Liver organoid compositions and methods of making and using the same

Abstract

To provide methods of inducing formation of liver organoids from precursor cells, such as iPSC cells, which can be used for screening for a serious adverse event (SAE), such as liver failure and / or drug induced liver injury (DILI), and / or drug toxicity.SOLUTION: This method comprises the steps of a) contacting definitive endoderm (DE) derived from iPSC cells with a FGF pathway activator and a GSK3 inhibitor, for a period of time sufficient to form posterior foregut spheroids, and b) incubating the posterior foregut spheroids obtained in the step a in the presence of retinoic acid (RA) for a period of time sufficient to form the liver organoid.SELECTED DRAWING: Figure 1

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 / 471,371, filed November 4, 2016, and No. 62 / 517,414, filed June 9, 2016, 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 reconstitution 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, mainly 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drug development due to failure of drug candidates identified in early screening, with nearly one-third of drugs being withdrawn from the market due to such failures (Takebe and Taniguchi, 2014). The failure of drug candidates results in a significant loss of therapeutic opportunities for patients. Preclinical testing generally consists of in vitro evaluation as the primary efficacy screen to identify “hit” compounds, followed by in vitro and in vivo safety testing to evaluate mechanisms of metabolism and toxicity. This inefficiency can be explained by the virtual lack of physiologically relevant preclinical models with high throughput to evaluate drug-induced liver injury (DILI) in humans, and thus the urgent need to develop in vitro human screening models to evaluate the vast numbers of the ever-growing compound library.

[0004] Primary hepatocytes are a highly polarized metabolic cell type that form bile canalicular structures with microvillous channels, separating the peripheral circulation from the bile acid secretory pathway. The most upstream aspect of DILI involves drug (or their reactive metabolites) detoxification by hepatocytes and 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, 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, as in the cases of troglitazone, nefazodone and tolcapone (https: / / livertox.nlm.nih.gov / index.html). Thus, the determination of toxicological properties mainly relies on animals as an essential step for drug development, but there is a notable lack of fidelity to human results due to the notable differences in physiology between humans and animals (Leslie et al., 2007; Yang et al., 2014). Furthermore, the development of idiosyncratic DILI (IDILI) is extremely rare, but is nevertheless 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). Taken together, there is a need for effective human cell models to screen compounds to test the detoxification and excretion of proposed drugs.

[0005] Despite the progressive advances in human hepatocyte differentiation methods from pluripotent stem cells (PSCs), clinical trials in a dish using human stem cells are still "hyped". There is a need for hepatocyte models for use in bioartificial liver devices, for example as a bridge for 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 above-mentioned needs in the art. Summary of the Invention

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

[0007] Those skilled in the art will understand that the drawings, described below, are for illustration 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 1]Generation of human liver organoids from iPSCs with luminal structures. A. Outline of differentiation method for liver organoids. B. Phase contrast images of human liver organoids. C. Immunostaining of albumin (ALB), type IV collagen (collagen IV) and ZO-1 in organoids. Nuclei were stained with hematoxylin (blue). Bar, 50 μm. 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 iPS cells, organoids at days 7, 11, 20 and 30 of differentiation, and primary hepatocytes (PH). Relative expression values ​​were compared in undifferentiated iPSCs (AFP, ALB, RBP4 and CK19) or in day 7 organoids (HNF6) or day 11 organoids (CYP3A4). Bars represent mean ± SD, n=3. E. Principal component analysis based on RNA-seq data in undifferentiated iPSCs (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. F. Albumin (ALB, n=10) and fibrinogen (FBG, n=4) secretion levels from organoids at days 25–30. Bars represent mean ± SEM. G. Complement factor secretion levels from organoids on days 25-30. FH: factor H, FB: factor B. Bars represent mean ± SEM, n=5. [Diagram 2]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. B. Transilluminal electron microscopy of organoids showing the microvilli (V) luminal surface; N: nuclei. Bar, 10 μm. 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 iPSC cells (ABCB11) or day 11 organoids (NTCP). Bars represent mean ± SD, n=3. D. Total bile acid secretion levels inside organoids at day 27. Bars represent mean ± SEM, n = 4. E. Bile acid uptake by organoids after 30 min of incubation in the presence of fluorescent bile acids (CGamF). F. CLF transport activity for organoids derived from four iPSC lines. T, W, 1, and F indicate the clonal names of the iPSC lines. Green: CLF. [Diagram 3] Bosentan-induced cholestasis is specific for CYP2C9*2 iPSC-liver organoids. A. Representative allele images of rs1799853 in CYP2C9*2 and rs4148323 in UGT1A1*6 show the risk SNPs for DILI with bosentan and irinotecan, respectively. The table shows the prevalence of risk alleles in each iPS cell line. B. Images of CLF transport activity and inhibition by bosentan. 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 indicate data from each organoid. [Figure 4]A high fidelity drug-induced cholestasis model using organoids. A. Sequential images of fluorescein diacetate efflux from the outside to the inside of the organoid. B. Comparison of fluorescein diacetate efflux transport. C. Quantification of fluorescein diacetate efflux transport into the organoid. Exemplary left figures are quantified ratios of fluorescein intensity between the inside and outside of the organoid. Right figures show the results of validation studies using control (DMSO), cyclosporine A (CSA), and streptomycin (STP) as a negative control. Bars represent mean ± SD, **: p<0.01, n=4. D. Images of fluorescein diacetate transport inhibition after 24 h treatment with nine training compounds. 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 of 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. [Diagram 5]High fidelity drug-induced mitochondrial toxicity screening using organoids. A. Images of mitochondrial membrane potential (MMP) on TMRM after treatment of nine training compounds. Bottom row: Quantification of transport inhibition after treatment of training compounds, bars represent mean ± SD, *: p<0.05, **: p<0.01, n=4–6. C. Quantification of MMP changes after treatment of training compounds, bars represent mean ± SD, *: p<0.05, **: p<0.01, n=4–5. CON: control samples, 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 reports for DILI in vivo, while those in class B are TCs with reports for drug-induced cholestasis in vivo. 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 for drug-induced cholestasis and potential for mitochondrial toxicity. Cholestatic and mitochondrial toxicity (Mito-tox) indices were derived from the data in Figure 3. The size of the circle indicated the magnitude of survival loss. [Figure 6] Modeling drug-induced liver injury in fragile states rescued by NAC exposure. A. Overview of drug-induced cytotoxicity assessment on fragile organoid model. B. Profiling of fragile model for lipid accumulation (blue: nuclei, green: lipids, red: F-actin). C. ROS production (blue: nuclei, green: ROS) and D. mitochondrial health (blue: nuclei, red: mitochondria). E. Images of organoids 24 hours after drug treatment. F. Viability assessment on fragile organoid model induced by lipid accumulation. 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 screen for toxicity prediction. [Figure 8] Optimization of retinoic acid treatment protocol A. Plan the timing and duration of retinoic acid treatment. RA: retinoic acid, HCM: hepatocyte culture medium. B. Albumin secretion levels in organoids at day 25 with different durations of RA treatment. [Figure 9] Morphology of organoids at D20. The total number of organoids at D20 was 305. Organoids with a lumen: 216, organoids without a lumen: 89. [Figure 10] Conversion formula for determining cell number in organoids A. Phase contrast image of a single organoid B. Diameter and cell number of each single organoid C. Correlation between diameter and cell number in a single organoid. [Figure 11] Supplemental Figure 4. Generation of organoids from multiple PSC lines. Phase contrast images and albumin secretion levels of organoids derived from different iPSC lines (317D6 and 1383D6). [Figure 12] Cell viability after 24 hours of treatment with 10 compounds. Viability assessment for lipid accumulation-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 13] Mitochondrial ROS generation and morphological changes in lipotoxic liver organoids. A. The ratio of ROS-producing cells to the total number of cells in the lipid accumulation-induced fragile organoid model by treatment with 800 μM oleic acid (OA). B. Images of mitochondria in organoids for the fragile organoid model. Red: mitochondria, purple: F-actin, blue: nuclei. C. The number and size of mitochondria for the fragile organoid model. Bars represent the 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 PREFERRED EMBODIMENTS

[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, for example, of 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, as per practice in the art. Alternatively, "about" can mean within 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. When a particular value is described in the present application and claims, the term "about" should be assumed to mean within an acceptable error range for the particular value, unless otherwise indicated.

[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 constructing a complete, viable organism. These cells are produced from the fusion of an egg cell and a sperm cell. The 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 stomach 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 obtained by induction of non-pluripotent cells, such as adult somatic cells, by forcing the expression of certain genes. Pluripotent stem cells can be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including human, rodent, porcine, and bovine.

[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 derived from normally non-pluripotent cells, such as adult somatic cells, by inducing "forced" expression of certain genes. hiPSC refers to human iPSCs.

[0015] As used herein, the term "embryonic stem cells (ESCs)" is also commonly abbreviated as ES cells, and refers to cells that are pluripotent and derived from the inner cell mass of an early embryo, the blastocyst. For the 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 may be used in the methods described herein, where one or more progenitor cells will acquire the ability to regenerate themselves or differentiate into one or more specialized cell types. In some embodiments, the progenitor cells are pluripotent or have the ability to become pluripotent. In some embodiments, the progenitor cells are subjected to treatment with external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, the progenitor cells may be 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, the progenitor cells may be from embryos, infants, children, or adults. In some embodiments, the progenitor cells may be somatic cells that undergo treatment such that pluripotency is imparted via genetic engineering 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, a 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 the totipotent cells of early mammalian embryos and are capable of unlimited undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early stage embryo. 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); 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 (Goteborg, Sweden); ES Cell International Pte Ltd (Singapore); 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] Further details on embryonic stem cells can be found in, 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 and "Chem. 1999, 14:131-132, 1999," 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 transfection of specific stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection is typically accomplished through a viral vector, such as a retrovirus. 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: Oct3 / 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 iPSC include but are not limited to Oct-3 / 4 (e.g. Pou5f1);Sox gene family specific members (e.g. Sox1, Sox2, Sox3 and Sox15);Klf family specific members (e.g. Klf1, Klf2, Klf4 and Klf5), Myc family specific members (e.g. 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 required genes into the DNA of mouse skin and liver cells, resulting in cells identical to embryonic stem cells. The risk of creating tumors is eliminated, since adenoviruses do not integrate any of their own genes into the target host. In some embodiments, reprogramming can be achieved via plasmids, albeit with very low efficiency, without the use of viral transfection systems at all. In other embodiments, direct delivery of proteins is used to generate iPSCs, thus eliminating the need for viruses or genetic modification. In some embodiments, generation of mouse iPSC cells is possible using a similar methodology: repeated treatment of cells with specific proteins directed into the cells 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 producing definitive endoderm from pluripotent cells (e.g., iPSCs or ESCs) is applicable to the methods described herein. Any method for producing 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 may include, but are not limited to, embryonic stem cells. Embryonic stem cells may be derived from the inner cell mass of an embryo or the gonadal ridge of an embryo. Embryonic stem cells or germ cells may be derived from a variety of animal species, including, but not limited to, a variety of 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] Applicant has found a method to create a 3D liver structure using human iPSCs. The structure contains a micro-liver structure including polarized liver epithelium, stellate cells, and canalicular structures. The disclosed composition shows 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 test and / or drug toxicity screening, transplantation, production of serum protein products, and development of personalized therapy. In one particular 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 have much higher functional activity such as albumin production (up to 10-fold increase compared to the previous gold standard model using iPSC-derived hepatocytes) and allow improved oxygen and / or nutrient supply due to the internal luminal structure, which allows for much longer culture (at least 60 days or more) and a long-term testing platform useful for drug testing. The disclosed compositions may be useful for the production of plasma products such as albumin, a clotting factor product for the treatment of hypoalbuminemia, and also 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 (individualization of treatment).

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

[0030] The method may include the steps of: a) contacting definitive endoderm (DE) derived from iPSC cells with an FGF pathway activator and a GSK3 inhibitor for a period of time 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 of time 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 signaling. Suitable FGF pathway activators will be readily understood by 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, 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 FGF signaling pathways may be used to activate these pathways.

[0032] In some embodiments, the 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 during 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 regimen described herein can be applied to any known molecule of the signaling pathway described herein, alone or in combination, including but not limited to any molecule in the FGF signaling pathway.

[0033] Suitable GSK3 inhibitors will be readily recognized by 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 carrying out 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.

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

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

[0036] In one embodiment, liver organoid can be characterized as 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.Such 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 embodiment, the liver organoids can be characterized as having bile transport activity.

[0038] In one embodiment, liver organoid can be derived from stem cells, and can comprise luminal structure further comprising internalized microvilli, 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: bile production capacity, bile transport activity, at least 50 ng / mL / 1xe 6 Complement factor H expression at least 40ng / mL / 1xe per cell / 24 hours 6 Complement factor B, at least 1000ng / mL / 1xe per cell / 24 hours 6 C3 expression at least 1000ng / mL / 1xe per cell / 24h 6 C4 expression at least 1000ng / mL / 1xe per cell / 24 hours 6 Fibrinogen production of at least 1000ng / mL / 1xe 6In one embodiment, the liver organoids have at least 10,000 ng / mL 1xe albumin production. 6 The liver organoid may be characterized by having total liver protein expression of cells / 24 hours.The liver organoid may be characterized by expressing one or more genes selected from PROX1, RBP4, CYP2C9, CYP3A4, ABCC11, CFH, C3, C5, ALB, FBG, MRP2, ALCAM, CD68, CD34, CD31.In one embodiment, the liver organoid may comprise cells that comprise drug-metabolizing cytochrome mutants, such as CY2C9*2 mutants.The liver organoid may comprise vasculature, such as those described in US 2016 / 0177270.

[0040] In one embodiment, the liver organoids can be characterized in that the liver organoids 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. The SAE can be liver failure and / or drug-induced liver injury (DILI). The method can include contacting a drug of interest whose toxicity is of interest with the liver organoid described herein. In one embodiment, the method can include measuring the uptake and / or efflux of fluorescein diacetate (FD), where impaired efflux indicates that the drug may induce serious adverse events. The toxicity of the drug of interest can be determined by measuring a parameter selected from mitochondrial membrane potential, measuring ROS, swelling of liver mitochondria, and combinations thereof, where damage to mitochondria indicates that the drug may induce serious adverse events. In one embodiment, the method can include analyzing organoid viability, where impaired or reduced organoid viability indicates that the drug of interest may induce serious adverse events.

[0042] In one aspect, disclosed is a method for treating the individual with liver damage, and this method can include 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, the method for identifying the preferred therapeutic agent for individual is disclosed.In this embodiment, the method can include contacting the liver organoid derived from target iPSC with candidate compound, where for example, target iPSC comprises one or more mutations found in such individual, or for example, target iPSC is derived from the same ethical background as such individual, or further, target iPSC is derived from such individual. EXAMPLES

[0044] In this study, applicants tested bile transport activity using fluorescein diacetate, which was excreted by MRP2 across the bile canalicular membrane into the bile canalicular network (Tian et al., 2004). It has been previously reported that troglitazone and cyclosporine inhibit MRP2 (Chang et al., 2013; Lechner et al., 2010). In addition, the efflux transporter MRP2 mediates the transport of bosentan (Fahrmayr et al., 2013). Although inhibition of MRP2 by nefazodone has not been reported, mitochondrial stress by nefazodone may be associated with a decrease in bile transport activity, fluorescein diacetate efflux, since MRP2 is an ATP-dependent bile salt transporter for canalicular excretion of bile acids in hepatocytes.

[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, applicants developed a human liver organoid (HLO) model to analyze clinical DILI pathology at organoid resolution. Differentiated HLO from human iPSCs contain polarized hepatocytes with a lumen lined by bile canaliculi-like structures, establishing a unidirectional bile acid transport pathway. Applicants exploited 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 for CYP2C9 poor metabolizer donor-derived HLO. 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 failure of drug candidates identified in early screening, and many (one third) drugs are withdrawn from the market due to such failures (Takebe and Taniguchi, 2014). Despite promising efficacy, failure of drug candidates results in a significant loss of treatment opportunities for patients. Preclinical testing generally consists of in vitro evaluation as the primary efficacy screen to identify “hit” compounds, followed by in vitro and in vivo safety testing to evaluate mechanisms of metabolism and toxicity. This inefficiency can be explained by the virtual lack of physiologically relevant preclinical models to evaluate drug-induced liver injury (DILI) in humans, and thus the urgent need to develop in vitro human screening models to evaluate the vast numbers of the ever-growing compound library.

[0047] Primary hepatocytes are a highly polarized metabolic cell type that form bile canalicular structures with microvillous channels, separating the peripheral circulation from the bile acid secretory pathway. The most upstream aspect of DILI involves drug (or their reactive metabolites) detoxification by hepatocytes and 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, 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, as in the cases of troglitazone, nefazodone and tolcapone (https: / / livertox.nlm.nih.gov / index.html). Thus, the determination of toxicological properties mainly relies on animals as an essential step for drug development, but there is a notable lack of fidelity to human results due to the notable differences in physiology between humans and animals (Leslie et al., 2007; Yang et al., 2014). Furthermore, the occurrence of idiosyncratic DILI (IDILI) is very rare, but is nevertheless involved in approximately 10-15% of acute liver failure cases in the United States (Reuben et al., 2010) and is almost impossible to predict (Kullak-Ublick et al., 2017). Taken together, effective human cell models are highly promising for screening compounds to test the detoxification and excretion of proposed drugs.

[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 "hyped". To some extent, this can be explained by challenges in previous cell-based approaches, including (1) overcoming lot-to-lot differences, (2) minimizing experimental batch differences, (3) increasing assay throughput, and (4) improving relevance to 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 have an intraluminal structure surrounded by polarized hepatocytes and have been shown to be capable of performing key human hepatocyte functions, including protein and bile acid production as well as transport functions. 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 allowed 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 liver organoid-based toxicity screen (LoT), is believed to be the first functional readout developed in human liver organoids, facilitating diagnostics, functional studies, drug development and personalized medicine.

[0049] result Generation and characterization of polarized liver organoids from multiple human iPSCs Applicant first established a novel liver organoid differentiation method by using foregut spheroids derived from human iPSCs (Spence et al., 2011) (Figure 1A). As a first step, Applicant used BMP and Activin A to promote differentiation into definitive endoderm as previously described (D'Amour et al., 2005). In addition, FGF4 and GSK3 inhibitor (CHIR99021) were used to induce foregut spheroids and budding spheroids were observed. After detaching mesenchymal cells seeded on the dish by gentle pipetting, the organoids were embedded in Matrigel. Retinoic acid (RA) has been reported to enhance cell polarity as indicated by an increase in the size and complexity of bile canaliculi and pericanalicular sheaths (Falasca et al., 1998). To generate polarized organoids suitable for bile transport modeling, the organoids were treated with RA. To optimize the organoid generation method, the applicant 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, and the 4-day RA treatment protocol tended to reach the highest level (Figure 8). Therefore, based on the level of albumin secretion, the duration of RA was set to 4 days. Morphologically, about 10 days after RA treatment, more than 300 organoids covered with epithelial cells were successfully generated, and the ratio of organoids with luminalized 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 had 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) that controls cholangiocyte differentiation, and cytochrome P450 3A4 (CYP3A4) during differentiation (Figure 1, panel D). However, expression levels of most hepatic genes extracted from bulk organoid-derived RNA were lower in organoids than in primary hepatocytes. Without 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. Applicant further profiled the organoids by global gene expression analysis using RNA sequencing (RNA-seq). Principal component analysis showed that gene expression in organoids was not similar to 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 by ELISA in the culture supernatant (Figure 1, panels F-G). To quantify the hepatic functionality of organoids, Applicant examined the albumin secretion level normalized by cell number (Figure 10). Albumin secretion level was 2133 ng / day / 10 6 cells (Figure 1, panel F) and other experiments in 2D and 3D differentiation of hPSCs into HLCs (150–1000 ng / day / 10 6 cells), whereas primary hepatocytes were at 30-40 μg / day / 10 in 3D scaffolds. 6These results showed that liver organoids contain hepatocytes with reasonable albumin secretion activity compared to stem cell-derived hepatocytes in published literature. Importantly, this organoid generation method is reproducible and therefore applicable to other PSC lines, as intraluminal organoids were generated from both 317D6 and 1383D6 iPS cell lines with albumin secretion capacity (Figure 11). In summary, applicants have established a protocol for generating a large number of polarized liver organoids with hepatocyte characteristics.

[0051] Microanatomical characterization of bile acid-producing human iPSC-liver organoids Next, to test whether liver organoids have bile transport activity, applicants first characterized the organoids by staining the major proteins involved in bile synthesis and excretory functions. Immunofluorescence 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 the tight junctions are located inside our liver organoids. Transmission electron microscopy revealed that the organoids contained microvilli oriented toward the lumen (Figure 2, panel B). Consistent with these anatomical features, qRT-PCR analysis revealed that the organoids had gene expression of ABCB11 and Na+-taurocholate cotransporting polypeptide (NTCP), although the levels were lower in the organoids than in the primary hepatocytes (Figure 2, panel C). Thus, the 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, applicants performed ELISA on the luminal fluid collected from the 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 higher than that of primary hepatocytes derived from sandwich culture in a previous report (Ni et al., 2016) (approximately 40 μg / day / 10 6Thus, the organoids not only had a bile canaliculus-like morphology, but also had bile acid production and secretion activity, suggesting that the bile acid transport pathway was correctly constructed.

[0053] Dynamic visualization of bile acid uptake and excretion in human liver organoids Bile acid excretion is a major determinant of bile flow, and therefore defects in this system may result in impaired bile secretion (cholestasis) associated with various liver disease pathologies (Nishida et al., 1991). Efflux transport proteins located in the apical (bile canalicular) membrane of hepatocytes play a key 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, applicants next wondered whether organoids could actively transport bile acids into their lumen. First, to examine the uptake of bile acids into the organoids, applicants challenged the organoids with cholylglycylamide-fluorescein (CGamF), a bile salt analog (Mork et al., 2012). After treatment with external CGamF, accumulation of CGamF in the lumen of organoids was successfully confirmed (Figure 2, panel E). Similarly, the fluorescent bile acid cholyl-lysyl-fluorescein (CLF) was found to be reproducibly excreted and accumulated in organoids from multiple human iPSC lines (Figure 2, panel F). To determine the specificity of this assay, applicants developed iPSC lines carrying a BSEP non-functionalized allele using a CRISPR-Cas9-based gene editing approach. BSEP is involved in bile transport, and consistent with this, BSEP-KO iPSC-organoids were unable to accumulate fluorescent bile acids compared to parental control organoids. Taken together, these data suggest that organoids have the ability to take up bile acids from the outside and excrete them inside the organoids.

[0054] Bosentan-induced cholestasis specific to CYP2C9*2 iPSC-liver organoids To test the clinical relevance of the organoid-based cholestatic phenotyping method, applicants employed pharmacogenomic insights into our system to address the issue of fidelity. Specifically, multiple iPSC lines harboring known susceptibility gene variants (i.e., for bosentan, 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 in the presence of bosentan was compared (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 towards 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-based cholestasis was not specific to CYP2C9*2 iPSC lines. These results demonstrated that the organoid-based cholestasis assay predicts some aspects of human diversity.

[0055] High-Throughput Drug-Induced Cholestasis Assessment in Organoids Given the important role of cholestasis in drug-induced DILI, applicants next considered whether this organoid model would reflect the pathology of DILI in the presence of specific compounds. Before testing a large number of compounds, applicants first sought to develop a high-throughput fluorescence-based assay, since both CLF and CGamF are not applicable to high-speed imaging due to several issues: 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, has been proposed (Barth and Schwarz, 1982; Bravo et al., 1998). The polar fluorescent metabolite fluorescein is trapped intracellularly until it is actively transported out of the cell into the bile canalicular lumen (Malinen et al., 2014). To determine whether FD can be used for live assessment of transport capacity without medium exchange or exposure dose adjustment, hepatobiliary transport activity over time was further examined using time-lapse imaging. Organoids were incubated with fluorescein diacetate for 45 min, and intraluminal accumulation was observed inside the organoids 20 min after treatment (Figure 4, panels A, B). The opposite direction of this transport flow was determined by microinjection of FD into the 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 evaluation of unidirectional excretory bile transport in liver organoids by simple fluorescent live imaging analysis.

[0056] Next, applicants verified the fidelity of the FD-based assay by evaluating the feasible doses of 10 FDA-approved drugs to measure secondary impairment due to cell damage. Applicants successfully found optimal doses for nine compounds with acceptable viability. In contrast, amiodarone (AMIO) was significantly toxic to organoids within the range tested, and therefore AMIO was excluded from further potential DILI evaluation studies (Figure 12). Applicants investigated the potential for cholestasis in organoids using FD with nine training compounds (TCs) that were classified as one of three types based on 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 inhibitory potential of FD excretion, applicant developed a simple but robust quantification method by determining the fluorescence intensity ratio between the outside and inside of the organoids by Image J (Figure 4, Panel B). As a validation study, applicant first confirmed the ability to evaluate the inhibition rate using cyclosporine A (CSA). At 5 min after FD treatment, a significant reduction (0.4 compared to the control) was observed in the group treated with CSA for 24 h compared to the control (DMSO) (Figure 4, Panel B). Next, applicant screened nine TCs at multiple concentrations to evaluate the fidelity of this approach. Interestingly, in this screening system, at 24 h after TC treatment, similar to clinical observations, FD excretion was significantly reduced (p<0.01 or 0.05) in class B compounds, bosentan, CSA, troglitazone and nefazodone, whereas no inhibitory effect was observed in class A and class C compounds (upper image of Figure 4, Panel D and Figure 4, Panel E). These results suggested that the liver organoid model is useful for classifying bile transport inhibitory potency for candidate compounds in drug development that are highly relevant to the human phenotype.

[0057] Assessing mitochondrial overload in organoids Furthermore, since mitochondrial toxicity plays a central role in DILI with multiple mechanisms related to the development of DILI (Pessayre et al., 2012), applicants investigated mitochondrial health assessment. In this study, to investigate mitochondrial health in organoids, an indicator of mitochondrial membrane potential (MMP) was used to monitor MMP in intact cells as a direct readout of mitochondrial health (Li et al., 2014). After 24 h of TC treatment, a dose-dependent increase in MMP was 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, images and graphs below panel A). Furthermore, troglitazone also increased MMP in organoids (3-5 fold change, p<0.05), although no dose-dependency was observed. On the other hand, after treatment with bosentan, entacapone and pioglitazone, no increase in MMP was clearly observed even at multiple doses. These results demonstrated that this live image-based assay, named liver organoid-based toxicity screening (LoT), distinguishes between compounds with and without mitochondrial toxicity.

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

[0059] Assessing vulnerability to DILI in human liver organoids It is known that the incidence of DILI is often confounded by numerous host factors. Indeed, there is growing evidence that the risk of hepatotoxicity from some drugs, such as acetaminophen, is highly increased due to obesity and NAFLD in both rodents and humans (APAP) (Fromenty, 2013; Michaut et al., 2016). Therefore, it is important to predict the DILI probability in such a "vulnerable" state for patients, even at the subclinical stage. In this study, the applicant established a lipotoxicity organoid model by simultaneous exposure to an unsaturated fatty acid, oleic acid (Figure 6, Panel A). Three days after oleic acid treatment on organoids, lipid accumulation in the organoids became intense (Figure 6, Panel B). Oxidation of fatty acids is an important source of reactive oxygen species (ROS), which leads to the depletion of ATP and nicotinamide dinucleotide, inducing 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 the published phenotype (Zborowski and Wojtczak, 1963). As hepatic mitochondrial dysfunction precedes the development of NAFLD in a rat model (Rector et al., 2010), these results indicate that hepatotoxic organoids model to some extent the in vivo fatty liver model.

[0060] Recognizing this lipotoxic organoid model as a vulnerable state with enhanced ROS production, we treated organoids with troglitazone (0-50 μM) for 24 h and assessed cell viability in the organoids. Treatment with 50 μM troglitazone alone resulted in 85% cell viability at 24 h, whereas it decreased to 67% at 72 h. However, after treatment with troglitazone for lipotoxic conditions, massive fragmentation of organoids was observed due to 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, applicants investigated whether organoids could be restored from DILI-like conditions by potential therapeutic compounds. Applicants used N-acetylcysteine ​​(NAC), an antioxidant, to inhibit ROS production, since 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). As expected, cell viability was significantly improved by NAC, suggesting that NAC rescued cell death in organoids even under vulnerable conditions (Figure 6, Panel E and 6, Panel 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 multi-drug regimens and drug discovery to treat DILI.

[0062] Serious adverse events (SAEs), including liver failure, are the main cause of drug attrition or discontinuation of marketed drugs during clinical development. 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 in pharmaceuticals. Although recent reports have shown promise for hepatocyte-based cholestasis models using transdifferentiated cells from human fibroblasts (Ni et al., 2016), these assay platforms still have reproducibility challenges as well as throughput issues due to the diverse and limited human hepatocyte sources, as well as the need for complex quantification algorithms. In addition, HepaRG cells, a human hepatocellular carcinoma cell line, are also useful for assessing cholestasis features, 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 to the pharmaceutical industry. Alternatively, the described method allows for 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 wells), 2. assay throughput (measurements possible in a single organoid), and 3. multiple readouts to analyze interactions between other factors, such as mitochondrial stress. Notably, as mentioned above, a retrospective study revealed that multiple cellular stress possibilities were associated with the incidence of DILI (Aleo et al., 2014), as cell viability was reduced in a dual readout; mitochondrial and cholestatic stress dependent manner, and the results using the LoT assay were comparable to those of this study. Oxidative stress plays an important role in cell death and was 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-induced apoptosis (Bernardi, 1996). Consistent with these findings, Applicant's correlation analysis of these dual readouts showed that cholestatic stress was a more dominant factor for liver injury compared to mitochondrial stress, as seen in Figure 5. Thus, the LoT system can be used as a model system to investigate DILI mechanisms.

[0063] Moreover, considering the recent establishment of iPSC panel populations, the potential assessment of differential susceptibility in individuals is also promising (Inoue et al., 2014). Prediction of SAE in traditional in vitro assay systems generally does not focus on individual differences, whereas SAE often occurs in small sub-patient groups that are prone to SAE (Stevens and Baker, 2009). Application of the LoT system to a diverse population iPSC panel can provide information on differential susceptibility to SAE that was previously unreachable. Considering the extremely rare nature of DILI, the use of cells from patients with specific genomic or ethnic factors would help elucidate the idiosyncratic mechanisms of DILI that are currently unknown. Thus, LoT can serve as a game-changing strategy for the pharmaceutical industry by providing essential insights to minimize the possibility of DILI (Figure 7).

[0064] One limitation in 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 evaluating drug-induced hypersensitivity are limited, sensitivity to troglitazone-induced cytotoxicity was enhanced using an in vitro coculture model with hepatic cell lines, Huh7 cells and THP-1 cells (Edling et al., 2009). Thus, advancing the LoT platform by focusing on the immune lineage will be interesting to evaluate hepatocellular inflammation. Nevertheless, the LoT testing platform appears to be 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 the analysis of intrahepatic cholestasis in various contexts, with potential for mechanistic studies as well as drug screening applications beyond DILI.

[0065] Investigating fragile human liver conditions with LoT assays Host factors such as obesity are known to have a significant impact on the development of DILI (Heidari et al., 2014), but are often not understood in clinical practice due to its complex nature. The presence of obesity or fatty liver may make patients vulnerable to liver damage caused by xenobiotics and non-toxic chemicals (e.g., drugs), which may become hepatotoxic at lower doses in the presence of risk factors (Fromenty, 2013). Nevertheless, the current clinical trial system is not designed to stratify volunteers under vulnerable liver conditions with a handful of biomarker (ALT, AST) levels. Since a number of patients with steatosis are asymptomatic and undetectable by biomarkers before medication, it is important to predict the outcome in this vulnerable condition before entering the clinical phase.

[0066] In an effort to develop the LoT system to evaluate toxicity in these fragile conditions in early drug screening stages such as lead compound generation / optimization, the applicant applied lipotoxic stress to liver organoids and demonstrated the profound synergistic effect of the antidiabetic drug troglitazone on DILI. Indeed, the organoid system nicely reflects this feature by showing massive hepatocyte death promoted by triglyceride accumulation in hepatocytes in organoids. One of the mechanisms of DILI in obesity can explain the reduction of glutathione (GSH) levels (Michaut et al., 2016). Drug-induced oxidative stress can have several causes, especially by GSH depletion and inhibition of the mitochondrial respiratory chain (Begriche et al., 2011; Pessayre et al., 2010). The fragile model may reflect the aggravation of troglitazone-induced oxidative stress through the reduction of intracellular GSH levels and mitochondrial dysfunction that 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 unanticipated window to study previously untested host factors, as isolated host factors can be effectively deployed in organoids.

[0067] Precision medicine based on LoT From the perspective of personalized medicine, the selection of optimal pharmacotherapy with LoT will be of major interest in clinical practice. For example, the strategy considered in the selection of antipsychotic drugs must take into account liver tolerance according to the not negligible incidence of liver injury in psychiatric treatment populations; 16% of possible DILI drugs are neuropsychiatric drugs (Dumortier et al., 2002). Considering that NASH is often accompanied by psychological disorders such as depression, a safer combination selection of antidepressants (tricyclics or SSRIs), mood stabilizers, and neuroleptics is necessary (Dumortier et al., 2002). Also, due to the increase in chronic conditions associated with aging, polypharmacy (i.e., polypharmacy) is a common consequence of providing healthcare to elderly people (Marcum and Gellad, 2012), making it very difficult to identify the causative drug in cases of suspected DILI. As patient-derived iPSC-organoids provide an infinite and reproducible source, LoT can serve as a panel to stratify the likelihood of DILI in patients and provide information for safer drug selection from an individualized perspective.

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

[0069] method Maintaining PSCs The TkDA3 harboring 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 and were 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 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, Laminin 411 from Mippi Co or Biolamina Co can be used.

[0070] Generation 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, colonies of hiPSCs 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 (>90% of 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). Cultures for cell differentiation were maintained at 37°C in an atmosphere of 5% CO2 / 95% air, and medium was changed daily. Differentiated definitive endoderm showed germination on the plate on day 7. If spheroids were not sufficient to embed in Matrigel, add 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 detached from the dish by gentle pipetting. The isolated spheroids were centrifuged at 800 rpm for 3 min, and the supernatant was removed before embedding in 100% Matrigel drop on the dish. The plate was placed at 37°C in an atmosphere of 5% CO2 / 95% air for 5–15 min. After 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) 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 an atmosphere of 5% CO2 / 95% air, and medium was changed every 3 days. Around days 20–30, organoids embedded in Matrigel drops were isolated by scratching and gentle pipetting for any analysis.

[0073] Matrigel sandwich method: On days 7–8, the 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 min, and after removing the supernatant, they were mixed with 100% Matrigel. At the same time, hepatocyte culture medium containing all supplements was mixed with the same volume of 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 an atmosphere of 5% CO2 / 95% air for 15–30 min. After the Matrigel was solidified, the spheroids mixed with Matrigel were seeded on the plate thickly coated with Matrigel. The plate was placed at 37°C in an atmosphere of 5% CO2 / 95% air for 5 min. 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, the organoids embedded in the 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 an atmosphere of 5% CO2 / 95% air and the medium was changed every 3 days. Around days 20–30, the organoids embedded in the Matrigel drops were isolated by scratching and gentle pipetting for any 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) retinoic acid (RA; Sigma, St. Louis, MO) 2 μM for 4 days. The medium was changed every other day. After 4 days of plate culture, the organoids begin to bud, while the 2D cells differentiate into hepatocytes. Both organoids and hepatocytes can be maintained for up to 60 days under 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 multi-well plates or 6-well plates and used for subsequent assays, if necessary. Cultures for cell differentiation were maintained at 37°C in an atmosphere of 5% CO2 / 95% air 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 staining. 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 h at room temperature. Nuclei were stained with 10 μg / mL Hoechst 33342 (Sigma) for 10 min at room temperature, after which the organoids were washed again three times with washing buffer. The specimens were observed under a fluorescent microscope or bright field. For whole-tissue immunohistochemical staining, liver organoids were fixed with 4% paraformaldehyde for 30 min and permeabilized with 2.5% Tween 20 (Sigma) at room temperature, after which the organoids were incubated overnight at 4 °C with the following primary antibodies diluted in PBS: polyclonal anti-BSEP antibody (1:200 Sigma). A fluorescent dye-conjugated secondary antibody, Alexa Fluor 568-conjugated donkey anti-rabbit immunoglobulin (IgG; 1:500; Invitrogen, A10042), was applied to the organoids for 2 h at room temperature. After the reaction, 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 stained with 10 μg / mL Hoechst After staining with 33342 (Sigma) for 10 min at room temperature, the organoids were washed again three times with washing buffer and the specimens were 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 SuperScriptIII First-Strand Sysnthesis Systen for RT-PCR (Invitrogen, CA, USA) according to the manufacturer's protocol. qPCR was performed using TaqMan Gene Expression Master Mix (Applied Biosystmes) 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 described previously (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 were 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 fragments mapped (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); 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. The gene expression levels were obtained from GSE85223 (Asai et al., 2017) and GSE85223 (Asai et al., 2016). Genes were used if all datasets had identical gene symbols after possible data preprocessing. Applicants performed quartile normalization of FPKM+1 and RPKM+1 data in log2 space, followed by selecting genes within the top 10,000 median expression levels. Principal component analysis was performed by using scaled gene expression levels by 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 of organoids, 200 μL of culture supernatant of organoids on ultra-low attachment 96-well plates (Corning) was collected. Culture supernatants were 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, supernatants were measured using a Luminex system (Luminex Corporation, Austin, TX) according to the manufacturer's instructions. A linear regression equation of cell number by organoid diameter was used to calculate the amount of albumin production per cell number. To measure the total bile acid secretion level of intraluminal organoids, microinjection Nanoject II (Drummond Scientific, Broomall, PA, USA) was used to absorb the liquid inside the organoids. 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 manner 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 0.1M sodium cacodylate buffer, and then incubated in 4% osmium tetroxide for 1 h. They were subsequently washed, then 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 with 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. Then, organoids were treated with 10 μM fluorescently labeled bile acid (CGamF; kindly donated by 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 fluorescein diacetate was treated into the organoids in the medium. Furthermore, to examine the direction of transport, fluorescein diacetate was injected into the organoids using a Nanoject III (Drummond Scientific). After treatment or injection of fluorescein diacetate, images were captured with a fluorescence microscope BZ-X710 (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 captured continuously using a fluorescence microscope BZ-9000 (Keyence). For the 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 consecutively using a fluorescence microscope BZ-X710. Analysis was performed by calculating the ratio of the intensity outside and inside the organoids 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 ultra-low attachment multi-well plates 6-well plates under each culture condition, organoids were picked and seeded on Microslide 8-well glass bottom (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 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), and pioglitazone (PIO, Sigma). After 30 min incubation, the 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. The arias and intensity of TMRM were calculated as MMP by IMARIS 8 (Bitplane AG, Switzerland). To evaluate cholestatic and mitochondrial stress, cell viability was measured by using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Mannheim, Germany) for each organoid at 24 hours after drug treatment, and viability was confirmed not to decrease at each dose to avoid secondary changes due to cell damage leading to cell death.

[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, an index was first established based on the values ​​obtained from the mitochondrial and cholestatic stress assays using the following formula: "index = - (sample value - control value) x 100". To analyze the cell damage associated with mitochondria and cholestatic stress, the 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 carried out as shown in Figure 5A. After being removed from Matrigel and washed, the organoids were treated with 800 μM oleic acid for 3 days on ultra-low attachment multi-well plates 6-well plates (Corning). They were then treated with 50 μM troglitazone in the presence or absence of 50 μM NAC for 24 h. Cell viability was performed using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega). Images were obtained continuously using a fluorescence microscope BZ-9000.

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

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

[0090] All percentages and ratios are calculated on a total composition basis 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 "about 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 to teach, suggest, or disclose any such invention, either alone or in combination with any other reference(s). Furthermore, if 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 control.

[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. An in vitro liver organoid comprising a luminal structure surrounded by polarized hepatocytes, said in vitro liver organoid comprising adherens zona.

2. An in vitro liver organoid as described in claim 1, wherein the in vitro liver organoid is derived from stem cells.

3. An in vitro liver organoid according to claim 1 or 2, wherein the stem cells are pluripotent stem cells (PSCs), induced pluripotent stem cells (iPSCs), or embryonic stem cells (ESCs).

4. An in vitro liver organoid as described in claim 2 or 3, wherein the stem cells are human iPSCs.

5. An in vitro liver organoid described in any one of claims 1 to 4, wherein the in vitro liver organoid expresses ABCB11 and Na+-taurocholate cotransporting polypeptide (NTCP).

6. An in vitro liver organoid as described in any one of claims 1 to 5, wherein the in vitro liver organoid comprises functional stellate cells and functional Kupffer cells.

7. An in vitro liver organoid as described in any one of claims 1 to 6, wherein the in vitro liver organoid comprises mesenchymal cells.

8. An in vitro liver organoid described in any one of claims 1 to 7, wherein the in vitro liver organoid comprises internalized microvilli.

9. An in vitro liver organoid described in any one of claims 1 to 8, wherein the in vitro liver organoid comprises bile production ability and / or bile transport activity.

10. An in vitro liver organoid according to any one of claims 1 to 9, characterized in that the in vitro liver organoid has one or more of the following: bile production ability, bile transport activity, complement factor H expression of at least 50 ng / mL / 1xe6 cells / 24 hours, complement factor B expression of at least 40 ng / mL / 1xe6 cells / 24 hours, C3 expression of at least 1000 ng / mL / 1xe6 cells / 24 hours, C4 expression of at least 1000 ng / mL / 1xe6 cells / 24 hours, fibrinogen production of at least 1000 ng / mL / 1xe6 cells / 24 hours, and albumin production of at least 1000 ng / mL / 1xe6 cells / 24 hours.

11. An in vitro liver organoid described in any one of claims 1 to 10, characterized in that the in vitro liver organoid has a total liver protein expression of at least 10,000 ng / mL 1xe6 cells / 24 hours.

12. An in vitro liver organoid according to any one of claims 1 to 11, wherein the in vitro liver organoid expresses one or more genes selected from PROX1, RBP4, CYP2C9, CYP3A4, ABCC11, CFH, C3, C5, ALB, FBG, MRP2, ALCAM, CD68, CD34, and CD31.

13. An in vitro liver organoid described in any one of claims 1 to 12, wherein the in vitro liver organoid comprises a drug-metabolizing cytochrome mutant.

14. An in vitro liver organoid as described in claim 13, wherein the drug-metabolizing cytochrome mutant is a CY2C9*2 mutant.

15. A method for screening for serious adverse events (SAEs), preferably liver failure and / or drug-induced liver injury (DILI), comprising a step of contacting a drug of interest with a liver organoid described in any one of claims 1 to 14.

16. The method of claim 15, comprising a step of measuring uptake and / or efflux of fluorescein diacetate (FD), wherein impairment of the efflux indicates that the drug may induce a serious adverse event.

17. The method of claim 15 or 16, wherein toxicity of the target drug is determined by measuring a parameter selected from mitochondrial membrane potential, measurement of ROS, hepatic mitochondrial swelling, and combinations thereof, and damage to the mitochondria indicates that the drug may induce serious adverse events.

18. A method according to any one of claims 15 to 17, the method comprising a step of analyzing organoid viability, wherein impairment of the determination of organoid viability indicates that the drug may induce a serious adverse event.

19. An in vitro liver organoid according to any one of claims 1 to 13 for use in treating an individual with liver damage.

20. The in vitro liver organoid of claim 19, wherein the liver disorder is selected from metabolic liver disease, end-stage liver disease, or both.

21. An in vitro method for testing a candidate compound on an individual, comprising contacting an in vitro liver organoid described in any one of claims 1 to 13 with the candidate compound.

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