figure 12). We then investigated the potential for cholestasis in organoids using FD with nine training compounds (TCs) classified into one of three types based on their DILI mechanism: DILI compounds without cholestasis (Class A), DILI compounds with cholestasis (Class B), and compounds not reported as DILI compounds (Class C) (Oorts et al., 2016). To quantify the potential for inhibition of FD excretion, we developed a simple yet robust quantification method by determining the fluorescence intensity ratio between the outside and inside of organoids using Image J (Figure 4, Panel B). As a validation study, we first confirmed the ability to evaluate the inhibition rate using cyclosporine A (CSA). At 5 minutes after FD treatment, a significant decrease (0.4% compared to the control) was observed in the group treated with CSA for 24 hours compared to the control (DMSO) (Figure 4, Panel B). Next, we screened nine TCs at multiple concentrations to evaluate the fidelity of this approach. Interestingly, in this screening system, 24 hours after TC treatment, similar to clinical observations, FD excretion was significantly reduced (p<0.01 or 0.05) by the class B compounds bosentan, CSA, troglitazone, and nefazodone, whereas no inhibitory effect was observed with class A and class C compounds (upper image of Figure 4, Panel D and Figure 4, Panel E). These results suggest that the liver organoid model is useful for classifying the bile transport inhibitory potency of candidate compounds in drug development that is highly relevant to the human phenotype.

[0057] Assessing mitochondrial overload in organoids Furthermore, because mitochondrial toxicity plays a central role in DILI through multiple mechanisms related to its development (Pessayre et al., 2012), we investigated mitochondrial health assessment. In this study, to examine mitochondrial health in organoids, we monitored mitochondrial membrane potential (MMP) in intact cells as a direct readout of mitochondrial health (Li et al., 2014). After 24 hours of TC treatment, dose-dependent increases in MMP were observed with tolcapone (2-8 fold change, p<0.01), diclofenac (7-13 fold change, p<0.05 or 0.01), CSA (3-7 fold change, p<0.01), and nefazodone (4-42 fold change, p<0.01) treatment (Figure 5, Panel A, bottom image and graph). Furthermore, troglitazone also increased MMP in organoids (3-5 fold change, p<0.05), although no dose-dependency was observed. On the other hand, no clear increase in MMP was observed after treatment with bosentan, entacapone, or pioglitazone, even at multiple doses. These results demonstrate that this live-image-based assay, named liver organoid-based toxicity screening (LoT), can distinguish compounds with and without mitochondrial toxicity.

[0058] Revisiting the mechanistic classification of DILI compounds using the LoT system The severe manifestations of human DILI are multifactorial and highly correlated with the combined effects of drugs specifically related to known mechanisms of DILI, such as mitochondrial and BSEP inhibition (Aleo et al., 2014). However, current in vitro functional models make it difficult to assess such multifactorial contributions. Given the advantages of multiplexed live functional readouts in the LoT system, we attempted to analyze the relationship between survival, cholestasis, and mitochondrial stress. Notably, drugs with dual effects (cholestasis and mitochondrial stress) at 24 hours, such as CSA, TRO, and NEFA, significantly reduced cell viability at 72 hours compared with TOL, DICLO, and BOS. These data are comparable to clinical data showing that dual toxicity is highly correlated with the severity of DILI and are consistent with previous reports (Aleo et al., 2014) (Figure 5, panels B and C). Furthermore, the present applicant also noted that entacapone treatment at 130 μM reduced organoid viability (from 85% at 24 hours to 64% at 72 hours). Entacapone requires extensive binding to plasma proteins, primarily albumin, to induce DILI (Fisher et al., 2002). However, based on available methods, it remains unclear how entacapone is toxic to the liver (Oorts et al., 2016). In summary, the LoT system is an advantageous human model system for classifying the key mechanisms of DILI and a useful testing platform for further elucidating the unknown complex mechanisms.

[0059] Assessing vulnerability to DILI in human liver organoids The incidence of DILI is known to be confounded by numerous host factors. Indeed, there is growing evidence that the risk of hepatotoxicity from some drugs, such as acetaminophen, is greatly increased due to obesity and nonalcoholic fatty liver disease (NAFLD) in both rodents and humans (Fromenty, 2013; Michaut et al., 2016). Therefore, it is important to predict the likelihood of DILI in patients who are in such a "vulnerable" state, even in the asymptomatic stage. In this study, we established a lipotoxic organoid model by simultaneous exposure to the unsaturated fatty acid, oleic acid (Figure 6, Panel A). Three days after oleic acid treatment, lipid accumulation in the organoids intensified (Figure 6, Panel B). Fatty acid oxidation is an important source of reactive oxygen species (ROS), which leads to the depletion of ATP and nicotinamide dinucleotide and induces DNA damage in fatty liver (Browning and Horton, 2004). Consistent with this, ROS production was observed in lipid-treated organoids (Figure 6, Panel C and Figure 13, Panel A). Furthermore, fatty acids induced massive swelling of liver mitochondria (Figure 6, Panel D and Figure 13, Panel B), similar to published phenotypes (Zborowski and Wojtczak, 1963). Because hepatic mitochondrial dysfunction precedes the development of NAFLD in rat models (Rector et al., 2010), these results indicate that hepatotoxic organoids model, to some extent, an in vivo fatty liver model.

[0060] Recognizing this lipotoxic organoid model as a vulnerable state accompanied by enhanced ROS production, we treated organoids with troglitazone (0–50 μM) for 24 hours and assessed cell viability. Treatment with 50 μM troglitazone alone resulted in 85% cell viability at 24 hours, whereas it decreased to 67% at 72 hours. However, after troglitazone treatment for lipotoxic conditions, massive fragmentation of organoids was observed, resulting in organoid death. Subsequent cell viability analysis confirmed this result (approximately 40% compared to control, p<0.05) (Figure 6, Panel E and 6, Panel F).

[0061] Next, we investigated whether potential therapeutic compounds could rescue organoids from a DILI-like state. Because intravenous NAC improved survival in patients with non-acetaminophen-related acute liver failure (Lee et al., 2009) and reduced troglitazone-induced cytotoxicity (Rachek et al., 2009), we used N-acetylcysteine ​​(NAC), an antioxidant, to inhibit ROS production. As expected, cell viability was significantly improved by NAC, suggesting that NAC rescued cell death in organoids even under vulnerable conditions (Figure 6, Panels E and F). In most cases of DILI, the only intervention is to remove the causative drug if it can be identified (Polson and Lee, 2005) (Bohan et al., 2001; Navarro and Senior, 2006). This LoT system could be a useful tool for identifying causative drugs relevant to multidrug regimens and drug discovery for treating DILI.

[0062] Serious adverse events (SAEs), including liver failure, are a major cause of drug attrition during clinical development or the discontinuation of marketed drugs. In particular, DILI is a significant challenge in drug development, where drug-induced cholestasis induced by the inhibition of transporter activity is one major cause. Sandwich culture using human primary hepatocytes is the current best choice for pharmaceutical development. Recent reports have shown promise for hepatocyte-based cholestasis models using transdifferentiated cells from human fibroblasts (Ni et al., 2016). However, due to the diverse and limited human hepatocyte sources and the need for complex quantification algorithms, these assay platforms still have reproducibility challenges and throughput issues. Furthermore, HepaRG cells, a human hepatocellular carcinoma cell line, are also useful for assessing cholestasis characteristics, but their low BSEP (bile salt export pump, or ABCB11, a key transporter for bile acid excretion and the main target of cholestatic drugs) activity and time-consuming differentiation procedures limit their use (Le Vee et al., 2013). More importantly, the lack of essential anatomical structures limits their practical application in the pharmaceutical industry. Alternatively, the described method enables a simple, robust, and high-throughput system for measuring bile transport activity by live fluorescence imaging in the presence of test compounds. The main advantages of the LoT assay include: 1. cost-effectiveness ($12.35 per 50 organoids, $94.85 per 384-well plate), 2. assay throughput (measurements can be performed with a single organoid), and 3. multiple readouts for analyzing interactions between other factors, such as mitochondrial stress. Notably, as mentioned above, a retrospective study revealed that multiple cellular stress possibilities are associated with the incidence of DILI (Aleo et al., 2014), as cell viability decreased in a dual-readout fashion, dependent on mitochondrial and cholestatic stress. The LoT assay demonstrated comparable results to this study. Oxidative stress plays an important role in cell death and has been associated with the development of cholestatic liver injury (Serviddio et al., 2004).Hydrophobic bile acids accumulate intracellularly during cholestasis, disrupting normal mitochondrial electron transport and inhibiting the activity of respiratory complexes I and III, resulting in decreased adenosine triphosphate synthesis (Krahenbuhl et al., 1994), leading to mitochondrial dysfunction and apoptosis (Bernardi, 1996). Consistent with these findings, our correlation analysis of these dual readouts showed that cholestatic stress is a more dominant factor in liver injury compared with mitochondrial stress, as seen in Figure 5. Therefore, the LoT system can be used as a model system to investigate DILI mechanisms.

[0063] Furthermore, given the recent establishment of iPSC panel populations, the potential assessment of differential susceptibility among individuals is also promising (Inoue et al., 2014). Prediction of SAEs in conventional in vitro assay systems generally does not focus on individual differences, whereas SAEs often occur in small subgroups of patients prone to SAEs (Stevens and Baker, 2009). Applying the LoT system to diverse population iPSC panels could provide previously unreachable insights into differential susceptibility to SAEs. Given the extremely rare nature of DILI, the use of cells from patients with specific genomic or ethnic factors could help elucidate currently unknown idiosyncratic mechanisms of DILI. Therefore, LoT could serve as a game-changing strategy for the pharmaceutical industry by providing essential insights into minimizing the potential for DILI (Figure 7).

[0064] One limitation of this organoid model is the lack of immunological response. Immunological effects resulting from hypersensitivity reactions are one possible mechanism for idiosyncratic DILI. Although in vitro models for assessing drug hypersensitivity are limited, sensitivity to troglitazone-induced cytotoxicity was enhanced using an in vitro coculture model with hepatocyte cell lines, Huh7 cells and THP-1 cells (Edling et al., 2009). Therefore, advancing the LoT platform by focusing on the immune system would be interesting for assessing hepatocellular inflammation. Nevertheless, the LoT testing platform appears superior in generating reproducible and large datasets from individual organoids, as inhibition of bile excretion function by multiple FDA-approved drugs is reproducibly observed in this assay. Considering that cholestasis can be induced by a wide range of liver diseases, including drug-induced, lipotoxic, infectious, and congenital conditions ( Chatterjee et al., 2014 ), organoid-based LoT assays are useful for analyzing intrahepatic cholestasis in various settings, with potential for mechanistic studies as well as drug screening applications beyond DILI.

[0065] Study of fragile human liver conditions by LoT assay Host factors such as obesity are known to have a significant impact on the development of DILI (Heidari et al., 2014), yet their complex nature often leaves them poorly understood in clinical practice. Obesity or fatty liver disease may render patients vulnerable to liver damage caused by xenobiotics and non-toxic chemicals (e.g., drugs), which can become hepatotoxic at lower doses in the presence of risk factors (Fromenty, 2013). Nevertheless, current clinical trial systems are not designed to stratify volunteers based on a handful of biomarkers (ALT, AST) for vulnerable liver conditions. Because many patients with steatosis are asymptomatic and undetectable by biomarkers before medication, predicting outcomes in this vulnerable condition before entering the clinical phase is crucial.

[0066] In an effort to develop a LoT system to assess toxicity in these vulnerable conditions during early drug screening stages, such as lead generation / optimization, we applied lipotoxic stress to liver organoids and demonstrated a significant synergistic effect of the antidiabetic drug troglitazone on DILI. Indeed, the organoid system successfully reflects this characteristic by showing massive hepatocyte death promoted by triglyceride accumulation in hepatocytes within the organoids. One mechanism of DILI in obesity may be reduced glutathione (GSH) levels (Michaut et al., 2016). Drug-induced oxidative stress can have several causes, particularly GSH depletion and inhibition of the mitochondrial respiratory chain (Begriche et al., 2011; Pessayre et al., 2010). The fragile model may reflect the exacerbation of troglitazone-induced oxidative stress through reduced intracellular GSH levels and mitochondrial dysfunction, which is ameliorated by providing NAC. Considering the dramatic rise in the prevalence of nonalcoholic steatohepatitis (NASH), it is noteworthy that there is still a minimal list of drugs to exacerbate existing NAFLD or induce more frequent acute hepatitis. Furthermore, the in vitro reduction system provides a previously unexpected window to study previously untested host factors, as isolated host factors can be effectively deployed in organoids.

[0067] LoT-based precision medicine From the perspective of personalized medicine, selecting optimal medications using LoT will be a major concern in clinical practice. For example, strategies for selecting antipsychotics must take into account hepatic tolerance due to the significant incidence of liver injury in psychiatric treatment populations; 16% of potential DILI medications are neuropsychiatric medications (Dumortier et al., 2002). Considering that NASH is often accompanied by psychological disorders such as depression, safer combinations of antidepressants (tricyclics or SSRIs), mood stabilizers, and neuroleptics are needed (Dumortier et al., 2002). Furthermore, due to the increase in chronic conditions associated with aging, polypharmacy (i.e., polypharmacy) is a common consequence of providing medical care to elderly patients (Marcum and Gellad, 2012), making it extremely difficult to identify the causative medication when DILI is suspected. Because patient-derived iPSC-organoids provide an unlimited and reproducible source, LoT can serve as a panel to stratify the likelihood of DILI in patients and provide information for the selection of safer drugs from an individualized perspective.

[0068] LoT-based drug discovery for DILI Equally important is the potential use of the LoT system for screening anti-DILI therapeutic compounds. Many drugs have adverse effects on the liver and DILI, which is a major clinical problem. In fact, acetaminophen accounts for approximately half of DILI cases in the United States (Russo et al., 2004). In other parts of the world, such as developing countries, other drugs, such as antituberculosis drugs, may be the primary cause of DILI (Bell and Chalasani, 2009). However, there are only a few symptomatic treatments available. Here, as a proof-of-concept experiment, we established an organoid survival experiment to evaluate the therapeutic efficacy of compounds that resist the toxic mechanisms of DILI, as evidenced by troglitazone. While NAC is the primary treatment option for paracetamol overdose (Makin et al., 1995; Verma and Kaplowitz, 2009), the focus of research has recently shifted to investigating its use in non-paracetamol-induced DILI (Chughlay et al., 2016). The LoT system is useful for evaluating the efficacy of NAC against non-paracetamol-induced DILI. Furthermore, this additional high-throughput approach serves as a powerful tool for screening large compound libraries for in vitro amelioration of DILI-like symptoms. The combined methods described herein can be used to identify and study cell-intrinsic and cell-extrinsic factors associated with clinical DILI phenotypes, facilitating lead optimization, mechanistic studies, and precision medicine, as well as anti-DILI therapy screening applications.

[0069] method Maintaining PSCs The TkDA3 CYP2C9*2 mutant human iPSC clone used in this study was kindly provided by K. Eto and H. Nakauchi. Other suitable lines include human iPSC lines donated by Kyoto University and lines purchased from the Coriell Biorepository, maintained as previously described (Takahashi et al., 2007). Undifferentiated hiPSCs were maintained under feeder-free conditions in mTeSR1 medium (StemCell Technologies, Vancouver, Canada). Other suitable media include E8 from Lonza or StemFit from Aijinomoto Co. Plates were coated with a 1 / 30 dilution of Matrigel (Corning Inc., New York, NY, USA) at 37°C in an incubator with 5% CO2 / 95% air. hPSC maintenance. Instead of Matrigel, Laminin 511 or Laminin 411 from Mippi Co. or Biolamina Co. can be used.

[0070] Production of liver organoids (HLOs) Differentiation of hiPSCs to definitive endoderm was induced using a previously described method with some modifications (Spence et al., 2011). Briefly, hiPSC colonies were isolated in Accutase (Thermo Fisher Scientific Inc., Waltham, MA, USA), and 150,000–300,000 cells were plated onto Matrigel- or laminin-coated tissue culture 24-well plates (VWR Scientific Products, West Chester, PA). When cells reached high density (more than 90% of the cells covered the well), the medium was changed to RPMI 1640 medium (Life Technologies, Carlsbad, CA) containing 100 ng / mL activin A (R&D Systems, Minnepolis, MN) and 50 ng / mL bone morphogenetic protein 4 (BMP4; R&D Systems) on day 1, to RPMI 1640 medium containing 100 ng / mL activin A and 0.2% fetal calf serum (FCS; Thermo Fisher Scientific Inc.) on day 2, and to RPMI 1640 medium containing 100 ng / mL activin A and 2% FCS on day 3. On days 4–6, cells were cultured in Advanced DMEM / F12 (Thermo Fisher Scientific Inc.) with B27 (Life Technologies) and N2 (Gibco, Rockville, MD) containing 500 ng / ml fibroblast growth factor (FGF4; R&D Systems) and 3 μM CHIR99021 (Stemgent, Cambridge, MA, USA). Differentiation cultures were maintained at 37°C in a 5% CO2 / 95% air atmosphere, and the medium was changed daily. Differentiated definitive endoderm showed budding on plates on day 7. If spheroids were not large enough to embed in Matrigel, add the medium from days 4–6 again and incubate overnight at 37°C.

[0071] Differentiation into Liver Organoids Three methods may be used to differentiate DE into liver organoids: the "Matrigel drop method," the "Matrigel sandwich method," and the "Matrigel-free method," each of which is described below.

[0072] Matrigel drop method: On days 7–8, definitive endoderm organoids with plated cells were gently pipetted to detach from the dish. The isolated spheroids were centrifuged at 800 rpm for 3 minutes, the supernatant removed, and then embedded in 100% Matrigel drops on the dish. The plate was placed in a 5% CO2 / 95% air atmosphere at 37°C for 5–15 minutes. After the Matrigel solidified, Advanced DMEM / F12 was supplemented with B27, N2, and 2 μM retinoic acid (RA; Sigma, St. Louis, MO) for 1–5 days. The medium was changed every other day. After RA treatment, organoids embedded in Matrigel drops were cultured in hepatocyte culture medium (HCM Lonza, Walkersville, MD) containing 10 ng / mL hepatocyte growth factor (HGF; PeproTech, Rocky Hill, NJ), 0.1 μM dexamethasone (Dex; Sigma), and 20 ng / mL oncostatin M (OSM; R&D Systems). Cultures for cell differentiation were maintained at 37°C in a 5% CO2 / 95% air atmosphere, and the medium was changed every 3 days. Around days 20–30, organoids embedded in Matrigel drops were isolated by scratching and gentle pipetting for further analysis.

[0073] Matrigel sandwich method: On days 7–8, definitive endoderm organoids with plated cells were gently pipetted to detach them from the dish. The isolated spheroids were centrifuged at 800 rpm for 3 minutes, and the supernatant was removed. They were then mixed with 100% Matrigel. At the same time, an equal volume of hepatocyte culture medium containing all supplements was mixed with 100% Matrigel. The HCM and Matrigel mixture was plated on the bottom of the dish to create a thick coating (0.3–0.5 cm) on the plate, which was then placed at 37°C in a 5% CO2 / 95% air atmosphere for 15–30 minutes. After allowing the Matrigel to solidify, the spheroids mixed with Matrigel were seeded onto the thickly coated plate. The plate was then placed at 37°C in a 5% CO2 / 95% air atmosphere for 5 minutes. Advanced DMEM / F12 was supplemented with B27, N2, and 2 μM retinoic acid (RA; Sigma, St. Louis, MO) for 1–5 days. The medium was changed every other day. After RA treatment, organoids embedded in Matrigel drops were cultured in hepatocyte culture medium (HCM Lonza, Walkersville, MD) with 10 ng / mL hepatocyte growth factor (HGF; PeproTech, Rocky Hill, NJ), 0.1 μM dexamethasone (Dex; Sigma), and 20 ng / mL oncostatin M (OSM; R&D Systems). Cultures for cell differentiation were maintained at 37°C in a 5% CO2 / 95% air atmosphere, and the medium was changed every 3 days. Around days 20–30, organoids embedded in Matrigel drops were isolated by scratching and gentle pipetting for further analysis.

[0074] Matrigel-free method: On days 7–8, definitive endoderm organoids with plated cells were plated in Advanced DMEM / F12 (Thermo Fisher Scientific Inc.) containing B27 (Life Technologies) and N2 (Gibco, Rockville, MD) with 2 μM retinoic acid (RA; Sigma, St. Louis, MO) for 4 days. The medium was changed every other day. After 4 days of plate culture, organoids begin to bud, while 2D cells differentiate into hepatocytes. Both organoids and hepatocytes can be maintained for up to 60 days in hepatocyte culture medium (HCM Lonza, Walkersville, MD) with 10 ng / mL hepatocyte growth factor (HGF; PeproTech, Rocky Hill, NJ), 0.1 μM dexamethasone (Dex; Sigma), and 20 ng / mL oncostatin M (OSM; R&D Systems) for 10 days. For organoid assays, floating organoids can be collected in ultra-low attachment multiwell plates (6-well plates) and used for subsequent assays, if necessary. Cultures for cell differentiation were maintained at 37°C in a 5% CO2 / 95% air atmosphere, and the medium was changed every 3 days.

[0075] H&E staining and immunohistochemistry Liver organoids were harvested from Matrigel, fixed in 4% paraformaldehyde, and then embedded in paraffin. Sections were subjected to H&E staining and immunohistochemistry. The following primary antibodies were used: anti-human albumin antibody (1:200 dilution, Abcam, Cambridge, UK), anti-type IV collagen antibody (1:200 dilution, eBioscience, San Diego, CA, USA), anti-ZO-1 antibody (1:200 dilution, BD Transduction Laboratories, San Jose, CA, USA), and anti-MRP2 antibody (1:200 dilution, Novus Biologicals, Littleton, CO). A dye-conjugated secondary antibody, Alexa Fluor 568-conjugated donkey anti-rabbit immunoglobulin (IgG; 1:1000; Invitrogen, A10042), was applied to the organoids for 2 hours at room temperature. Nuclei were stained with 10 μg / mL Hoechst. Organoids were stained with 33342 (Sigma) at room temperature for 10 minutes, after which the organoids were washed three times with washing buffer. The specimens were observed under a fluorescent microscope or brightfield. For whole-tissue immunohistochemical staining, liver organoids were fixed with 4% paraformaldehyde for 30 minutes and permeabilized with 2.5% Tween 20 (Sigma) at room temperature. The organoids were then incubated overnight at 4°C with the following primary antibody: polyclonal anti-BSEP antibody (1:200, Sigma) diluted in PBS. A fluorochrome-conjugated secondary antibody, Alexa Fluor 568-conjugated donkey anti-rabbit immunoglobulin (IgG; 1:500; Invitrogen, A10042), was applied to the organoids for 2 hours at room temperature. After the incubation, the cells were washed three times with washing buffer (PBS containing 0.5% Triton-X 100 [Sigma] and 0.5% bovine serum albumin [BSA; Sigma]). Nuclei were then stained with 10 μg / mL Hoechst. After staining with 33342 (Sigma) for 10 minutes at room temperature, the organoids were washed again three times with washing buffer and observed under confocal imaging performed on a Nikon A1Rsi inverted confocal microscope.

[0076] RNA isolation, RT-qPCR RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed using the SuperScript III First-Strand System for RT-PCR (Invitrogen, CA, USA) according to the manufacturer's protocol. qPCR was performed using TaqMan Gene Expression Master Mix (Applied Biosystems) on a QuantStudio 3 Real-Time PCR System (Thermo). All primer and probe information for each target gene was obtained from the Universal ProbeLibrary Assay Design Center (https: / / qpcr.probefinder.com / organism.jsp).

[0077] Principal component analysis of RNA-seq data RNA isolation, cDNA synthesis, and sequencing on an Illumina HiSeq 2500 were previously described (Asai et al., 2017). RNA-Seq reads were aligned to the human genome (GRCh37 / hg19) using TopHat (version 2.0.13). Alignment data from TopHat was fed into the assembler Cufflinks (version 2.2.1) to assemble aligned RNA-Seq reads into transcripts. Annotated transcripts were obtained from the UCSC Genome Browser (http: / / genome.ucsc.edu) and the Ensembl database. Transcript abundance was measured in fragments per kilobase of exon per million mapped fragments (FPKM).

[0078] To compare pHLO lineages, applicants combined in-house RNA-seq data (pFG and organoids) with preprocessed public data as follows: transcript abundances for iPSCs, DE, HS, HP, iDH, and NHC were obtained from GSE86007 (Jalan-Sakrikar et al., 2016); transcript abundances for pediatric liver tissue, adult liver tissue, adult right lobe tissue, fetal liver tissue, and primary hepatocytes were obtained from ENCODE (ENCFF418BVF, ENCFF804QWF, ENCFF965IQH, ENCFF918SJO, ENCFF367FJJ, ENCFF029IUF, ENCFF280YNO, ENCFF347TXW, ENCFF724CQI, ENCFF624LQL, ENCFF962SOD, ENCFF170AEC) (Consortium, 2012; Sloan et al. Genes were used if all datasets had the same gene symbol after possible data preprocessing. Applicants performed quartile normalization of the FPKM+1 and RPKM+1 data in log2 space, and then selected genes within the top 10,000 median expression levels. Principal component analysis was performed using scaled gene expression levels using the R package FactoMineR (version 1.35) (Sebastien Le, 2008).

[0079] Protein secretion analysis To measure the secretion levels of albumin, fibrinogen, and complement factors, 200 μL of organoid culture supernatant was collected from ultra-low-attachment 96-well plates (Corning). The culture supernatant was collected and stored at -80°C until use. Supernatants were assayed using a human albumin ELISA quantification set (Bethyl Laboratories, Inc., TX, USA) and fibrinogen (Thermo Fisher Scientific) according to the manufacturer's instructions. To analyze complement factors, the supernatant was measured using a Luminex system (Luminex Corporation, Austin, TX) according to the manufacturer's instructions. A linear regression equation was used to calculate albumin production per cell count by organoid diameter. To measure the total bile acid secretion levels of intraluminal organoids, the liquid inside the organoids was absorbed using a microinjection Nanoject II (Drummond Scientific, Broomall, PA, USA). The absorbed liquid was diluted with PBS and then assayed using a total bile acid ELISA kit (Antibodies-online, Inc., GA, USA).To calculate the total bile acid content, the number of cells in the organoid was calculated using a linear regression equation in the same way as for albumin production, and the molecular weight of cholic acid was used for calculation and compared with the content in previous reports.

[0080] Transmission electron microscopy For transmission electron microscopy, briefly, organoids were fixed in 3% glutaraldehyde overnight at 4°C, washed in 0.1 M sodium cacodylate buffer, and then incubated in 4% osmium tetroxide for 1 hour. They were subsequently washed, dehydrated in an ethanol series, and finally embedded in propylene oxide / LX112. Tissues were then sectioned and stained with 2% uranyl acetate followed by lead citrate. Images were visualized using a Hitachi transmission electron microscope.

[0081] CGamF assay Briefly, organoids were preincubated with transport buffer (118 mM NaCl, 23.8 mM NaHCO3, 4.83 mM KCl, 0.96 mM KH2PO4, 1.20 mM MgSO4, 12.5 mM HEPES, 5 mM glucose, 1.53 mM CaCl2, adjusted to pH 7.4) for 30 min. Next, organoids were treated with 10 μM fluorescently labeled bile acid (CGamF; a gift from Dr. Hofmann) for 1 h, after which the organoids were washed three times with PBS. Images were obtained on a fluorescence microscope BZ-X710 (Keyence, Osaka, Japan).

[0082] Assessment of biliary transport inhibition Fluorescein diacetate was used to evaluate bile transport activity in organoids. Around day 25, organoids were rinsed with PBS and treated with fluorescein diacetate in the culture medium. To further investigate the direction of transport, fluorescein diacetate was injected into organoids using a Nanoject III (Drummond Scientific). After treatment or injection of fluorescein diacetate, images were captured using a BZ-X710 fluorescence microscope (Keyence). Next, to confirm the feasibility of the test system, 20 μM cyclosporine A (CSA; Sigma) was added to 10 mg / mL fluorescein diacetate (Sigma) in HCM for 45 min, and images were continuously captured using a BZ-9000 fluorescence microscope (Keyence). For evaluation of bile transport inhibition, 10 mg / mL fluorescein diacetate in HCM was added after treatment with dimethyl sulfoxide (DMSO; Sigma), streptomycin (STP; Sigma) as a negative control, tolcapone (Tol; Sigma), diclofenac (Diclo; Sigma), bosentan (BOS; Sigma), CSA, troglitazone (Tro; Sigma), nefadzone (Nefa; Sigma), entacapone (Enta; Sigma), and pioglitazone (PIO; Sigma). After 5 min of incubation, organoids were rinsed three times with PBS, and images were captured sequentially using a BZ-X710 fluorescence microscope. Analysis was performed by calculating the ratio of intensity outside to inside the organoid using Imagej 1.48k software (Wayne Rasband, NIHR, USA, http: / / imagej.nih.gov / ij). Changes in brightness or contrast during treatment were applied equally across the entire image.

[0083] Assessment of mitochondrial toxicity potential After culturing in 6-well ultra-low attachment multiwell plates under each culture condition, organoids were picked and seeded onto 8-well glass-bottom Microslide plates (Ibidi, WI, USA). For evaluation of mitochondrial membrane potential (MMP), 250 nM tetramethylrhodamine, methyl ester, perchlorate (TMRM; Thermo Fisher Scientific) was added after 24 h of treatment with dimethyl sulfoxide (DMSO; Sigma), streptomycin (STP; Sigma) as a negative control, tolcapone (Tol; Sigma), diclofenac (Diclo; Sigma), bosentan (BOS; Sigma), cyclosporine A (CSA; Sigma), troglitazone (Tro; Sigma), nefadzone (Nefa; Sigma), entacapone (Enta; Sigma), or pioglitazone (PIO; Sigma). After 30 minutes of incubation, organoids were rinsed three times with PBS and images were scanned on a Nikon A1 inverted confocal microscope (Japan) using a 60x water-immersion objective. TMRM arithmetic and intensity were calculated as MMP using IMARIS 8 (Bitplane AG, Switzerland). To assess cholestatic and mitochondrial stress, cell viability was measured for each organoid 24 hours after drug treatment using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Mannheim, Germany). To avoid secondary changes due to cell damage leading to cell death, viability was confirmed to be unaffected at each dose.

[0084] Analysis of the relationship between mitochondrial and cholestatic stress and cell viability in organoids To demonstrate the relationship between cell viability and mitochondria and cholestatic stress, we first established an index based on the values ​​obtained from the mitochondrial and cholestatic stress assays using the following formula: "index = - (sample value - control value) × 100". To analyze cellular damage associated with mitochondria and cholestatic stress, ATP content per organoid was measured 72 hours after drug treatment using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega). These data were presented as Figure 4, Panel B, using Infogr.am (http: / / infogr.am), a free web-based tool.

[0085] Assessing organoid viability under fragile conditions The experiment was performed as shown in Figure 5A. After removal from Matrigel and washing, organoids were treated with 800 μM oleic acid for 3 days on an ultra-low attachment multi-well plate (Corning 6-well). Next, they were treated with 50 μM troglitazone in the presence or absence of 50 μM NAC for 24 hours. Cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega). Images were acquired serially using a fluorescence microscope BZ-9000.

[0086] Lipid-induced mitochondrial stress assessment After culturing in 6-well ultra-low attachment multiwell plates under each culture condition, 20 organoids were picked and seeded onto 8-well glass-bottom Microslides (Ibidi, WI, USA) for live cell staining. The following reagents or kits were used: BODIPY® 493 / 503 (Thermo Fisher Scientific) for lipids, and the SiR Actin Kit (USA Scientific, FL, USA) for cytoskeleton staining. CellROX® Green Reagent (Fisher Scientific) for ROS staining, and TMRM (Thermo Fisher Scientific) for mitochondria staining. Organoids were visualized and scanned using a Nikon A1 inverted confocal microscope (Japan) with a 60x water-immersion objective. ROS production, mitochondrial size, and number were analyzed using IMARIS8.

[0087] statistics Statistical significance was determined using unpaired Student's t-test or one-way ANOVA with Dunnett's multiple comparison post-hoc test. P<0.05 was considered significant.

[0088] References Aleo, MD, Luo, Y., Swiss, R., Bonin, PD, Potter, DM, and Will, Y. (2014).Human drug-induced liver injury severity is highly associated with dual inhibition of liver mitochondrial function and bile salt export pump.Hepatology 60,1015-1022. Asai,A.,Aihara,E.,Watson,C.,Mourya,R.,Mizuochi,T.,Shivakumar,P.,Phelan,K.,Mayhew,C.,Helmrath,M.,Takebe,T.,et al.(2017).Paracrine signals regulate human liver organoid maturation from induced pluripotent stem cells.Development 144,1056-1064. Barth,C.A.,and Schwarz,L.R.(1982).Transcellular transport of fluorescein in hepatocyte monolayers:evidence for functional polarity of cells in culture.Proc Natl Acad Sci U S A 79,4985-4987. Begriche,K.,Massart,J.,Robin,M.A.,Borgne-Sanchez,A.,and Fromenty,B.(2011).Drug-induced toxicity on mitochondria and lipid metabolism:mechanistic diversity and deleterious consequences for the liver.J Hepatol 54,773-794. Bell,L.N.,and Chalasani,N.(2009).Epidemiology of idiosyncratic drug-induced liver injury.Semin Liver Dis 29,337-347. Bernardi,P.(1996).The permeability transition pore.Control points of a cyclosporin A-sensitive mitochondrial channel involved in cell death.Biochim Biophys Acta 1275,5-9. Bohan,T.P.,Helton,E.,McDonald,I.,Konig,S.,Gazitt,S.,Sugimoto,T.,Scheffner,D.,Cusmano,L.,Li,S.,and Koch,G.(2001).Effect of L-carnitine treatment for valproate-induced hepatotoxicity.Neurology 56,1405-1409. Bravo,P.,Bender,V.,and Cassio,D.(1998).Efficient in vitro vectorial transport of a fluorescent conjugated bile acid analogue by polarized hepatic hybrid WIF-B and WIF-B9 cells.Hepatology 27,576-583. Browning,J.D.,and Horton,J.D.(2004).Molecular mediators of hepatic steatosis and liver injury.J Clin Invest 114,147-152. Chang,J.H.,Plise,E.,Cheong,J.,Ho,Q.,and Lin,M.(2013).Evaluating the in vitro inhibition of UGT1A1,OATP1B1,OATP1B3,MRP2,and BSEP in predicting drug-induced hyperbilirubinemia.Mol Pharm 10,3067-3075. Chatterjee,S.,Richert,L.,Augustijns,P.,and Annaert,P.(2014).Hepatocyte-based in vitro model for assessment of drug-induced cholestasis.Toxicol Appl Pharmacol 274,124-136. Chughlay,M.F.,Kramer,N.,Spearman,C.W.,Werfalli,M.,and Cohen,K.(2016).N-acetylcysteine for non-paracetamol drug-induced liver injury:a systematic review.Br J Clin Pharmacol 81,1021-1029. Consortium,E.P.(2012).An integrated encyclopedia of DNA elements in the human genome.Nature 489,57-74. Cutrin,J.C.,Cantino,D.,Biasi,F.,Chiarpotto,E.,Salizzoni,M.,Andorno,E.,Massano,G.,Lanfranco,G.,Rizzetto,M.,Boveris,A.,et al.(1996).Reperfusion damage to the bile canaliculi in transplanted human liver.Hepatology 24,1053-1057. D'Amour,K.A.,Agulnick,A.D.,Eliazer,S.,Kelly,O.G.,Kroon,E.,and Baetge,E.E.(2005).Efficient differentiation of human embryonic stem cells to definitive endoderm.Nat Biotechnol 23,1534-1541. Davidson,M.D.,Ballinger,K.R.,and Khetani,S.R.(2016).Long-term exposure to abnormal glucose levels alters drug metabolism pathways and insulin sensitivity in primary human hepatocytes.Sci Rep 6,28178. Dumortier,G.,Cabaret,W.,Stamatiadis,L.,Saba,G.,Benadhira,R.,Rocamora,J.F.,Aubriot-Delmas,B.,Glikman,J.,and Januel,D.(2002).[Hepatic tolerance of atypical antipsychotic drugs].Encephale 28,542-551. Dvir-Ginzberg,M.,Gamlieli-Bonshtein,I.,Agbaria,R.,and Cohen,S.(2003).Liver tissue engineering within alginate scaffolds:effects of cell-seeding density on hepatocyte viability,morphology,and function.Tissue Eng 9,757-766. Edling,Y.,Sivertsson,L.K.,Butura,A.,Ingelman-Sundberg,M.,and Ek,M.(2009).Increased sensitivity for troglitazone-induced cytotoxicity using a human in vitro co-culture model.Toxicol In Vitro 23,1387-1395. Fahrmayr,C.,Konig,J.,Auge,D.,Mieth,M.,Munch,K.,Segrestaa,J.,Pfeifer,T.,Treiber,A.,and Fromm,M.(2013).Phase I and II metabolism and MRP2-mediated export of bosentan in a MDCKII-OATP1B1-CYP3A4-UGT1A1-MRP2 quadruple-transfected cell line.Br J Pharmacol 169,21-33. Falasca,L.,Favale,A.,Serafino,A.,Ara,C.,and Conti Devirgiliis,L.(1998).The effect of retinoic acid on the re-establishment of differentiated hepatocyte phenotype in primary culture.Cell Tissue Res 293,337-347. Fisher,A.,Croft-Baker,J.,Davis,M.,Purcell,P.,and McLean,A.J.(2002).Entacapone-induced hepatotoxicity and hepatic dysfunction.Mov Disord 17,1362-1365;discussion 1397-1400. Fromenty,B.(2013).Drug-induced liver injury in obesity.J Hepatol 58,824-826. Heidari,R.,Niknahad,H.,Jamshidzadeh,A.,and Abdoli,N.(2014).Factors affecting drug-induced liver injury:antithyroid drugs as instances.Clin Mol Hepatol 20,237-248. Inoue,H.,Nagata,N.,Kurokawa,H.,and Yamanaka,S.(2014).iPS cells:a game changer for future medicine.EMBO J 33,409-417. Jalan-Sakrikar,N.,De Assuncao,T.M.,Lu,J.,Almada,L.L.,Lomberk,G.,Fernandez-Zapico,M.E.,Urrutia,R.,and Huebert,R.C.(2016).Hedgehog Signaling Overcomes an EZH2-Dependent Epigenetic Barrier to Promote Cholangiocyte Expansion.PLoS One 11,e0168266. Kock,K.,and Brouwer,K.L.(2012).A perspective on efflux transport proteins in the liver.Clin Pharmacol Ther 92,599-612. Krahenbuhl,S.,Talos,C.,Fischer,S.,and Reichen,J.(1994).Toxicity of bile acids on the electron transport chain of isolated rat liver mitochondria.Hepatology 19,471-479. Le Vee,M.,Noel,G.,Jouan,E.,Stieger,B.,and Fardel,O.(2013).Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells.Toxicol In Vitro 27,1979-1986. Lechner,C.,Reichel,V.,Moenning,U.,Reichel,A.,and Fricker,G.(2010).Development of a fluorescence-based assay for drug interactions with human Multidrug Resistance Related Protein(MRP2;ABCC2) in MDCKII-MRP2 membrane vesicles.Eur J Pharm Biopharm 75,284-290. Lee,W.M.,Hynan,L.S.,Rossaro,L.,Fontana,R.J.,Stravitz,R.T.,Larson,A.M.,Davern,T.J.,2nd,Murray,N.G.,McCashland,T.,Reisch,J.S.,et al.(2009).Intravenous N-acetylcysteine improves transplant-free survival in early stage non-acetaminophen acute liver failure.Gastroenterology 137,856-864,864 e851. Leslie,E.M.,Watkins,P.B.,Kim,R.B.,and Brouwer,K.L.(2007).Differential inhibition of rat and human Na+-dependent taurocholate cotransporting polypeptide(NTCP / SLC10A1)by bosentan:a mechanism for species differences in hepatotoxicity.J Pharmacol Exp Ther 321,1170-1178. Li,N.,Oquendo,E.,Capaldi,R.A.,Robinson,J.P.,He,Y.D.,Hamadeh,H.K.,Afshari,C.A.,Lightfoot-Dunn,R.,and Narayanan,P.K.(2014).A systematic assessment of mitochondrial function identified novel signatures for drug-induced mitochondrial disruption in cells.Toxicol Sci 142,261-273. Makin,A.J.,Wendon,J.,and Williams,R.(1995).A 7-year experience of severe acetaminophen-induced hepatotoxicity(1987-1993).Gastroenterology 109,1907-1916. Malinen,M.M.,Kanninen,L.K.,Corlu,A.,Isoniemi,H.M.,Lou,Y.R.,Yliperttula,M.L.,and Urtti,A.O.(2014).Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels.Biomaterials 35,5110-5121. Marcum,Z.A.,and Gellad,W.F.(2012).Medication adherence to multidrug regimens.Clin Geriatr Med 28,287-300. Michaut,A.,Le Guillou,D.,Moreau,C.,Bucher,S.,McGill,M.R.,Martinais,S.,Gicquel,T.,Morel,I.,Robin,M.A.,Jaeschke,H.,et al.(2016).A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease:Application to acetaminophen.Toxicol Appl Pharmacol 292,40-55. Miki,T.,Ring,A.,and Gerlach,J.(2011).Hepatic differentiation of human embryonic stem cells is promoted by three-dimensional dynamic perfusion culture conditions.Tissue Eng Part C Methods 17,557-568. Mork,L.M.,Isaksson,B.,Boran,N.,Ericzon,B.G.,Strom,S.,Fischler,B.,and Ellis,E.(2012).Comparison of culture media for bile Acid transport studies in primary human hepatocytes.J Clin Exp Hepatol 2,315-322. Navarro,V.J.,and Senior,J.R.(2006).Drug-related hepatotoxicity.N Engl J Med 354,731-739. Ni,X.,Gao,Y.,Wu,Z.,Ma,L.,Chen,C.,Wang,L.,Lin,Y.,Hui,L.,and Pan,G.(2016).Functional human induced hepatocytes(hiHeps) with bile acid synthesis and transport capacities:A novel in vitro cholestatic model.Sci Rep 6,38694. Nishida,T.,Gatmaitan,Z.,Che,M.,and Arias,I.M.(1991).Rat liver canalicular membrane vesicles contain an ATP-dependent bile acid transport system.Proc Natl Acad Sci U S A 88,6590-6594. Oorts,M.,Baze,A.,Bachellier,P.,Heyd,B.,Zacharias,T.,Annaert,P.,and Richert,L.(2016).Drug-induced cholestasis risk assessment in sandwich-cultured human hepatocytes.Toxicol In Vitro 34,179-186. Pessayre,D.,Fromenty,B.,Berson,A.,Robin,M.A.,Letteron,P.,Moreau,R.,and Mansouri,A.(2012).Central role of mitochondria in drug-induced liver injury.Drug Metab Rev 44,34-87. Pessayre,D.,Mansouri,A.,Berson,A.,and Fromenty,B.(2010).Mitochondrial involvement in drug-induced liver injury.Handb Exp Pharmacol,311-365. Polson,J.,and Lee,W.M.(2005).AASLD position paper:the management of acute liver failure.Hepatology 41,1179-1197. Rachek,L.I.,Yuzefovych,L.V.,Ledoux,S.P.,Julie,N.L.,and Wilson,G.L.(2009).Troglitazone,but not rosiglitazone,damages mitochondrial DNA and induces mitochondrial dysfunction and cell death in human hepatocytes.Toxicol Appl Pharmacol 240,348-354. Rector,R.S.,Thyfault,J.P.,Uptergrove,G.M.,Morris,E.M.,Naples,S.P.,Borengasser,S.J.,Mikus,C.R.,Laye,M.J.,Laughlin,M.H.,Booth,F.W.,et al.(2010).Mitochondrial dysfunction precedes insulin resistance and hepatic steatosis and contributes to the natural history of non-alcoholic fatty liver disease in an obese rodent model.J Hepatol 52,727-736. Russo,M.W.,Galanko,J.A.,Shrestha,R.,Fried,M.W.,and Watkins,P.(2004).Liver transplantation for acute liver failure from drug induced liver injury in the United States.Liver Transpl 10,1018-1023. Sebastien Le,J.J.,Francois Husson(2008).FactoMineR:An R Package for Multivariate Analysis.Journal of Statistical Software 25. Serviddio,G.,Pereda,J.,Pallardo,F.V.,Carretero,J.,Borras,C.,Cutrin,J.,Vendemiale,G.,Poli,G.,Vina,J.,and Sastre,J.(2004).Ursodeoxycholic acid protects against secondary biliary cirrhosis in rats by preventing mitochondrial oxidative stress.Hepatology 39,711-720. Sloan,C.A.,Chan,E.T.,Davidson,J.M.,Malladi,V.S.,Strattan,J.S.,Hitz,B.C.,Gabdank,I.,Narayanan,A.K.,Ho,M.,Lee,B.T.,et al.(2016).ENCODE data at the ENCODE portal.Nucleic Acids Res 44,D726-732. Song,W.,Lu,Y.C.,Frankel,A.S.,An,D.,Schwartz,R.E.,and Ma,M.(2015).Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation.Sci Rep 5,16884. Song,Z.,Cai,J.,Liu,Y.,Zhao,D.,Yong,J.,Duo,S.,Song,X.,Guo,Y.,Zhao,Y.,Qin,H.,et al.(2009).Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells.Cell Res 19,1233-1242. Spence,J.R.,Mayhew,C.N.,Rankin,S.A.,Kuhar,M.F.,Vallance,J.E.,Tolle,K.,Hoskins,E.E.,Kalinichenko,V.V.,Wells,S.I.,Zorn,A.M.,et al.(2011).Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.Nature 470,105-109. Stevens,J.L.,and Baker,T.K.(2009).The future of drug safety testing:expanding the view and narrowing the focus.Drug Discov Today 14,162-167. Takahashi,K.,Tanabe,K.,Ohnuki,M.,Narita,M.,Ichisaka,T.,Tomoda,K.,and Yamanaka,S.(2007).Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell 131,861-872. Takebe,T.,and Taniguchi,H.(2014).Human iPSC-derived miniature organs:a tool for drug studies.Clin Pharmacol Ther 96,310-313. Tian,X.,Zamek-Gliszczynski,M.J.,Zhang,P.,and Brouwer,K.L.(2004).Modulation of multidrug resistance-associated protein 2(Mrp2) and Mrp3 expression and function with small interfering RNA in sandwich-cultured rat hepatocytes.Mol Pharmacol 66,1004-1010. Tsukada,N.,Ackerley,C.A.,and Phillips,M.J.(1995).The structure and organization of the bile canalicular cytoskeleton with special reference to actin and actin-binding proteins.Hepatology 21,1106-1113. Verma,S.,and Kaplowitz,N.(2009).Diagnosis,management and prevention of drug-induced liver injury.Gut 58,1555-1564. Vosough,M.,Omidinia,E.,Kadivar,M.,Shokrgozar,M.A.,Pournasr,B.,Aghdami,N.,and Baharvand,H.(2013).Generation of functional hepatocyte-like cells from human pluripotent stem cells in a scalable suspension culture.Stem Cells Dev 22,2693-2705. Yang, K., Woodhead, JL, Watkins, PB, Howell, BA, and Brouwer, KL (2014). Systems pharmacology modeling predicts delayed presentation and species differences in bile acid-mediated troglitazone hepatotoxicity. Clin Pharmacol Ther 96,589-598. Zborowski, J., and Wojtczak, L. (1963).Induction of Swelling of Liver Mitochondria by Fatty Acids of Various Chain Length.Biochim Biophys Acta 70,596-598.

[0089] Unless otherwise indicated, all percentages and ratios are calculated by weight.

[0090] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[0091] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0092] The dimensions and values ​​disclosed herein should not be understood as being strictly limited to the exact numerical values ​​recited. Rather, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "20 mm" is intended to mean "approximately 20 mm."

[0093] All documents cited herein, including any cross-referenced or related patents or applications, are incorporated herein by reference in their entirety, unless expressly excluded or otherwise limited. The citation of any document shall not be deemed to be prior art to any invention disclosed or claimed herein, or that it, alone or in combination with any other reference(s), teaches, suggests, or discloses any such invention(s). Furthermore, to the extent that any meaning or definition of a term in this document conflicts with a meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0094] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

1. A method for inducing the formation of liver organoids from definitive endoderm (DE) cells, comprising: a) contacting definitive endoderm cells with an FGF pathway activator and a Wnt signaling pathway activator for 1-3 days to form posterior foregut spheroids; b) incubating the posterior foregut spheroids of step a) in the presence of retinoic acid (RA) for 1 to 5 days to form the liver organoids.

2. 10. The method of claim 1, wherein the DE cells are derived from stem cells.

3. 3. The method of claim 2, wherein the stem cells are human ESCs or human iPSCs.

4. In any one of claims 1 to 3, the method further comprises incubating the liver organoid of step b) with hepatocyte growth factor, dexamethasone, and / or oncostatin M for at least 10 days.

5. The method according to any one of claims 1 to 4, further comprising the step of embedding the foregut spheroids in a basement membrane matrix.

6. 6. The method according to any one of claims 1 to 5, wherein the liver organoid expresses alpha-fetoprotein (AFP), albumin (ALB), retinol-binding protein (RBP4), cytokeratin 19 (CK19), hepatocyte nuclear factor 6 (HNF6), and cytochrome P450 3A4 (CYP3A4).

7. The method according to any one of claims 1 to 6, wherein the liver organoid has bile transport activity.

8. 8. The method of any one of claims 1-3 or 5-7, wherein the FGF pathway activator is selected from a small molecule or protein FGF signaling pathway activator, FGF1, FGF2, FGF3, FGF4, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, or a combination thereof.

9. The method of any one of claims 1 to 3 or 5 to 8, wherein the FGF pathway activator is FGF4.

10. 10. The method of any one of claims 1-9, wherein the FGF pathway activator is provided at a concentration of 200 ng / mL or greater.

11. 11. The method of any one of claims 1-10, wherein the FGF pathway activator is provided at a concentration of 500 ng / mL.

12. The method of any one of claims 1 to 11, wherein the Wnt signaling pathway activator is selected from a small molecule or protein Wnt signaling pathway activator.

13. 13. The method of any one of claims 1 to 12, wherein the Wnt signaling pathway activator is Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, or any combination thereof.

14. 13. The method of any one of claims 1 to 12, wherein the Wnt signaling pathway activator is a small molecule selected from lithium chloride; 2-amino-4,6-disubstituted pyrimidine (hetero)arylpyrimidine; IQ1; QS11; NSC668036; and 2-amino-4-[3,4-(methylenedioxy)-benzyl-amino]-6-(3-methoxyphenyl)pyrimidine, a GSK3 inhibitor, CHIR99021, or a combination thereof.

15. The method according to any one of claims 1 to 12, wherein the Wnt signalling pathway activator is a GSK3 inhibitor.

16. 16. The method of any one of claims 1 to 15, wherein the Wnt signaling pathway activator is CHIR99021.

17. 17. The method of claim 16, wherein the CHIR99021 is provided at a concentration of 1 μM to 100 μM.

18. 17. The method of claim 15 or 16, wherein the CHIR99021 is 3 μM.

19. The method of any one of claims 1 to 14, wherein the Wnt signaling pathway activator is not a GSK3 inhibitor.

20. 15. The method of any one of claims 1 to 14, wherein the Wnt signaling pathway activator is not CHIR99021.

21. A liver organoid produced by the method according to any one of claims 1 to 20.

22. 22. The liver organoid of claim 21, said liver organoid comprises mesenchymal cells and luminal structure comprising internalized microvilli, said luminal structure being surrounded by polarized hepatocytes and basement membrane.

23. 23. The liver organoid of claim 21 or 22, wherein said liver organoid comprises stellate cells and Kupffer cells.

24. The liver organoid according to any one of claims 21 to 23, wherein the liver organoid has the following: bile production ability, bile transport activity, at least 50 ng / mL / 1xe ６ Complement factor H expression at least 40 ng / mL / 1xe per cell / 24 h ６ Complement factor B expression at least 1000 ng / mL / 1xe cells / 24 hours ６ C3 expression in cells / 24 hours; at least 1000 ng / mL / 1xe ６ C4 expression at least 1000 ng / mL / 1xe cells / 24 hours ６ fibrinogen production of at least 1000 ng / mL / 1xe ６ Liver organoids characterized by having one or more of the following: cells / 24-hour albumin production.

25. The liver organoid of any one of claims 21 to 24, wherein said liver organoid expresses one or more genes selected from PROX1, RBP4, CYP2C9, CYP3A4, ABCC11, CFH, C3, C5, ALB, FBG, MRP2, ALCAM, CD68, CD34, CD31, liver organoid.

26. 26. The liver organoid according to any one of claims 21 to 25, wherein the liver organoid comprises a drug-metabolizing cytochrome mutant.

27. 27. The liver organoid of claim 26, wherein the drug-metabolizing cytochrome mutant is CYP2C9. ＊ 2 mutant, liver organoids.

28. 28. The liver organoid according to any one of claims 21 to 27, wherein the liver organoid does not contain inflammatory cells.

29. A method for screening for serious adverse events (SAE), comprising contacting a drug of interest with the liver organoid of any one of claims 21 to 28.

30. 30. The method of claim 29, wherein the SAE is liver failure and / or drug-induced liver injury (DILI).

31. 31. The method of claim 29 or 30, wherein the method comprises measuring the uptake and / or excretion of fluorescein diacetate (FD), and impaired excretion indicates that the drug may induce serious adverse events.

32. The method of any one of claims 29 to 31, further comprising a step of measuring a parameter selected from mitochondrial membrane potential, measurement of ROS, swelling of liver mitochondria, and a combination thereof, performed in addition to or separately from the step of claim 31, wherein the toxicity of the drug of interest is determined by the step of measuring a parameter selected from mitochondrial membrane potential, measurement of ROS, swelling of liver mitochondria, and a combination thereof, and damage to the mitochondria indicates that the drug may induce a serious adverse event.

33. The method according to any one of claims 29 to 32, further comprising a step of measuring organoid viability, which is performed in addition to or separately from the steps of claims 31 or 32, wherein a failure in determining organoid viability indicates that the drug may induce a serious adverse event.

34. A liver organoid according to any one of claims 21 to 27 for use in treating an individual with liver damage.

35. 35. The liver organoid of claim 34, wherein said liver disorder is selected from metabolic liver disease, end-stage liver disease, or a combination thereof.

36. A method for identifying a therapeutic agent preferred for an individual, comprising contacting the liver organoid of any one of claims 21 to 27 with a candidate compound, wherein the liver organoid is derived from iPSC.

37. 37. The method of claim 36, wherein the iPSCs comprise one or more mutations found in the individual.

38. 38. The method of claim 36 or 37, wherein the iPSCs are derived from the same ethnic background as the individual.

39. 39. The method of any one of claims 36 to 38, wherein the iPSCs are derived from the individual.

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