Liver organoid composition and method of preparing 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 of related applications This application claims priority and interest to U.S. Provisional Patent Application No. 62 / 471,371, filed on 4 November 2016, and U.S. Provisional Patent Application No. 62 / 517,414, filed on 9 June 2016, the contents of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] The liver is a vital organ that performs many metabolic functions essential to life, including detoxification and coagulation of exogenous compounds, as well as the production of lipids, proteins, ammonium, and bile. In vitro reconstruction of patient livers may offer applications including regenerative therapy, drug discovery, and pharmacotoxicology research. 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 the failure of candidate drugs identified in early screening, and nearly one-third of drugs are recalled from the market due to such failures (Takebe and Taniguchi, 2014). The failure of drug candidates results in a tremendous loss of treatment opportunities for patients. Preclinical trials generally consist of in vitro evaluations as the primary efficacy screening to identify “hit” compounds, followed by in vitro and in vivo safety studies to assess metabolic and toxic mechanisms. This inefficiency can be explained by the virtually nonexistent physiologically relevant preclinical models with high throughput for evaluating drug-induced liver injury (DILI) in humans, and therefore there is an urgent need to develop in vitro human screening models to evaluate the enormous number of ever-growing compound libraries.

[0004] Primary hepatocytes are a highly polarized metabolic cell type, forming bile canaliculi structures with microvilli channels that separate peripheral circulation from the bile acid secretory pathway. The most upstream phase of DILI involves drug (or their reactive metabolites) detoxification by hepatocytes and excretion into bile canaliculi via transporters such as multidrug resistance-associated protein (MRP) transporters. This suggests the need to reconstruct these uniquely organized structures as key in vivo characteristics of hepatocytes to predict DILI pathology. However, as in the case of troglitazone, nefazodone, and tolcapone (https: / / livertox.nlm.nih.gov / index.html), there are considerable differences in drug toxicity profiles between current simplified culture models and in vivo physiology, involving the use of isolated primary human hepatocytes or hepatocyte lines. Therefore, while the determination of toxicological properties relies primarily on animals as an essential step in drug development, fidelity to human results is significantly lacking due to the substantial physiological differences between humans and animals (Leslie et al., 2007; Yang et al., 2014). Furthermore, although the development of idiopathic DILI (IDILI) is extremely rare, it nevertheless accounts for 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). In summary, there is a need for effective human cell models to screen compounds for testing the detoxification and excretion of proposed drugs.

[0005] Despite novel advances in methods for differentiating human hepatocytes from pluripotent stem cells (PSCs), clinical trials using human stem cells in a dish remain a "hype." Beyond drug screening for efficacy and / or toxicity, there is a need for hepatocyte models for use in bioprosthetic liver devices, for example, as a bridge for transplantation and as a bridge for precision (personalized medicine). This disclosure attempts to address one or more of the aforementioned needs in the art. The following are prior art documents related to the invention of this application (including documents cited in the international phase after the international filing date and documents cited when the application entered the national phase in other countries): (Prior art document) (Patent Document) (Patent Document 1) International Publication No. 2015 / 183920 (Patent Document 2) U.S. Patent Application Publication No. 2013 / 0189327 (Patent Document 3) U.S. Patent No. 2,393,917 (Patent Document 4) International Publication No. 2010 / 094694 (Patent Document 5) U.S. Patent Application Publication No. 2016 / 0237400 (Patent Document 6) International Publication No. 2017 / 048193 (Patent Document 7) International Publication No. 2017 / 153992 (Patent Document 8) International Publication No. 2015 / 183920 (Patent Document 9) International Publication No. 2017 / 048193 (Patent Document 10) International Publication No. 2016 / 056999 (Patent Document 11) International Publication No. 2011 / 116930 (Non-patent literature) (Non-patent document 1) Ramachandran et al. "In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells" PLOS One, 21 October 2015, Vol.10, No.10, p g.1-14. (Non-Patent Document 2) Ijpenberg et al. "Wt1 and retinoic acid signaling are essential for stellate cell development and liver morphogenesis" Developmental Biology.Vol 312, No. 1, Pg. 157-170. (Non-patent document 3) Thomas J. Cunningham et al. "Mechanisms of retinoic acid signaling and its roles in organ and limb development".Nat.Rev.Mol.Cell.Biol.Vol.16,No.2,Pg.110-123. (Non-patent document 4) Yuan Guan et al. "Human hepatic organoids for the analysis of human genetic diseases" JCI Insight.Vol.2, No.17, e94954. (Non-patent Literature 5) Richard Siller et al. "Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells" Stem Cell Reports. Vol.4, No.5, Pg.939-952 (Non-patent document 6) Takanori Takebe et al. "Vascularized and functional human liver from an iPSC-derived organ bud transplant" Nature, Vol. 499, No. 7459, Pg. 481-484. [Overview of the Initiative]

[0006] A method for inducing the formation of liver organoids from progenitor cells such as iPSC cells is disclosed. The 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. The disclosed liver organoids can also be used to treat individuals with liver damage or to identify preferred therapeutic agents. [Brief explanation of the drawing]

[0007] Those skilled in the art will understand that the drawings shown below are for illustrative purposes only. The drawings are not intended in any way to limit the scope of this instruction.

[0008] The patent or application file contains at least one drawing created in color. A copy of this patent or published patent application with the 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. Schematic of the differentiation method for human liver organoids. B. Phase-contrast image of human liver organoids. C. Immunostaining of albumin (ALB), collagen type IV (Collagen IV) and ZO-1 in organoids. Nuclei were stained with hematoxylin (blue). Scale 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 on days 7, 11, 20 and 30 of differentiation, and primary hepatocytes (PH). Relative expression levels were compared to undifferentiated iPSCs (for AFP, ALB, RBP4 and CK19), day 7 organoids (for HNF6) or day 11 organoids (for CYP3A4). Bars represent mean ± SD, n=3. E. Principal component analysis based on RNA sequencing data in undifferentiated iPS cells (iPSCs), definitive endoderm (DE), hepatic specified cells (HS), hepatic progenitor cells (HP), iPSC-derived cholangiocytes (iDC), normal human cholangiocytes (NHC), iPSC-derived posterior foregut (pFG), iPSC-derived human liver organoids, primary hepatocytes, fetal liver tissue, and liver tissue and right lobe of human liver. F. Secretion levels of albumin (ALB, n=10) and fibrinogen (FBG, n=4) from organoids on days 25 to 30. Bars represent mean ± SEM. G. Complement factor secretion levels from organoids on days 25 to 30. FH: Factor H, FB: Factor B. Bars represent mean ± SEM, n=5. [Figure 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. Transmission electron micrograph of an organoid showing the luminal surface of microvilli (V); N: nucleus. Bar, 10 μm. C. Quantitative RT-PCR of ATP-binding cassette subfamily B member 11 (ABCB11) and sodium taurocholate co-transporting polypeptide (NTCP) in undifferentiated iPS cells, organoids at day 20 of differentiation (NTCP) and day 30 of differentiation (ABCB11), and primary hepatocytes (PH). Relative expression values were compared against undifferentiated iPS cells (ABCB11) or day 11 organoids (NTCP). Bars represent mean ± SD, n=3. D. Total bile acid secretion level inside organoids at day 27. Bars represent mean ± SEM, n=4. E. Bile acid uptake by organoids after 30 minutes of culture in the presence of fluorescent bile acid (CGamF). F. CLF transport activity in organoids derived from four iPSC lines. T, W, 1, and F indicate clone names of iPS cell lines. Green: CLF. [Figure 3] Bosentan-induced cholestasis is specific to CYP2C9*2 iPSC-liver organoids. A. Representative allele images of rs1799853 in CYP2C9*2 and rs4148323 in UGT1A1*6, which represent risk SNPs for DILI induced by bosentan and irinotecan, respectively. This table shows the carrier rate of risk alleles in each iPS cell line. B. Images of CLF transport activity and its 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 the box plot, the top and bottom of the box represent the 75th percentile and 25th percentile, respectively, the central line represents the median. Dots show 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 organoids. B. Comparison of fluorescein diacetate efflux transport. C. Quantification of fluorescein diacetate efflux transport to organoids. The exemplary left figure shows the quantified ratio of fluorescein intensity between the inside and outside of organoids. The right figure shows the results of validation tests using a 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 hours of 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 with training compounds. Bars represent mean ± SD, *: p<0.05, **: p<0.01, n=4-5. CON: control sample, STP: streptomycin, TOL: tolcapone, DICLO: diclofenac, BOS: bosentan, CSA: cyclosporine. [Figure 5]High-fidelity drug-induced mitochondrial toxicity screening using organoids. A. Images of mitochondrial membrane potential (MMP) on TMRM after treatment with nine training compounds. Bottom panel: Quantification of transport inhibition after treatment with training compounds, bars represent mean ± SD, *: p<0.05, **: p<0.01, n=4-6. C. Quantification of MMP changes after treatment with training compounds, bars represent mean ± SD, *: p<0.05, **: p<0.01, n=4-5. CON: control sample, STP: streptomycin, TOL: tolcapone, DICLO: diclofenac, BOS: bosentan, CSA: cyclosporine A, TRO: troglitazone, NEFA: nefazodone, ENTA: entacapone, PIO: pioglitazone. B. Classification of the set of nine training compounds (TC) mentioned in Oorts et al., 2016. Class A represents TCs with known reports of drug-induced cholestasis (DILI) in vivo, while Class B represents TCs with reported drug-induced cholestasis in vivo. Mechanisms of toxicity based on literature data are also provided. Class C compounds are generally considered safe with respect to DILI. C. Analysis of 72-hour post-drug treatment survival rate and dual risk parameters: the likelihood of drug-induced cholestasis and the likelihood of mitochondrial toxicity. Cholestatic and mitochondrial toxicity (Mito-tox) indices were derived from the data in Figure 3. The size of the circle indicates the magnitude of the survival reduction. [Figure 6] Modeling of drug-induced liver injury in a vulnerable state rescued by NAC exposure. A. Overview of drug-induced cytotoxicity assessment of a vulnerable organoid model. B. Profiling of the vulnerable model for lipid accumulation (blue: nucleus, green: lipids, red: F-actin). C. ROS production (blue: nucleus, green: ROS) and D. Mitochondrial health (blue: nucleus, red: mitochondria). E. Image of organoids 24 hours after drug treatment. F. Survival assessment of a vulnerable 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] Screening based on multiple liver organoids for toxicity prediction. [Figure 8] Optimization of retinoic acid treatment protocol A. Planning of the timing and duration of retinoic acid treatment. RA: retinoic acid, HCM: hepatocyte culture medium. B. Albumin secretion levels in organoids at day 25 in RA treatments of different durations. [Figure 9] Morphology of organoids in D20. The total number of organoids in D20 was 305. Organoids with lumens: 216, organoids without lumens: 89. [Figure 10] Conversion formula for determining the number of cells in an organoid: A. Phase contrast image of a single organoid. B. Diameter and number of cells in each single organoid. C. Correlation between diameter and number of cells in a single organoid. [Figure 11] Supplement to Figure 4. Organoid generation from multiple PSC lines. Phase-contrast images and albumin secretion levels of organoids derived from different iPS cell lines (317D6 and 1383D6). [Figure 12] Cell viability 24 hours after treatment with 10 different compounds. Survival evaluation for a 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 production and morphological changes in lipotoxic liver organoids. A. Ratio of ROS-producing cells to total cells in a lipid accumulation-induced fragility organoid model induced 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: nucleus. C. Number and size of mitochondria for the fragile organoid model. Bars represent mean ± SD, *: p<0.05, n=5~6. [Figure 14] Schematic diagram of the cell Matrigel-free method. A schematic diagram of a liver organoid generation method that does not use Matrigel to generate organoids is shown. [Modes for carrying out the invention]

[0010] Unless otherwise specified, terms should be understood in accordance with their conventional usage by those skilled in the art.

[0011] The terms “approximately” or “about” mean, according to the determination of those skilled in the art, that a particular value is within an acceptable margin of error, depending, for example, on the limits of the measuring system, how its value is measured or determined. For example, “approximately” may mean within one or more standard deviations, according to the practice in the art. Alternatively, “approximately” may mean within 20%, 10%, 5%, or 1% of a given value. Or, particularly with respect to biological systems or biological processes, the term may mean within 10 times, preferably 5 times, and more preferably 2 times, a certain value. Where a particular value is described in this application and claims, unless otherwise specified, the term “approximately” should be assumed to mean within an acceptable margin of error for that particular value.

[0012] As used herein, the term “totipotent stem cell” (also known as omnipotent stem cell) refers to a stem cell capable of differentiating into embryonic and extraembryonic cell types. Such cells can construct a complete and viable organism. These cells are produced from the fusion of an egg cell and a sperm cell. Cells produced by the first few divisions of a fertilized egg are also totipotent.

[0013] As used herein, the term “pluripotent stem cells (PSCs)” encompasses any cells capable of differentiating into any of the body’s nearly all cell types, namely any cells derived from any of the three germ layers (embryonic epithelium), including the endoderm (stomach wall, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, genitourinary tract), and ectoderm (epidermal tissue and nervous system). PSCs may be descendants of inner cell mass cells of a pre-implantation blastocyst, or they may be obtained by inducing non-pluripotent cells, such as adult somatic cells, by forcing the expression of specific genes. Pluripotent stem cells may originate from any suitable source. Examples of sources for pluripotent stem cells include mammalian sources, including humans, rodents, pigs, and cattle.

[0014] As used herein, the term “induced pluripotent stem cells (iPSCs)” is commonly abbreviated as iPS cells and refers to a type of pluripotent stem cell artificially induced from normally non-pluripotent cells, such as adult somatic cells, by inducing the “forced” expression of specific 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 pluripotent cells derived from the inner cell mass of a blastocyst, which is an early embryo. For the purposes of this invention, the term “ESC” is used more broadly to also include embryonic germ cells, as may be the case.

[0016] As used herein, the term “progenitor cell” encompasses any cells that may be used in the methods herein, in which one or more progenitor cells acquire the ability to regenerate themselves or to differentiate into one or more specialized cell types. In some embodiments, progenitor cells are pluripotent or capable of becoming pluripotent. In some embodiments, progenitor cells are subjected to treatment with an extrinsic factor (e.g., a growth factor) to acquire pluripotency. In some embodiments, progenitor cells may be totipotent or omnipotent stem cells; pluripotent stem cells (inducible or uninducible); multipotent stem cells; oligopotent stem cells and monopotent stem cells. In some embodiments, progenitor cells may be derived from an embryo, infant, child, or adult. In some embodiments, progenitor cells may be somatic cells that are treated such that pluripotency is conferred 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 “targeted differentiation” refers to the process by which less specialized cells become a specific specialized target cell type. The specificity of the specialized target cell type can be determined by any applicable method that can be used to define or alter the fate of the initial cell. Exemplary methods include, but are not limited to, genetic engineering, chemical treatment, protein treatment, and nucleic acid treatment.

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

[0019] Further stem cells that may be used in embodiments of the present invention include, but are not limited to, those provided by or listed in the databases of the National Stem Cell Bank (NSCB), 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, California); Cellartis AB (Goteborg, Sweden); ES Cell International Pte Ltd (Singapore); Technion at the Israel Institute of Technology (Haifa, Israel); and the Stem Cell Databases held by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that may be used in embodiments of 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, for example, in Thomson et al., 1998, "Embryonic Stem Cell Lines Derived from Human Blastocysts," Science 282(5391):1145-1147; Andrews et al., 2005, "Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin," Biochem Soc Trans 33:1526-1530; Martin 1980, "Teratocarcinomas and mammalian embryogenesis," Science 209(4458):768-776; Evans and Kaufman, 1981, "Establishment in culture of pluripotent cells from mouse embryos," Nature 292(5819):154-156; Klimanskaya et al., 2005, "Human embryonic stem cells derived without feeder Cells can be found in "Lancet 365(9471):1636-1641," but each of those descriptions is incorporated herein by reference in its entirety.

[0021] Induced pluripotent stem cells (iPSCs) In some embodiments, iPSCs are induced by transfection of non-pluripotent cells, such as adult fibroblasts, with specific stem cell-related genes. Transfection is typically achieved through a viral vector, such as a retrovirus. Transfected genes include the master transcription factors Oct-3 / 4 (Pouf51) and Sox2, but other genes have been suggested to enhance 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 a lentiviral system. Genes whose expression is induced in iPSCs include, but are not limited to, Oct-3 / 4 (e.g., Pou5f1); specific members of the Sox gene family (e.g., Sox1, Sox2, Sox3, and Sox15); specific members of the Klf family (e.g., Klf1, Klf2, Klf4, and Klf5); specific members of the Myc family (e.g., C-myc, L-myc, and N-myc); Nanog, and LIN28.

[0022] In some embodiments, non-viral techniques are used to produce iPSCs. In some embodiments, adenoviruses are used to deliver four required genes to the DNA of mouse skin and liver cells, resulting in cells identical to embryonic stem cells. Since adenoviruses do not incorporate any of their own genes into the target host, the risk of tumor formation is eliminated. In some embodiments, reprogramming can be achieved via plasmids without the use of any viral transfection system, although with very low efficiency. In other embodiments, iPSCs are produced using direct protein delivery, thus eliminating the need for viruses or genetic modification. In some embodiments, the production of mouse iPSC cells is possible using a similar methodology: repeated treatment of cells with specific proteins guided into the cell via polyarginine anchors was sufficient to induce pluripotency. In some embodiments, the expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions.

[0023] Further details regarding embryonic stem cells can be found in 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 These can be found in "Proteins," Cell Stem Cell 4(5):381-384; and each of these descriptions 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-DF4-3;iPS-DF6-9;iPS(precipitous);iPS(IMR90); and iPS(IMR90).

[0025] Further details regarding the function of signaling pathways related to DE development 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 This can be found in 60(7):1322–1332; and each of these descriptions is incorporated herein by reference in its entirety.

[0026] Any method for producing 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, pluripotent cells are derived from a morula. In some embodiments, pluripotent stem cells are stem cells. The 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 crest of an embryo. Embryonic stem cells or germ cells may be derived from various animal species, including, but not limited to, various mammalian species, including humans. In some embodiments, human embryonic stem cells are used to produce endoderm. In some embodiments, human embryonic germ cells are used to produce endoderm. In some embodiments, iPSCs are used to produce endoderm.Further methods for obtaining or producing DE cells that can be used in the present invention include, but are not limited to, those described in U.S. Patent No. 7,510,876 (D'Amour et al.); U.S. Patent No. 7,326,572 (Fisk et al.); Kubo et al., 2004, "Development of definitive endoderm from embryonic stem cells in culture," Development 131:1651-1662; D'Amour et al., 2005, "Efficient differentiation of human embryonic stem cells to definitive endoderm," Nature Biotechnology 23:1534-1541; and Ang et al., 1993, "The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3 / forkhead proteins," Development 119:1301-1315.

[0027] The applicant has found a method for fabricating a 3D liver structure using human iPSCs. This structure includes a microliver structure comprising polar hepatic epithelium, stellate cells, and tubule structures. The disclosed composition shows improvements in liver function, bile transport activity, and durability compared to existing models. The 3D structural model can be used as a novel and robust model for drug screening tests and / or drug toxicity screening, transplantation, serum protein product production, and the development of personalized therapies. In one particular application, the composition and method can be used to screen drug compounds for hepatotoxicity.

[0028] While 3D aggregated hepatocytes have been reported, the disclosed composition exhibits extremely high functional activity, including albumin production (up to 10-fold increase compared to conventional best-standard models using iPSC-derived hepatocytes), and allows for improved oxygen and / or nutrient delivery due to its internal lumen structure, thus enabling much longer culture times (at least 60 days) and a long-term testing platform useful for drug testing. The disclosed composition is useful for the production of plasma products such as albumin, a coagulation factor product for the treatment of hypoalbuminemia, and may also be useful for therapeutic transplantation, where a miniature liver derived from human iPSCs can be transplanted to treat disorders in vivo. Finally, the disclosed composition can be used in personalized medicine (personalized treatment).

[0029] In one embodiment, a method for inducing liver organoid formation from iPSC cells is disclosed. The method is as follows:

[0030] The procedure may include: a) contacting 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 factor (FGF) is a family of growth factors involved in angiogenesis, wound healing, and embryonic development. FGF is a heparin-binding protein, and its interaction with cell surface-associated heparan sulfate proteoglycans has been shown to be essential for FGF signaling. Suitable FGF pathway activators will be readily apparent to those skilled in the art. Exemplary FGF pathway activators include, but are not limited to, one or more molecules selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. In some embodiments, these pathways may be activated using siRNA and / or shRNA targeting cellular components associated with the FGF signaling pathway.

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

[0033] Appropriate GSK3 inhibitors will be readily apparent to those skilled in the art. Examples of GSK3 inhibitors, but not limited to, include Chiron / CHIR99021, which inhibits GSK3β. Those skilled in the art will recognize a suitable GSK3 inhibitor for carrying out the disclosed method. GSK3 inhibitors may be administered in amounts of approximately 1 μM to approximately 100 μM, or approximately 2 μM to approximately 50 μM, or approximately 3 μM to approximately 25 μM. Those skilled in the art will readily recognize appropriate doses and durations.

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

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

[0036] In one embodiment, liver organoids may be characterized by the expression of 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 may occur, for example, between day 40 and day 50. The expression levels may be similar to those observed in human hepatocytes, for example, adult hepatocytes.

[0037] In one embodiment, liver organoids may be characterized by having bile transport activity.

[0038] In one embodiment, the liver organoid may be derived from stem cells and may further include luminal structures containing internalized microvilli and mesenchymal cells. The luminal structures may be surrounded by polar hepatocytes and a basement membrane. The liver organoid may include functional stellate cells and functional Kupffer cells.

[0039] In one embodiment, liver organoids possess the following characteristics: bile production capacity, bile transport activity, and at least 50 ng / mL / 1xe 6 Cell / 24-hour complement factor H expression, at least 40 ng / mL / 1xe 6 Cells / 24-hour complement factor B, at least 1000 ng / mL / 1xe 6 Cell / 24-hour C3 expression, at least 1000 ng / mL / 1xe 6 Cell / 24-hour C4 expression, at least 1000 ng / mL / 1xe 6 Cellular fibrinogen production per 24 hours, and at least 1000 ng / mL / 1xe 6It may be characterized by having one or more of the following: cell / 24-hour albumin production. In one embodiment, the liver organoid has at least 10,000 ng / mL 1xe 6 The liver organoids may be characterized by having total liver protein expression per cell / 24 hours. The liver organoids 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, and CD31. In one embodiment, the liver organoids may include cells containing drug-metabolizing cytochrome mutants, such as the CY2C9*2 mutant. The liver organoids may include vascular organoids, such as those described in US 2016 / 0177270.

[0040] In one embodiment, the liver organoid may be characterized by the absence of inflammatory cells, such as T cells or other inflammatory secretory proteins.

[0041] In one embodiment, a method for screening for serious adverse events (SAEs) is disclosed. SAEs may be hepatic failure and / or drug-induced liver injury (DILI). The method may include the step of bringing a drug of interest, for which toxicity is the objective, into contact with liver organoids described herein. In one embodiment, the method may include the step of measuring the ingestion and / or efflux of fluorescein diacetate (FD), where impaired efflux indicates that the drug may induce a serious adverse event. The toxicity of the drug of interest may be determined by measuring parameters selected from mitochondrial membrane potential, ROS measurement, hepatic mitochondrial swelling, and combinations thereof, where damage to mitochondria indicates that the drug may induce a serious adverse event. In one embodiment, the method includes the step of analyzing organoid survival, where impaired or reduced organoid survival indicates that the drug of interest may induce a serious adverse event.

[0042] In one embodiment, a method for treating an individual with liver damage is disclosed, which may include the step of transplanting a liver organoid described herein into an individual in need. Liver damage may include, for example, metabolic liver disease, end-stage liver disease, or a combination thereof.

[0043] In one embodiment, a method for identifying a preferred therapeutic agent for an individual is disclosed. In this embodiment, the method may include the step of contacting a liver organoid derived from an iPSC of interest with a candidate compound, where, for example, the iPSC of interest contains one or more mutations found in such individual, or, for example, the iPSC of interest originates from the same ethical background as such individual, or furthermore, the iPSC of interest is derived from such individual. [Examples]

[0044] In this study, the applicant tested bile transport activity using fluorescein diacetate effluxed into the bile canal network by MRP2 across the bile canal membrane (Tian et al., 2004). Inhibition of MRP2 by troglitazone and cyclosporine has been previously reported (Chang et al., 2013; Lechner et al., 2010). Furthermore, the efflux transporter MRP2 mediates the transport of bosentan (Fahrmayr et al., 2013). While inhibition of MRP2 by nefazodone has not been reported, mitochondrial stress induced by nefazodone may be associated with reduced bile transport activity and decreased fluorescein diacetate efflux, as MRP2 is an ATP-dependent bile salt transporter for bile acid efflux in hepatocytes.

[0045] Preclinical detection of drug-induced liver injury (DILI) risk compounds remains a critical challenge in drug development, highlighting the need for predictive human systems. Here, the applicant developed a human liver organoid (HLO) model for analyzing clinical DILI pathology at organoid resolution. Differentiated HLOs from human iPSCs contain polar hepatocytes with lumens lined with canaliculi-like structures, establishing a unidirectional bile acid transport pathway. The applicant leveraged the structural features of organoids by modeling DILI using liver organoid imaging called LoT (Liver Organoid-Based Toxicity Screening). LoT is functionally validated in 10 commercially available drugs and 5 different donors based on cholestatic and / or mitochondrial toxicity. Bosentan-induced cholestatics are specific to HLOs derived from CYP2C9 low-metabolism donors. Interestingly, steatotic organoids were vulnerable to rosiglitazone toxicity as suggested in the clinic, followed by chemical rescue from mass organoid deaths. Therefore, LoT is a high-fidelity organoid model that can be used to analyze drug safety, is also a cost-effective platform that facilitates compound optimization, provides mechanistic research, and leads to personalized medicine and anti-DILI treatment screening applications.

[0046] In the pharmaceutical industry, billions of dollars are lost annually from drug development due to the failure of candidate drugs identified in early screening, and many (one-third) of drugs are recalled from the market due to such failures (Takebe and Taniguchi, 2014). Despite promising efficacy, the failure of drug candidates results in a tremendous loss of treatment opportunities for patients. Preclinical trials generally consist of in vitro evaluations as the primary efficacy screening to identify “hit” compounds, followed by in vitro and in vivo safety studies to assess metabolic and toxic mechanisms. This inefficiency can be explained by the virtually nonexistent preclinical models for evaluating drug-induced liver injury (DILI) in humans, and therefore there is an urgent need to develop in vitro human screening models to evaluate the enormous number of ever-growing compound libraries.

[0047] Primary hepatocytes are a highly polarized metabolic cell type, forming bile canaliculi structures with microvilli channels that separate peripheral circulation from the bile acid secretory pathway. The most upstream phase of DILI involves drug (or their reactive metabolites) detoxification by hepatocytes and excretion into bile canaliculi via transporters such as multidrug resistance-associated protein (MRP) transporters. This suggests the need to reconstruct these uniquely organized structures as key in vivo characteristics of hepatocytes to predict DILI pathology. However, as in the case of troglitazone, nefazodone, and tolcapone (https: / / livertox.nlm.nih.gov / index.html), there are considerable differences in drug toxicity profiles between current simplified culture models and in vivo physiology, involving the use of isolated primary human hepatocytes or hepatocyte lines. Therefore, while the determination of toxicological properties relies primarily on animals as an essential step in drug development, fidelity to human results is significantly lacking due to the substantial physiological differences between humans and animals (Leslie et al., 2007; Yang et al., 2014). Furthermore, although the development of idiopathic DILI (IDILI) is extremely rare, it nevertheless accounts for 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). In summary, effective human cell models are highly desirable for screening compounds to test the detoxification and excretion of proposed drugs.

[0048] Despite groundbreaking advances in methods for differentiating human hepatocytes from pluripotent stem cells (PSCs), clinical trials using human stem cells in a dish remain a "hype." To some extent, this can be attributed to challenges in previous cell-based approaches, including (1) overcoming lot-to-lot variability, (2) minimizing experimental batch variability, (3) improving assay throughput, and (4) improving relevance to clinical trial data. The applicant addresses these issues by developing a relatively simple and robust organoid-based testing platform using stably scalable human stem cells, i.e., iPSCs. The applicant first directed human PSCs into posterior foregut organoids and continued gradual hepatocyte differentiation through polarized culture with defined factors and matrices. The resulting human liver organoids have been shown to possess intraluminal structures surrounded by polarized hepatocytes and to be capable of performing important human hepatocyte functions, including protein and bile acid production and transport. Interestingly, the applicant found that dynamic detection of fluorescent diacetate uptake and excretion based on live imaging accurately models cholestasis induced by a range of DILI drugs characterized as inhibitors of bile excretion, with a high level of reproducibility. Separately, mitochondrial membrane potential assessment enabled independent risk assessment for each compound, reflecting the conventional classification of DILI drugs established by clinical trials. Furthermore, the applicant extended the approach to model conditions induced by lipotoxic stress, confirming the enhancement of the potential for DILI by reactive oxygen species (ROS) production. Survival assessment based on organoids confirmed the reversal of DILI by N-acetylcysteine, highlighting the potential of our approach for anti-DILI drug screening. Taken together, this robust assay, called liver organoid-based toxicity screening (LoT), is considered the first functional readout developed in human liver organoids, facilitating diagnosis, functional research, drug development, and personalized medicine.

[0049] result Generation and Characterization of Polar Liver Organoids from Multiple Human iPSCs The applicant first established a novel method for liver organoid differentiation by using foregut spheroids derived from human iPSCs (Spence et al., 2011) (Figure 1A). As a first step, the applicant used BMP and activin A to promote differentiation into endoderm, as previously described (D'Amour et al., 2005). Furthermore, foregut spheroids were induced using FGF4 and GSK3 inhibitors (CHIR99021), and budding spheroids were observed. After detaching mesenchymal cells seeded on a dish by gentle pipetting, the organoids were embedded in Matrigel. Retinoic acid (RA) has been reported to enhance cell polarity, as indicated by increased size and complexity of bile canaliculi and peritubulosus sheaths (Falasca et al., 1998). To generate polar 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. Albumin secretion levels of organoids were 1160, 1054, 3092, 4709, and 3865 ng / mL at D25 for 1, 2, 3, 4, and 5 days of RA treatment, respectively, with the 4-day RA treatment protocol tending to reach the highest levels (Figure 8). Therefore, based on the level of albumin secretion, the duration of RA was set to 4 days. Morphologically, approximately 10 days after RA treatment, more than 300 organoids covered with epithelial cells were successfully generated, with a proportion of organoids having tubular structures of 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 (adhesion occlusion) stained the inner luminal layer, suggesting that these organoids possess polar properties (Figure 1, Panel C).

[0050] Quantitative polymerase chain reaction (qPCR) analysis revealed that the expression of liver marker genes, including alpha-fetoprotein (AFP), albumin (ALB), retinol binding protein 4 (RBP4), cytokeratin 19 (CK19), hepatocyte nuclear factor 6 (HNF6) that regulates cholangiocyte differentiation, and cytochrome P450 3A4 (CYP3A4) during differentiation, was significantly increased in organoid cells (Figure 1, Panel D). However, the expression level of the most hepatic genes extracted from bulk organoid-derived RNA was lower in organoids than in primary hepatocytes. Without wishing to be bound by theory, these differing mRNA profiles are thought to be partly due to the presence of stromal lineages, since approximately 30% of cells identified by stromal cell markers are non-parenchymal cells, which makes organoids closer to in vivo liver tissue than primary hepatocytes. Applicants further profiled organoids by comprehensive gene expression analysis using RNA sequencing (RNA-seq). Principal component analysis showed that gene expression in organoids was not similar to that of iPSC-derived cholangiocytes and normal human cholangiocytes (Figure 1, Panel E). Furthermore, hepatocyte-specific proteins such as ALB, fibrinogen (Fbg) and complement factors were confirmed in the culture supernatant by ELISA (Figure 1, Panels F-G). To quantify the hepatic functionality of organoids, applicants examined albumin secretion levels normalized by cell number (Figure 10). Albumin secretion level was 2133 ng / day per 10 6 cells (Figure 1, Panel F), which is higher than other experiments on 2D and 3D differentiation of hPSCs into HLCs related to published iPSC-derived hepatocytes (Miki et al., 2011; Song et al., 2015; Song et al., 2009; Vosough et al., 2013) (150-1000 ng / day per 10 6 cells), whereas primary hepatocytes have 30-40 μg / day per 10 6Cells are produced (Davidson et al., 2016; Dvir-Ginzberg et al., 2003). These results demonstrate that, in the published literature, the liver organoids contain hepatocytes with reasonable albumin-secreting activity compared to stem cell-derived hepatocytes. 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 that possess albumin-secreting ability (Figure 11). In summary, the applicant has established a protocol for generating a large number of polar liver organoids with hepatocyte characteristics.

[0051] Microanatomical characterization of bile acid-producing human iPSC-liver organoids Next, to test whether the liver organoids possess bile transport activity, the applicant first characterized the organoids by staining the major proteins involved in bile synthesis and excretion. Immunofluorescence staining of BSEP and MRP2 demonstrated that these proteins preferentially localize to the intraluminal region (Figure 2, Panel A). The bile duct is the smallest hepatic endocrine channel, and the bile canaliculi 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 demarcated by the collisiobyl complex, and the microvilli are located inside the bile canaliculi lumen (Tsukada et al., 1995). ZO-1 staining is known to stain the bile canaliculi region of the liver, and Figure 1, Panel C, suggested that the collisiobyl complex is located inside our liver organoid. Transmission electron microscopy revealed that the organoids contained microvilli directed toward the lumen (Figure 2, Panel B). Consistent with these anatomical features, qRT-PCR analysis revealed that the organoids expressed genes for ABCB11 and Na+-taurocholic acid cotransport polypeptide (NTCP), but at lower levels in the organoids than in primary hepatocytes (Figure 2, Panel C). Thus, the organoids contained polar human hepatocytes separated from the lumen by adhesion ridges, reflecting a unique microanatomical structure that mimics in vivo hepatic bile canaliculi.

[0052] Next, to determine bile acid (BA) production capacity, the applicant performed ELISA on intraluminal fluid collected from organoid cultures. The total BA pool level in the intraluminal fluid was 26.7 μg / day / 10 6 The BA concentration was approximately 125 μmol / L in a 200 μm diameter organoid (Figure 2, Panel D), and surprisingly, the BA concentration was similar to that of primary hepatocytes derived from sandwich cultures in a previous report (Ni et al., 2016) (approximately 40 μg / day / 10 6The concentration was comparable to that of cells (10 μmol / L in the culture supernatant). Thus, the organoids not only possess a morphology similar to bile canales, but also exhibit bile acid production and secretion activity, suggesting that the bile acid transport pathway is 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 can lead to biliary secretion disorders (cholestasis) associated with various liver disease pathologies (Nishida et al., 1991). Efflux transport proteins located in the apical (bile canal) membrane of hepatocytes play a crucial role in the hepatic removal of many endogenous and exogenous compounds, including drugs and metabolites (Kock and Brouwer, 2012). BSEP and MRP2 mediate bile salt transport in bile canales in humans. After demonstrating positive expression of key proteins for bile transport, the applicant then considered whether organoids could actively transport bile acids into their lumen. Firstly, to investigate bile acid uptake into organoids, the applicant challenged organoids with cholylglycyrrhizamide-fluorescein (CGamF), a bile salt analog (Mork et al., 2012). Following external treatment with CGamF, the accumulation of CGamF in the lumen of organoids was successfully confirmed (Figure 2, Panel E). Similarly, the fluorescent bile acid choryl-lysyl-fluorescein (CLF) was reproducibly excreted and found to accumulate in organoids from multiple human iPSC strains (Figure 2, Panel F). To determine the specificity of this assay, the applicant developed iPSC strains with BSEP-unfunctionalized alleles 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. In summary, these data suggest that organoids have the ability to take in bile acids from the outside and excrete them into the organoid interior.

[0054] CYP2C9*2 iPSC-liver organoid-specific bosentan-induced cholestasis To test the clinical relevance of an organoid-based method for determining cholestatic phenotyping, the applicant employed pharmacogenetic insights into our system to address fidelity issues. Specifically, multiple iPSC strains possessing well-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 was compared in the presence of bosentan (Figure 3, Panel B). Interestingly, CLF excretion into organoids was significantly impaired in CY2C9*2-possessing organoids but not in non-possessing organoids. This is consistent with the clinical tendency toward bosentan-induced cholestasis shown in three different iPSC-derived organoids in the absence of CYP2C9*2, as shown in Figure 3, Panel C. In contrast, irinotecan-based cholestasis was not specific to CYP2C9*2 iPSC strains. These results demonstrate that organoid-based cholestasis assays predict several aspects of human diversity.

[0055] High-throughput drug-induced cholestasis evaluation in organoids Given the crucial role of cholestasis in drug-induced DILI, the applicant then considered whether this organoid model would reflect the pathology of DILI in the presence of specific compounds. Before testing numerous compounds, the applicant first attempted to develop a high-throughput fluorescence-based assay, as both CLF and CGamF were unsuitable for high-speed imaging due to several issues: 1. strong background requiring a manual washing process; 2. weak signal intensity requiring careful acquisition setup. Alternatively, the use of fluorescein diacetate (FD), which has been reported as 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 from the cell into the canal lumen (Malinen et al., 2014). To determine whether FD could be used for a raw assessment of transport capacity without medium changes or exposure adjustments, the hepatobiliary transport activity over time was further investigated using slow-speed imaging. Organoids were incubated with fluorescein diacetate for 45 minutes, and intraluminal accumulation was observed inside the organoids 20 minutes post-treatment (Figure 4, Panels A and 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 high-throughput potential for evaluating unidirectional bile efflux transport in liver organoids by simple fluorescence live imaging analysis.

[0056] Next, the applicant validated the fidelity of the FD-based assay by evaluating viable doses of 10 FDA-approved drugs and measuring secondary damage due to cell injury. The applicant successfully found optimal doses for nine compounds with acceptable viability. In contrast, amiodarone (AMIO) was significantly toxic to organoids within the tested range and was therefore excluded from further potential DILI evaluation tests (Figure 12). The applicant used FD with nine training compounds (TC) classified as one of three types based on the 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 investigate the potential for cholestasis in organoids. To quantify the potential for inhibition of FD excretion, the applicant developed a simple but robust quantification method by determining the fluorescence intensity ratio between the outer and inner parts of the organoid using image J (Figure 4, Panel B). As a validation study, the applicant first confirmed the ability to assess the inhibition rate using cyclosporine A (CSA). At 5 minutes after FD treatment, a significant reduction (0.4 compared to control) was observed in the group treated with CSA for 24 hours compared to the control (DMSO) (Figure 4, Panel B). Next, the applicant screened nine TCs at multiple concentrations to assess the fidelity of this approach. Interestingly, in this screening system, at 24 hours after TC treatment, FD excretion was significantly reduced with class B compounds, bosentan, CSA, troglitazone, and nefazodone (p<0.01 or 0.05), similar to clinical observations, while no inhibitory effect was observed with class A and class C compounds (image above Figure 4, Panel D and Figure 4, Panel E). These results suggest that liver organoid models are useful for classifying the bile transport inhibitory efficacy of candidate compounds in drug development that are highly correlated with human phenotypes.

[0057] Evaluation of mitochondrial overload in organoids Furthermore, since mitochondrial toxicity plays a central role in DILI through multiple mechanisms associated with its development (Pessayre et al., 2012), the applicant investigated mitochondrial health assessment. In this study, to examine mitochondrial health in organoids, MMP in intact cells was monitored using mitochondrial membrane potential (MMP) as an indicator by directly reading mitochondrial health (Li et al., 2014). After 24-hour TC treatment, dose-dependent increases in MMP were observed with treatment of tolcapone (2-8x change, p<0.01), diclofenac (7-13x change, p<0.05 or 0.01), CSA (3-7x change, p<0.01), and nefazodone (4-42x change, p<0.01) (Figure 5, image and graph below panel A). Furthermore, although dose-dependent, troglitazone also increased MMPs in organoids (3- to 5-fold change, p<0.05). In contrast, after treatment with bosentan, entacapone, and pioglitazone, no clear increase in MMPs was observed, even with multiple doses. These results demonstrate that this live-image-based assay, named Liver Organoid-Based Toxicity Screening (LoT), can distinguish compounds with or without mitochondrial toxicity.

[0058] Re-examination of the mechanistic classification of DILI compounds using the LoT system Severe symptom development in human DILI is multifactorial and highly correlated with combinations of drug effects particularly relevant to known mechanisms of DILI, such as mitochondrial and BSEP inhibition (Aleo et al., 2014). However, current in vitro functional models struggle to assess such multifactorial contributions. Considering the advantages of multiplexed biological functional readout in LoT systems, the applicant attempted to analyze the relationships between survival, cholestatics, and mitochondrial stress. Notably, drugs with dual effects (cholestasis and mitochondrial stress) at 24 hours, such as CSA, TRO, and NEFA, significantly reduced cell viability at 72 hours compared to TOL, DICLO, and BOS. These data are comparable to clinical data showing a strong correlation between dual toxicity and DILI severity and are consistent with previous reports (Aleo et al., 2014) (Figure 5, Panels B and C). Furthermore, the applicant also noted that entacapone treatment at 130 μM reduced organoid survival (from 85% at 24 hours to 64% at 72 hours). Entacapone requires extensive binding to plasma proteins, primarily albumin, to induce DILI (Fisher et al., 2002). However, based on available methods, how entacapone is toxic to the liver remains unknown (Oorts et al., 2016). In summary, the LoT system is a favorable human model system for classifying the major mechanisms of DILI and a useful experimental platform for further detailing unknown and complex mechanisms.

[0059] Evaluation of vulnerability to DILI in human liver organoids The incidence of DILI is known to be often complicated by numerous host factors. Indeed, there is growing evidence that the hepatotoxic risk from certain drugs, such as acetaminophen, is greatly increased in both rodents and humans due to obesity and NAFLD (APAP) (Fromenty, 2013; Michaut et al., 2016). Therefore, it is important to predict the possibility of DILI in patients who are in such a “vulnerable” state, even at the asymptomatic stage. In this study, the applicant established a lipotoxic organoid model by co-exposure to the unsaturated fatty acid, oleic acid (Figure 6, Panel A). Three days after oleic acid treatment of organoids, lipid accumulation in the organoids became severe (Figure 6, Panel B). Oxidation of fatty acids is a significant source of reactive oxygen species (ROS), which leads to the depletion of ATP and nicotinamide dinucleotides and induces DNA damage in fatty liver (Browning and Horton, 2004). Consistent with this, ROS production was observed in lipid-treated organoids (Figure 6, Panel C and Figure 13, Panel A). Furthermore, fatty acids induced massive hepatic mitochondria swelling, similar to the published phenotype (Figure 6, Panel D and Figure 13, Panel B) (Zborowski and Wojtczak, 1963). Since hepatic mitochondrial dysfunction precedes the development of NAFLD in rat models (Rector et al., 2010), these results suggest that hepatotoxic organoids model a fatty liver model to some extent in vivo.

[0060] Recognizing this lipotoxic organoid model as a vulnerable state with enhanced ROS production, troglitazone (0–50 μM) was treated for 24 hours, and cell viability in organoids was evaluated. Treatment with 50 μM troglitazone alone resulted in a cell viability of 85% at 24 hours, while it decreased to 67% at 72 hours. However, after troglitazone treatment for the lipotoxic state, significant organoid fragmentation was observed due to organoid death. This result was confirmed by subsequent cell viability analysis (approximately 40% compared to control, p<0.05) (Figure 6, panels E and 6, panel F).

[0061] Next, the applicant investigated whether organoids could be restored from a DILI-like state by potential therapeutic compounds. Since intravenous NAC improved survival in patients with acetaminophen-independent acute liver failure (Lee et al., 2009) and reduced troglitazone-induced cytotoxicity (Rachek et al., 2009), the applicant used N-acetylcysteine ​​(NAC) and antioxidants to inhibit ROS production. As expected, cell viability was significantly improved by NAC, suggesting that NAC rescued cell death in organoids even under fragile conditions (Figure 6, panels 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 multidrug regimens and drug discovery for treating DILI.

[0062] Serious adverse events (SAEs), including hepatic failure, are a major cause of drug reductions in clinical development or discontinuation of marketed drugs. DILI, in particular, is a significant challenge in drug development, where drug-induced cholestasis induced by transporter activity inhibition is a major cause. Sandwich cultures using primary human hepatocytes are currently the best choice for drug development. Recent reports have shown the potential of hepatocyte-based cholestasis models using differentiated human fibroblasts (Ni et al., 2016), however, these assay platforms still face reproducibility and throughput challenges due to the diverse and limited human hepatocyte sources, as well as the need for complex quantification algorithms. Furthermore, while HepaRG cells, a human hepatocellular carcinoma cell line, are also useful for evaluating cholestasis features, their use is limited due to their low BSEP (bile salt efflux pump, or ABCB11, a key transporter for bile acid efflux, and a major target of cholestatic agents) activity and time-consuming differentiation procedures (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 enables a simple, robust, and high-throughput system for measuring bile transport activity by live fluorescence imaging in the presence of the test compound. The main advantages of the LoT assay include: 1. Cost-effectiveness ($12.35 per 50 organoids, $94.85 per 384 wells), 2. Assay throughput (measurable in a single organoid), and 3. Multiple readouts for analyzing interactions between other factors such as mitochondrial stress. In particular, as mentioned above, retrospective studies have shown that multiple cellular stress possibilities are associated with the incidence of DILI (Aleo et al., 2014), as cell viability decreased in a dual readout; dependent on mitochondrial and cholestatic stress, and the LoT assay is comparable to the results obtained using this study. Oxidative stress plays a significant role in cell death and has been linked to the development of cholestatic liver injury (Serviddio et al., 2004).Hydrophobic bile acids accumulate intracellularly during cholestasis, disrupting normal mitochondrial electron transport, inhibiting the activity of respiratory complexes I and III, and consequently reducing adenosine triphosphate synthesis (Krahenbuhl et al., 1994), leading to mitochondrial dysfunction-induced apoptosis (Bernardi, 1996). Consistent with these findings, the applicant's correlation analysis of these dual readouts, as shown in Figure 5, indicated that cholestatic stress is a more dominant factor for liver damage compared to mitochondrial stress. Therefore, the LoT system can be used as a model system for investigating the DILI mechanism.

[0063] Furthermore, given the recent establishment of iPSC panel populations, a potential assessment of individual susceptibility is also promising (Inoue et al., 2014). While conventional in vitro assay systems generally do not focus on individual variability, SAEs often occur in small, quasi-patient populations susceptible to SAE (Stevens and Baker, 2009). Applying the LoT system to diverse population iPSC panels can provide insights into divergent susceptibility to SAE that were previously unattainable. Given the extremely rare nature of DILI, the use of cells from patients with specific genomic or ethnic factors would help elucidate the currently unknown idiosyncratic mechanisms of DILI. Therefore, LoT could serve as a game-changing strategy for the pharmaceutical industry by providing essential insights to minimize the likelihood of DILI (Figure 7).

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

[0065] Study of vulnerable human liver status using LoT assays Host factors such as obesity are known to significantly influence the development of DILI (Heidari et al., 2014), but their complex nature often prevents them from being fully understood in clinical practice. 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 can become hepatotoxic at lower doses in the presence of risk factors (Fromenty, 2013). Nevertheless, current clinical trial systems are not designed to stratify volunteers under vulnerable liver conditions at the level of a handful of biomarkers (ALT, AST). Since the number of patients with steatosis is asymptomatic and undetectable by biomarkers before drug administration, it is crucial to predict outcomes under this vulnerable condition before entering the clinical stage.

[0066] To assess toxicity under these vulnerable conditions during the early stages of drug screening, such as lead compound generation / optimization, the applicant has made efforts to develop a LoT system and applied lipotoxic stress to liver organoids, demonstrating a significant synergistic effect of the antidiabetic drug troglitazone on DILI. Indeed, the organoid system reflects this characteristic well by exhibiting massive hepatocyte death facilitated by the accumulation of triglycerides in hepatocytes within the organoids. One mechanism of DILI in obesity can explain decreased glutathione (GSH) levels (Michaut et al., 2016). Drug-induced oxidative stress can have several causes, particularly GSH depletion and inhibition of the mitochondrial respiratory chain (Begriche et al., 2011; Pessayre et al., 2010). The vulnerable model may reflect decreased intracellular GSH levels and exacerbation of troglitazone-induced oxidative stress via mitochondrial dysfunction improved by providing NAC. Given the dramatic increase in the incidence of non-alcoholic steatohepatitis (NASH), it is worth noting that there is still a minimal list of drugs that exacerbate pre-existing NAFLD or more frequently induce acute hepatitis. Furthermore, in vitro reduction systems offer a previously unforeseen window for studying previously untested host factors, as isolated host factors can be effectively deployed into organoids.

[0067] Lot-based precision medicine From the perspective of personalized medicine, the selection of optimal pharmacotherapy using LoT will be a major clinical concern. For example, strategies to consider in the selection of antipsychotics must take liver resistance into account, given the non-negligible incidence of liver damage in the psychiatric treatment population; 16% of possible DILI drugs are neuropsychotropic drugs (Dumortier et al., 2002). Given that NASH is often accompanied by psychological disorders such as depression, safer combinations of antidepressants (tricyclic or SSRIs), mood stabilizers, and neuroleptics are needed (Dumortier et al., 2002). Furthermore, due to the increasing prevalence of chronic conditions with age, polypharmacy (i.e., multiple drugs) is a common consequence of providing healthcare to the elderly (Marcum and Gellad, 2012), making it extremely difficult to identify the causative drug when DILI is suspected. Because patient-derived iPSC organoids provide an unlimited and reproducible source, LoT can serve as a panel for stratifying the potential for DILI in patients and provide information for selecting safer drugs from an individualized perspective.

[0068] Lot-based drug discovery for DILI Equally important is the potential use of LoT systems for screening anti-DILI therapeutic compounds. Many drugs have adverse effects on the liver and DILI, which is a significant clinical problem. In fact, acetaminophen accounts for about half of DILI cases in the United States (Russo et al., 2004). In other parts of the world, such as developing countries, other drugs, such as anti-tuberculosis drugs, may be the main cause of DILI (Bell and Chalasani, 2009). However, there are only a handful of symptomatic treatments available. Here, as a proof-of-concept experiment, the applicant established an organoid survival experiment to evaluate the therapeutic effect of compounds that resist the toxic mechanisms of DILI, as demonstrated by troglitazone. While NACs are the primary treatment option for paracetamol overdose (Makin et al., 1995; Verma and Kaplowitz, 2009), recently the focus of research has shifted to investigating the use of NACs in non-paracetamol DILI (Chughlay et al., 2016). The LoT system is useful for evaluating the efficacy of NACs against DILI with non-paracetamol drugs. Furthermore, this higher-throughput approach serves as a powerful tool for screening large compound libraries that restore DILI-like symptoms in vitro. The methods described herein can be combined to identify and study intracellular and exogenous factors associated with the clinical DILI phenotype, facilitating lead compound optimization, mechanism studies, and refinement medicine, as well as anti-DILI therapy screening applications.

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

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

[0071] DEs may be differentiated into liver organoids using three methods: 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, endoderm organoids containing plated cells were gently pipetteed and detached from the dish. The isolated spheroids were centrifuged at 800 rpm for 3 minutes, the supernatant was removed, and they were embedded in 100% Matrigel drops on the dish. The plates were placed in a 5% CO2 / 95% air atmosphere at 37°C for 5-15 minutes. After the Matrigel solidified, B27, N2, and 2 μM retinoic acid (RA; Sigma, St. Louis, MO) were added to Advanced DMEM / F12 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). The cultures for cell differentiation were maintained at 37°C in a 5% CO2 / 95% air atmosphere, and the medium was changed every 3 days. Around 20–30 days, organoids embedded in Matrigel drops were isolated by scratching and gentle pipetting for any analysis.

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

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

[0075] H&E staining and immunohistochemistry Liver organoids were collected from Matrigel, fixed in 4% paraformaldehyde, and then embedded in paraffin. Sections were subjected to H&E staining and immunohistochemical 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 at room temperature for 2 hours. The nuclei were stained with 10 μg / mL Hoechst The organoids were stained with 33342 (Sigma) at room temperature for 10 minutes, and then washed three times again with washing buffer. The specimens were observed under a fluorescence microscope or in bright field. For whole tissue immunohistochemical staining, liver organoids were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 2.5% Tween 20 (Sigma) at room temperature, and then incubated overnight at 4°C with the following primary antibody diluted in PBS: polyclonal anti-BSEP antibody (1:200 Sigma). The organoids were then treated with a fluorescent dye-conjugated secondary antibody, Alexa Fluor 568-conjugated donkey anti-rabbit immunoglobulin (IgG; 1:500; Invitrogen, A10042), at room temperature for 2 hours. 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]). The nuclei were stained with 10 μg / mL Hoechst The organoids were stained with 33342 (Sigma) at room temperature for 10 minutes, and then washed three times again with washing buffer. The specimens were observed under confocal imaging performed with a Nikon A1Rsi inverted confocal microscope.

[0076] RNA isolation, RT-qPCR RNA was isolated using the RNeasy mini-kit (Qiagen, Hilden, Germany). Reverse transcription was performed using the SuperScript III First-Strand System for RT-PCR (Invitrogen, CA, USA) according to the manufacturer's protocol. qPCR was performed on a QuantStudio 3 real-time PCR system (Thermo) using the TaqMan gene expression master mix (Applied Biosystems). 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 sequence data RNA isolation, cDNA synthesis, and sequencing on Illumina HiSeq 2500 have been previously described (Asai et al., 2017). RNA-Seq reads were aligned to the human genome (GRCh37 / hg19) using TopHat (version 2.0.13). Alignment data from TopHat was fed into the assembler Cufflinks (version 2.2.1) to assemble the 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 as the number of fragments per kilobase (FPKM) of exons per million mapped fragments.

[0078] To compare pHLO strains, the applicant combined in-house RNA sequence data (pFG and organoids) with pre-processed public data as follows: Transcript abundances of iPSC, DE, HS, HP, iDH, and NHC were obtained from GSE86007 (Jalan-Sakrikar et al., 2016); those from pediatric liver tissue, adult liver tissue, adult right lobe tissue, fetal liver tissue, and primary hepatocytes were encoded (ENCODE) (ENCFF418BVF, ENCFF804QWF, ENCFF965IQH, ENCFF918SJO, ENCFF367FJJ, ENCFF029IUF, ENCFF280YNO, ENCFF347TXW, ENCFF724CQI, ENCFF624LQL, ENCFF962SOD, ENCFF170AEC) (Consortium, 2012; Sloan et al.) The genes were obtained from al., 2016 and GSE85223 (Asai et al., 2017). After possible data preprocessing, genes were used if all datasets had the same gene symbol. The applicant performed quartile normalization of the FPKM+1 and RPKM+1 data in log2 space, and then selected genes within the top 10,000 of the median expression levels. Principal component analysis was performed 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 from organoids, 200 μL of the culture supernatant from organoids on ultra-low adhesion 96-well plates (Corning) was collected. The culture supernatant was collected and stored at -80°C until use. The supernatant was assayed using a human albumin ELISA quantification set (Bethyl Laboratories, Inc., TX, USA) and fibrinogen (Thermo Fisher Scientific) according to the manufacturer's instructions. To analyze complement factors, the supernatant was measured using the Luminex system (Luminex Corporation, Austin, TX) according to the manufacturer's instructions. A linear regression equation based on the cell number and organoid diameter was used to calculate albumin production per cell number. To measure the total bile acid secretion level of intraluminal organoids, the fluid inside the organoids was absorbed using a microinjector, Nanoject II (Drummond Scientific, Broomall, PA, USA). The absorbed liquid was diluted with PBS and then assayed using a total bile acid ELISA kit (Antibodies-online, Inc., GA, USA). To calculate the total bile acid volume, the number of cells in the organoids was calculated using a linear regression equation in the same manner as for albumin production, and the molecular weight of cholic acid was used to calculate the volume, which was then compared with previously reported volumes.

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

[0081] CGamF assay Briefly, the organoids were pre-incubated for 30 minutes with a 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, pH 7.4). Next, the organoids were treated with 10 μM fluorescently labeled bile acid (CGamF; donated by Dr. Hofmann) for 1 hour, and then washed three times with PBS. Images were obtained using a fluorescence microscope BZ-X710 (Keyence, Osaka, Japan).

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

[0083] Assessment of mitochondrial toxicity potential After culturing in ultra-low adhesion 6-well multi-well plates under various culture conditions, organoids were picked up and seeded on Microslide 8-well glass-bottom plates (Ibidi, WI, USA). To evaluate mitochondrial membrane potential (MMP), dimethyl sulfoxide (DMSO; Sigma) was used, followed by 24-hour treatment with negative controls including streptomycin (STP; Sigma), tolcapone (Tol; Sigma), diclofenac (Diclo; Sigma), bosentan (BOS; Sigma), cyclosporine A (CSA; Sigma), troglitazone (Tro; Sigma), nefadozone (Nefa; Sigma), entacapone (Enta; Sigma), and pioglitazone (PIO, Sigma), after which 250 nM tetramethylrhodamine, methyl ester, perchlorate (TMRM; Thermo Fisher Scientific) was added. After incubation for 30 minutes, 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 lens. The arithmetic activity (TMRM) and intensity were calculated as MMP using IMARIS 8 (Bitplane AG, Switzerland). To assess cholestatic and mitochondrial stress, cell viability was measured for each organoid 24 hours after drug treatment using the CellTiter-Glo® bioluminescent cell viability assay (Promega, Mannheim, Germany). It was confirmed that viability did not decrease at each dose to avoid secondary changes resulting from 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 mitochondrial and cholestatic stress, an index was first established based on values ​​obtained from mitochondrial and cholestatic stress assays using the following formula: "Index = -(Sample Value - Control Value) × 100". To analyze cellular damage associated with mitochondrial and cholestatic stress, ATP content per organoid was measured 72 hours after drug treatment using the CellTiter-Glo® luminescent cell viability assay (Promega). These data are shown as Figure 4, Panel B, using the free web-based tool Infogr.am (http: / / infogr.am).

[0085] Evaluation of organoid survival rates under vulnerable conditions The experiment was carried out as shown in Figure 5A. After removal from Matrigel and washing, the organoids were treated with 800 μM oleic acid for 3 days on a 6-well ultra-low adhesion multi-well plate (Corning). Next, they were treated with 50 μM troglitazone for 24 hours in or without 50 μM NAC. Cell viability was performed using the CellTiter-Glo® luminescent cell viability assay (Promega). Images were obtained sequentially using a fluorescence microscope BZ-9000.

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

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

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[0089] Unless otherwise specified, all percentages and proportions are calculated in weight.

[0090] All percentages and proportions are calculated based on the whole composition unless otherwise specified.

[0091] It should be understood that any maximum numerical limitation described throughout this specification includes all smaller numerical limitations as if they were explicitly stated herein. Any minimum numerical limitation described throughout this specification includes all larger numerical limitations as if they were explicitly stated herein. Any numerical range described throughout this specification includes all narrower numerical ranges that fall within such wider ranges as if all such narrower numerical ranges were explicitly stated herein.

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

[0093] All documents referenced herein, including any cross-referenced or related patents or applications, are incorporated herein by reference in their entirety unless expressly excluded or particularly limited. No reference to any document shall be deemed prior art to any invention disclosed or claimed herein, nor shall it be deemed to teach, suggest or disclose all such inventions, either alone or in combination with any other reference(s). Furthermore, if any meaning or definition of a term in this document conflicts with the 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 prevail.

[0094] While specific embodiments of the present invention have been illustrated and described, it will be apparent 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. Therefore, it is intended that all such changes and modifications within the scope of the invention be covered by the appended claims.

Claims

1. An in vitro liver organoid derived from stem cells, comprising a tubular structure surrounded by polar hepatocytes, wherein the in vitro liver organoid comprises an adhesion zone, and the in vitro liver organoid comprises bile production capacity and / or bile transport activity.

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

3. An in vitro liver organoid according to claim 1 or 2, wherein the stem cells are human iPSCs.

4. An in vitro liver organoid according to any one of claims 1 to 3, wherein the in vitro liver organoid expresses ABCB11 and Na+-taurocholic acid cotransport polypeptide (NTP).

5. An in vitro liver organoid according to any one of claims 1 to 4, wherein the in vitro liver organoid comprises functional stellate cells and functional Kupffer cells.

6. An in vitro liver organoid according to any one of claims 1 to 5, wherein the in vitro liver organoid comprises mesenchymal cells.

7. An in vitro liver organoid according to any one of claims 1 to 6, wherein the in vitro liver organoid includes internalized microvilli.

8. An in vitro liver organoid according to any one of claims 1 to 7, wherein the in vitro liver organoid has bile production capacity, bile transport activity, and at least 50 ng / mL / 1xe ６ Cell / 24-hour complement factor H expression, at least 40 ng / mL / 1xe ６ Cells / 24-hour complement factor B, at least 1000 ng / mL / 1xe ６ Cell / 24-hour C3 expression, at least 1000 ng / mL / 1xe ６ Cell / 24-hour C4 expression, at least 1000 ng / mL / 1xe ６ Cellular fibrinogen production over 24 hours, and at least 1000 ng / mL / 1xe ６ An in vitro liver organoid characterized by having one or more of the following: cells / 24-hour albumin production.

9. An in vitro liver organoid according to any one of claims 1 to 8, wherein the in vitro liver organoid contains at least 10,000 ng / mL 1xe ６ An in vitro liver organoid characterized by having total liver protein expression over 24 hours.

10. An in vitro liver organoid according to any one of claims 1 to 9, 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.

11. An in vitro liver organoid according to any one of claims 1 to 10, wherein the in vitro liver organoid comprises a drug-metabolizing cytochrome variant.

12. In the in vitro liver organoid according to claim 11, the drug-metabolizing cytochrome mutant is a CY2C9*2 mutant.

13. A method for screening for serious adverse events (SAEs), comprising the step of bringing a drug of interest into contact with a liver organoid described in any one of claims 1 to 12.

14. A method according to claim 13, the method comprising the step of measuring the ingestion and / or excretion of fluorescein diacetate (FD), wherein impaired excretion indicates that the drug may induce a serious adverse event.

15. A method according to claim 13 or 14, wherein the toxicity of the drug in question is determined by measuring parameters selected from mitochondrial membrane potential, ROS, hepatic mitochondrial swelling, and combinations thereof, and the damage to the mitochondria indicates that the drug may induce a serious adverse event.

16. A method according to any one of claims 13 to 15, the method comprising the step of analyzing organoid survival rates, wherein an impairment in determining the organoid survival rates indicates that the drug may induce a serious adverse event.

17. An in vitro liver organoid according to any one of claims 1 to 11 for use in the treatment of an individual with liver damage.

18. In the in vitro liver organoid according to claim 17, the liver impairment is selected from metabolic liver disease, end-stage liver disease, or both.

19. An in vitro method for testing a candidate compound on an organism, comprising contacting the candidate compound with an in vitro liver organoid according to any one of claims 1 to 11.

20. The method according to claim 13, wherein the SAE is hepatic failure and / or drug-induced liver injury (DILI).

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