Liver organoid disease models and methods of making the same

Abstract

To provide methods of making and using lipotoxic organoid models.SOLUTION: A method of making a lipotoxic organoid model comprises the steps of contacting a liver organoid with a free fatty acid (FFA) composition, wherein the FFA composition comprises oleic acid, linoleic acid, palmitic acid, or combinations thereof, preferably oleic acid.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 471,371, filed November 4, 2016, and No. 62 / 517,414, filed June 9, 2016, the contents of each of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] Irreversible epithelial organ remodeling is a major driver of death and disease worldwide, costing the healthcare system billions of dollars annually (Hynds and Giangreco, 2013). Diseases of epithelial remodeling include lung and gastrointestinal cancers, as well as chronic diseases such as liver cirrhosis, chronic obstructive pulmonary disease (COPD), and inflammatory bowel disease (Hynds and Giangreco, 2013). Unfortunately, most epithelial organ research, primarily conducted in animals, has failed to yield new treatments for these diseases, and mortality rates remain unacceptably high. This is due to the lack of predictive human systems to test the efficacy of vast compound libraries in the pharmaceutical industry, imposing a fundamental challenge to develop high-fidelity systems to model human inflammation and fibrosis toward clinically relevant therapeutic development.

[0003] Nonalcoholic fatty liver disease (NAFLD) is one of the major challenges to overcome in developed countries as it increases the likelihood of developing fatal liver damage, but as of yet there is no effective treatment. Similarly, iatrogenic parenteral nutrition-associated liver disease (PNALD) is a disorder resulting from parenteral nutrition, for which there is currently no effective treatment. There is a need for models with clinicopathological features of liver disease, such as steatohepatitis and / or lipid droplet accumulation and bladder skeletal filament disorganization, steatosis, and hepatocyte swelling, that can be used to address these and other liver disorders or pathologies. Despite the promise of disease models using patient stem cells, current approaches are limited in their application to single-cell, single-gene, and relatively simple pathologies, failing to capture more common and complex disease pathologies, such as epithelial organ fibrosis.

[0004] The human liver is a vital organ that provides many metabolic functions essential for life, such as lipid metabolism, ammonium and bile production, coagulation, and detoxification of exogenous compounds. Using induced pluripotent stem cell (iPSC) technology, in vitro reconstitution of patients' hepatic responses is attractive to the pharmaceutical industry due to numerous promising applications, including regenerative therapy, drug discovery, and drug toxicity studies. To this end, currently, traditional in vitro approaches have investigated two-dimensional (2-D) and 3-D differentiation platforms to generate hepatocytes. However, most of the reported methods differentiate cells primarily into target epithelial cell types and completely lack essential supporting components, such as profibrotic and / or inflammatory cell types. Alternatively, applicants have proposed co-culture-based approaches by mixing epithelial and supporting lineages, but these assays are highly variable and often confounded by many artificial changes, such as the epithelial cell medium (ECM) and the difficulty in selecting a medium that can simultaneously sustain them. Thus, there is a need for the establishment of novel and robust assay systems that co-develop supporting lineages for disease modeling and further screening applications. Summary of the Invention

[0005] Disclosed herein is the method of making and using lipotoxicity organoid model.In certain embodiments, the method can comprise contacting liver organoid with free fatty acid (FFA) composition.In one embodiment, the FFA composition can comprise oleic acid, linoleic acid, palmitic acid, or combinations thereof. [Brief description of the drawings]

[0006] Those skilled in the art will understand that the drawings, described below, are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0007] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0008] [Figure 1]Co-differentiation of profibrotic and inflammatory lineages in human iPSC-liver organoids. A. Schematic of retinoic acid (RA)-based liver organoid differentiation method and brightfield image of liver organoids at day 20. Scale bar, 100 μm. B. Brightfield image of liver organoids in Matrigel at day 20 after 4 days of culture in the presence and absence of RA. C. Organoid numbers were measured manually at day 20 after 4 days of culture in the presence (black bars) and absence (gray bars) of RA. D. Organoid diameters were recognized and measured by Image J at day 20 after 4 days of culture in the presence (black bars) and absence (gray bars) of RA. E. Albumin production was measured from culture supernatants collected 24 hours after 22-25 days of culture of liver organoids in Matrigel with (black bars) and without (gray bars) RA for 4 days. F. Organoids with internal luminal structures were counted manually on day 25 after 4 days of culture in the presence and absence of RA. Red and grey bars indicate the percentage of organoids with (HLO) / without (spheroids) internal lumen, respectively. G. Bile transport activity was monitored by fluorescein diacetate (FD), which turns to fluorescein (green) after esterase hydrolysis in hepatocytes and tracks the flow of fluorescence in the culture medium. Results are presented as mean ± standard deviation, n = 3. Percentages of Epcam, CD166, and CD68 positive populations were determined by flow cytometry. Results are presented as mean ± standard deviation, n = 2–3. H. FPKM (log2) values ​​for hepatocyte-, stellate cell-, Kupffer cell-, and endothelial-related genes expressed by single-cell RNA sequencing human iPSCs, human iPSC-derived definitive endoderm, foregut spheroids, HLO, human adult liver tissue, and human fetal liver tissue. I. The percentage of Epcam, CD166, CD68, and F4 / 80 positive populations was determined by flow cytometry. Results are presented as mean ± standard deviation, n=3. J. Immunofluorescence (IF) staining of HLOs on day 25 for albumin, CD68, vimentin, GFAP, and Epcam. White arrows indicate the localization of cells positive for CD68, GFAP, and vimentin. Results are presented as mean ± standard deviation, n=3.K. Phagocytic activity was analyzed by monitoring intracellular pH using pHrodo indicator, which reflects phagocytosis (red). Fluorescence expression was captured under a confocal microscope. Results are expressed as mean ± standard deviation, n=6. [Diagram 2]Generation of steatohepatitis HLOs (sHLOs) by fatty acid treatment. A. Schematic of the method for generating steatohepatitis HLOs (sHLOs). B. Live cell imaging of lipid droplets (green), membranes (red), and nuclei (blue). Images were taken by overlaying 10–20 Z-stack images. Increased lipid droplet accumulation as well as enlargement of lipid droplets and cells were observed in a dose-dependent manner. C. Representative total lipid volumes normalized by each organoid size. Bars indicate the average of total lipid volumes. Lipid droplets increased in a dose-dependent manner in response to 0 (black), 200 μM OA (red), 400 μM OA (green), and 800 μM OA (blue). D. Quantification of triglycerides in HLOs. HLOs were isolated from one Matrigel droplet, split into HCM medium in the presence (blue bars) or absence (black bars) of oleic acid (800 μM), and cultured for 3 days. E. ELISA measurement of IL-6 using culture supernatants obtained from wells containing 20–30 HLOs cultured in the presence or absence of oleic acid (800 μM) and cultured for 3 days. Final values ​​were normalized by the number of organoids in each well. IL-6 was released 2.2-fold in 800 μM OA treatment (blue bar) when compared to untreated (black bar). F. Gene expression of proinflammatory cytokines TNF-alpha and IL-8. They were normalized by 18S. Both TNF-alpha and IL-8 gene expression were upregulated in 200 μM OA (red bar) and 800 μM OA (blue bar) compared to untreated (black bar). G. 10–20 HLOs were cultured in HCM medium containing 0, 400, and 800 μM OA for 3 days. Culture supernatants were collected from each well, and THP-1 migration was measured by using membrane penetration with those supernatants. Migrated cells were counted and normalized by the exact number of organoids in each well. H. Trichrome staining of HLOs on day 25. Percentage of trichrome stained HLOs in HLO (black bars), sHLO (red bars) and cHLO (blue bars) populations. Results are expressed as mean ± standard deviation, n = 8–20 organoids. I. IF staining for Epcam and Vimentin of HLOs on day 25. Percentage of Epcam and Vimentin positive HLOs in HLO (black bars), sHLO (red bars) and cHLO (blue bars) populations.Results are expressed as mean ± standard deviation, n = 8–20 organoids. J. 20–30 HLOs were cultured for 5 days in the presence (blue bars) or absence of oleic acid (800 μM). P3NP was measured by ELISA using these supernatants. Final values ​​were normalized by the exact number of organoids in each well. P3NP was increased 2.8-fold in 800 μM OA treatment (blue bars) when compared to untreated (black bars). [Diagram 3] Cirrhotic transition of steatohepatitis HLOs measured by AFM. A. Schematic diagram for measuring stiffening of HLOs by AFM. The top area of ​​each single HLO (14 × 14 matrix in 25 × 25 square µm) was scanned with the AFM cantilever, which can provide spatial mapping of topographical and mechanical information of HLOs. Scale bar, 100 µm. B. Representative histograms of calculated Young's modulus (stiffening of HLOs; E, kPa) of single HLOs showed a Gaussian-like distribution, whose peak value and width increased in a dose-dependent manner in response to 0 (black), 200 (red), 400 (green) and 800 (blue) µM OA. C. Young's modulus (stiffening of HLOs) was determined from 7–12 organoids and summarized by dot plots accompanied by boxplots. An increase in the median value was observed at 0 (black), 200 (red), 400 (green) and 800 (blue) µM OA in a dose-dependent manner. [Figure 4] cHLO stiffness recapitulates the clinical phenotype of Wolman disease. A. Brightfield images of HLOs and cHLOs established from several iPSC lines, including healthy subjects (317D6), NAFLD patients (NAFLD150, NAFLD77, and NAFLD27), and Wolman disease patients (WD90, WD92, and WD91). B. Average Young's modulus (HLO stiffness: Pa) of single HLOs and cHLOs derived from several iPSC lines. [Figure 5A] Modeling human phenotypic variation in steatosis using iPSC-sHLOs A. Representative allele functions of PNPLA3, GCKR, and TM6SF2 involved in increasing hepatic TG content. [Figure 5B]B. Pie chart showing the percentage distribution of 2,504 publicly available cell lines based on the total polygenic scoring of three risk alleles: 0.6 < x (green region), 0.3 < x ≤ 0.6 (yellow region), x <= 0.3 (brown region). [Figure 5C] C. This table summarizes the characteristics of donors with polygenic scores assigned in further studies. [Figure 5D] D. Live cell imaging of lipid droplets (green) and nuclei (blue) in the total polygenic scoring of three groups of three alleles: 0.6 < x, 0.3 < x <= 0.6, x <= 0.3. The images were taken by overlaying 10 - 20 Z-stack images. [Figure 5E] E. Representative total lipid volume normalized by the size of each organoid in the total polygenic scoring of three groups of three alleles: 0.6 < x, 0.3 < x <= 0.6, x <= 0.3. The red and dark blue bars indicate sHLO treated with 800 μM OA and 200 μM OA, respectively. [Figure 5F] F. Representative gene expression of inflammatory cytokines in the total polygenic scoring of three groups of three alleles: 0.6 < x (black bars), 0.3 < x < 0.6 (red bars), x <= 0.3 (blue bars). [Figure 5G] G. Average Young's modulus of a single cLO in the total polygenic scoring of three groups of three alleles (hardening of HLO: Pa): 0.6 < x (black dots), 0.3 < x <= 0.6 (red dots), x <= 0.3 (blue dots) H. OCA response [Figure 6] Phagocytic activity of THP-1 [Figure 7] Lipid accumulation comparison of 800 μM OA, PA, LA, and SA. [Figure 8] Actual number of migratory cells of THP-1 with 400 and 800 μM OA [Figure 9]A. Photographs of E-cad positive and negative sorted reconstituted spheroids from A-cad-mruby organoids, HepG2, LX-2, and THP-1. B. Gene expression of pro-inflammatory cytokines TNF-alpha and IL-8. C. ELISA measurement of P3NP. [Figure 10] Lack of effect of resveratrol on ROS production in liver organoids. [Figure 11] The percentage of BODIPY-positive cells (lipids) was measured by flow cytometry. Results are shown as mean ± standard deviation, n = 5. [Figure 12] Lipid accumulation in organoids in the presence and absence of 400 μM Intralipid [Figure 13] Brightfield images of liver organoids in untreated cultures, 800 μM OA alone cultures, and 800 μM OA and 40 ng / ml FGF19 cultures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Unless otherwise specified, terms are to be understood according to conventional usage by those of ordinary skill in the art.

[0010] The term "about" or "approximately" means within an acceptable error range for a particular value, depending on how the value is measured or determined, for example, of the limitations of the measurement system, as determined by one of ordinary skill in the art. For example, "about" can mean within one or more standard deviations, as per practice in the art. Alternatively, "about" can mean within 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within 10-fold, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in the present application and claims, the term "about" should be assumed to mean within an acceptable error range for the particular value, unless otherwise indicated.

[0011] As used herein, the term "totipotent stem cells" (also known as omnipotent stem cells) are stem cells that can differentiate into embryonic and extraembryonic cell types. Such cells are capable of constructing a complete, viable organism. These cells are produced from the fusion of an egg cell and a sperm cell. The cells produced by the first few divisions of a fertilized egg are also totipotent.

[0012] As used herein, the term "pluripotent stem cells (PSCs)" encompasses any cell that can differentiate into almost any cell type in the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (inner stomach wall, digestive tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), and ectoderm (epidermal tissue and nervous system). PSCs can be the descendants of inner cell mass cells of preimplantation blastocysts, or can be obtained by induction of non-pluripotent cells, such as adult somatic cells, by forcing the expression of certain genes. Pluripotent stem cells can be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including human, rodent, porcine, and bovine.

[0013] As used herein, the term "induced pluripotent stem cells (iPSCs)", commonly abbreviated as iPS cells, refers to a type of pluripotent stem cell that is artificially derived from normally non-pluripotent cells, such as adult somatic cells, by inducing "forced" expression of certain genes. hiPSC refers to human iPSCs.

[0014] As used herein, the term "embryonic stem cells (ESCs)" is also commonly abbreviated as ES cells, and refers to cells that are pluripotent and derived from the inner cell mass of an early embryo, the blastocyst. For the purposes of the present invention, the term "ESCs" is used broadly to optionally include embryonic germ cells.

[0015] As used herein, the term "progenitor cell" encompasses any cell that may be used in the methods described herein, where one or more progenitor cells will acquire the ability to regenerate themselves or differentiate into one or more specialized cell types. In some embodiments, the progenitor cells are pluripotent or have the ability to become pluripotent. In some embodiments, the progenitor cells are subjected to treatment with external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, the progenitor cells may be totipotent or omnipotent stem cells; pluripotent stem cells (induced or non-induced); multipotent stem cells; oligopotent stem cells and unipotent stem cells. In some embodiments, the progenitor cells may be from embryos, infants, children, or adults. In some embodiments, the progenitor cells may be somatic cells that undergo treatment such that pluripotency is imparted via genetic engineering or protein / peptide treatment.

[0016] Embryonic cell-derived pluripotent stem cells In some embodiments, a step is to obtain stem cells that are pluripotent or can be induced to become pluripotent. In some embodiments, the pluripotent stem cells are derived from embryonic stem cells, which are derived from the totipotent cells of early mammalian embryos and are capable of unlimited undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early stage embryo. Methods for deriving embryonic stem cells from blastocysts are well known in the art. Human embryonic stem cells H9 (H9-hESC) are used in the exemplary embodiments described herein, but those skilled in the art will understand that the methods and systems described herein are applicable to any stem cells.

[0017] Additional stem cells that may be used in embodiments according to the present invention include, but are not limited to, those provided by or described in databases maintained by the National Stem Cell Bank (NSCB), the Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF); WISC cell Bank at the Wi Cell Research Institute; the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW-SCRMC); Novocell, Inc. (San Diego, Calif.); Cellartis AB (Goteborg, Sweden); ES Cell International Pte Ltd (Singapore); Technion at the Israel Institute of Technology (Haifa, Israel); and the Stem Cell Database maintained by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that may be used in embodiments according to the present invention include, but are not limited to, SA01 (SA001); SA02 (SA002); ES01 (HES-1); ES02 (HES-2); ES03 (HES-3); ES04 (HES-4); ES05 (HES-5); ES06 (HES-6); BG01 (BGN-01); BG02 (BGN-02); BG03 (BGN-03); TE03 (13); TE04 (14); TE06 (16); UC01 (HSF1); UC06 (HSF6); WA01 (H1); WA07 (H7); WA09 (H9); WA13 (H13); WA14 (H14).

[0018] Further details on embryonic stem cells can be found in, for example, Thomson et al., 1998, "Embryonic Stem Cell Lines Derived from Human Blastocysts", Science 282 (5391): 1145-1147; Andrews et al., 2005, "Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin", Biochem Soc Trans 33: 1526-1530; Martin 1980, "Teratocarcinomas and mammalian embryogenesis", Science 209 (4458): 768-776; Evans and Kaufman, 1981, "Establishment in culture of pluripotent cells from mouse embryos", Nature 292 (5819): 154-156; Klimanskaya et al., 2005, "Human embryonic stem cells derived without feeder ", Lancet 365(9471):1636-1641, each of which is incorporated herein by reference in its entirety.

[0019] Induced pluripotent stem cells (iPSC) In some embodiments, iPSCs are derived by transfection of specific stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection is typically accomplished through a viral vector, such as a retrovirus. Transfected genes include the master transcriptional regulators Oct-3 / 4 (Pouf51) and Sox2, although other genes have been suggested to increase the efficiency of induction. After 3-4 weeks, a small number of transfected cells become morphologically and biochemically similar to pluripotent stem cells and are typically isolated through morphological selection, doubling time, or reporter gene and antibiotic selection. As used herein, iPSCs include, but are not limited to, first generation iPSCs, second generation iPSCs in mice, and human induced pluripotent stem cells. In some embodiments, a retroviral system is used to transform human fibroblasts into pluripotent stem cells using four central genes: Oct3 / 4, Sox2, Klf4, and c-Myc. In another embodiment, somatic cells are transformed with OCT4, SOX2, NANOG and LIN28 using lentivirus system.The genes whose expression is induced in iPSC include but are not limited to Oct-3 / 4 (e.g. Pou5fl);Sox gene family specific members (e.g. Sox1, Sox2, Sox3 and Sox15);Klf family specific members (e.g. Klf1, Klf2, Klf4 and Klf5), Myc family specific members (e.g. C-myc, L-myc and N-myc), Nanog and LIN28.

[0020] In some embodiments, non-viral techniques are used to generate iPSCs. In some embodiments, adenoviruses are used to deliver the four required genes into the DNA of mouse skin and liver cells, resulting in cells identical to embryonic stem cells. The risk of creating tumors is eliminated, as adenoviruses do not integrate any of their own genes into the target host. In some embodiments, reprogramming can be achieved via plasmids, albeit with very low efficiency, without the use of viral transfection systems at all. In other embodiments, direct delivery of proteins is used to generate iPSCs, thus eliminating the need for viruses or genetic modification. In some embodiments, generation of mouse iPSC cells is possible using similar methodologies, and repeated treatment of cells with specific proteins directed into the cells via polyarginine anchors was sufficient to induce pluripotency. In some embodiments, expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions.

[0021] For further details regarding embryonic stem cells, see Kaji et al., 2009, "Virus free induction of pluripotency and subsequent excision of reprogramming factors", Nature 458:771-775; Woltjen et al., 2009, "piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells", Nature 458:766-770; Okita et al., 2008, "Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors", Science 322(5903):949-953; Stadtfeld et al., 2008, "Induced Pluripotent Stem Cells Generated without Viral Integration", Science 322(5903):945-949; and Zhou et al., 2009, "Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins", Cell Stem Cell 4(5):381-384, each of which is incorporated herein by reference in its entirety.

[0022] In some embodiments, exemplary iPS cell lines include, but are not limited to, iPS-DF19-9; iPS-DF19-9; iPS-DF4-3; iPS-DF6-9; iPS(foreskin); iPS(IMR90); and iPS(IMR90).

[0023] Further details regarding the function of signaling pathways involved in the development of DE can be found in, for example, Zorn and Wells, 2009, "Vertebrate endoderm development and organ formation", Annu Rev Cell Dev Biol 25:221-251; Dessimoz et al., 2006, "FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo", Mech Dev 123:42-55; McLin et al., 2007, "Repression of Wnt / β-catenin signaling in the anterior endoderm is essential for liver and pancreas development. Development", 134:2207-2217; Wells and Melton, 2000, Development 127:1563-1572; de Santa Barbara et al., 2003, "Development and differentiation of the intestinal epithelium", Cell Mol Life Sci. 60(7):1322-1332, each of which is incorporated herein by reference in its entirety.

[0024] Any method for generating definitive endoderm from pluripotent cells (e.g., iPSCs or ESCs) is applicable to the methods described herein. Exemplary methods are described, for example, in US97 / 19068B2 (Wells et al.), "Methods and systems for converting precursor cells into intestinal tissues through directed differentiation", and US2017 / 0240866A1 (Wells et al.), "Methods and systems for converting precursor cells into gastric tissues through directed differentiation". In some embodiments, the pluripotent cells may be derived from a morula. In some embodiments, the pluripotent stem cells may be stem cells. Stem cells used in these methods may include, but are not limited to, embryonic stem cells. Embryonic stem cells may be derived from the inner cell mass of an embryo or the gonadal 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 definitive endoderm. In some embodiments, human embryonic germ cells are used to produce definitive endoderm. In some embodiments, iPSCs are used to produce definitive endoderm.Additional methods for obtaining or generating DE cells that can be used in the present invention include, but are not limited to, those described in U.S. Pat. No. 7,510,876 (D'Amour et al.); U.S. Pat. No. 7,326,572 (Fisk et al.); Kubol et al., 2004, "Development of definitive endoderm from embryonic stem cells in culture", Development 131:1651-1662; D'Amour et al., 2005, "Efficient differentiation of human embryonic stem cells to definitive endoderm", Nature Biotechnology 23:1534-1541; and Ang et al., 1993, "The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3 / forkhead proteins", Development 119:1301-1315.

[0025] Nonalcoholic fatty liver disease (NAFLD) is one of the major challenges to overcome in developed countries to increase the chances of developing fatal liver damage, but so far there is no effective treatment. Similarly, iatrogenic parental nutrition-associated liver disease (PNALD) is a disease that currently lacks effective treatment. Models with clinicopathological features of liver disease such as steatohepatitis and / or lipid droplet accumulation and vesicular skeletal filament disorganization, steatosis, and hepatocyte swelling are needed. Despite the promise of disease models using patient stem cells, current approaches are limited in their application to single-cell, single-gene, and relatively simple pathologies and fail to capture more common and complex pathological conditions such as epithelial organ fibrosis.

[0026] In one aspect, disclosed is a method for making lipotoxic organoid model.This method can comprise contacting the liver organoid made according to the method described herein with free fatty acid (FFA) composition.FFA composition can comprise oleic acid, linoleic acid, palmitic acid or combination thereof, preferably oleic acid.The amount of FFA can be determined by those skilled in the art. In one embodiment, the FFA, preferably oleic acid, can be contacted with the liver organoids in an amount of about 10 μM to about 10,000 μM, or about 20 μM to about 5,000 μM, or about 30 μM to about 2500 μM, or about 40 μM to about 1250 μM, or about 50 μM to about 1000 μM, or about 75 μM to about 900 μM, or about 80 μM to about 800 μM, or about 90 μM to about 700 μM, or about 100 μM to about 500 μM, or about 200 μM to about 400 μM. The FFA can be contacted with the liver organoid for about 1 hour to about 10 days, or about 2 hours to about 9 days, or about 3 hours to about 8 days, or about 4 hours to about 7 days, or about 5 hours to about 6 days, or about 6 hours to about 5 days, or about 7 hours to about 4 days, or about 8 hours to about 3 days, or about 9 hours to about 2 days, or about 10 hours to about 1 day. In one embodiment, the range is about 3 to about 5 days, ±24 hours.

[0027] In one aspect, the lipotoxicity organoid model is a model of fatty liver disease.

[0028] In one aspect, the lipotoxicity organoid model is a model of steatohepatitis.

[0029] In one aspect, the lipotoxic organoid model is a model of liver cirrhosis.

[0030] In one aspect, the lipotoxic organoid model is a model of parenteral nutrition associated liver disease (PNALD).

[0031] In one embodiment, the lipotoxic organoid model is a model of NAFLD.

[0032] In one aspect, the lipotoxicity organoid model may be characterized by cytoskeletal filament disorganization, increased ROS, mitochondrial swelling, triglyceride accumulation, fibrosis, hepatocyte ballooning, IL6 secretion, steatosis, inflammation, ballooning and Mallory's body-like, tissue stiffening, cell death and combinations thereof.

[0033] In one embodiment, disclosed is the method for screening the drug for treating liver disease, including NAFLD and / or cholestasis.This method can include contacting candidate drug with lipotoxicity organoid model as disclosed herein.

[0034] In one embodiment, disclosed is the method for assaying the effectiveness of nutritional supplements / TPN.This method can include contacting nutritional supplements / TPN with lipotoxicity organoid model as disclosed herein.

[0035] In one aspect, a three-dimensional (3D) liver organoid model of fatty liver disease is disclosed, in which the organoids are characterized by steatosis, inflammation, ballooning and Mallory bodies, ROS accumulation and mitochondrial overload, fibrosis and tissue stiffening, and cell death.

[0036] In one embodiment, the three-dimensional (3D) liver organoid model is a model of drug-induced liver toxicity and inflammation / fibrosis.

[0037] In one aspect, the three-dimensional (3D) liver organoid model is a model of parenteral nutrition-associated liver disease (PNALD).

[0038] In one embodiment, the three-dimensional (3D) liver organoid model does not contain inflammatory cells, such as T cells or other inflammatory secreted proteins.

[0039] Also disclosed is the method for inducing the formation of liver organoid from iPSC cell, which can be used in the above-mentioned method and / or can be used to obtain the above-mentioned composition.The method can include the following steps:

[0040] The method may include the steps of: a) contacting definitive endoderm (DE) derived from iPSC cells with an FGF pathway activator and a GSK3 inhibitor for a period of time sufficient to form posterior foregut spheroids, preferably about 1 to about 3 days; and b) incubating the posterior foregut spheroids obtained in step a in the presence of retinoic acid (RA) for a period of time sufficient to form liver organoids, preferably about 1 to about 5 days, preferably about 4 days.

[0041] Fibroblast growth factors (FGFs) are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. FGFs are heparin-binding proteins, and interaction with cell surface-associated heparan sulfate proteoglycans has been shown to be essential for FGF signaling. Suitable FGF pathway activators will be readily understood by those skilled in the art. Exemplary FGF pathway activators include, but are not limited to, one or more molecules selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. In some embodiments, siRNA and / or shRNA targeting cellular components related to FGF signaling pathways may be used to activate these pathways.

[0042] In some embodiments, the DE culture is treated with one or more molecules of the signaling pathway described herein at a concentration of 10 ng / ml or more; 20 ng / ml or more; 50 ng / ml or more; 75 ng / ml or more; 100 ng / ml or more; 120 ng / ml or more; 150 ng / ml or more; 200 ng / ml or more; 500 ng / ml or more; 1,000 ng / ml or more; 1,200 ng / ml or more; 1,500 ng / ml or more; 2,000 ng / ml or more; 5,000 ng / ml or more; 7,000 ng / ml or more; 10,000 ng / ml or more; or 15,000 ng / ml or more. In some embodiments, the concentration of the signaling molecule is kept constant during the treatment. In other embodiments, the concentration of the signaling pathway molecule changes during the course of the treatment. In some embodiments, the signaling molecule according to the present invention is suspended in a medium comprising DMEM and fetal bovine serum (FBS). FBS can be at a concentration of 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, or 50% or more. One of skill in the art will understand that the regimens described herein are applicable to any known molecule of the signaling pathways described herein, alone or in combination, including but not limited to any molecule in the FGF signaling pathway.

[0043] Suitable GSK3 inhibitors will be readily recognized by those skilled in the art. Exemplary GSK3 inhibitors include, but are not limited to, Chiron / CHIR99021, which inhibits GSK3β. Those skilled in the art will recognize suitable GSK3 inhibitors for carrying out the disclosed methods. GSK3 inhibitors may be administered in amounts of about 1 μM to about 100 μM, or about 2 μM to about 50 μM, or about 3 μM to about 25 μM. Those skilled in the art will readily recognize appropriate amounts and durations.

[0044] In one embodiment, the stem cells may be mammalian, or human iPSCs.

[0045] In one embodiment, the foregut spheroids can be embedded in a basement membrane matrix, such as, for example, the commercially available basement membrane matrix sold under the trademark Matrigel.

[0046] In one embodiment, the liver organoid may be characterized as expressing alpha-fetoprotein (AFP), albumin (ALB), retinol binding protein (RBP4), cytokeratin 19 (CK19), hepatocyte nuclear factor 6 (HNF6), cytochrome P450 3A4 (CYP3A4), HNF4a, E-cadherin, DAPI, and Epcam. Such expression may occur, for example, on days 40 to 50. The expression level may be similar to that observed in human hepatocytes, for example, that of adult hepatocytes.

[0047] In one embodiment, the liver organoids can be characterized as having bile transport activity.

[0048] In one embodiment, liver organoid can be derived from stem cells and can comprise tubular structure further comprising internalized microvilli cells and mesenchymal cells.Tubular structure can be surrounded by polarized hepatocytes and basement membrane.Liver organoid can comprise functional stellate cells and functional Kupffer cells.

[0049] In certain embodiments, the liver organoids have the following: bile production capacity, bile transport activity, at least 50 ng / mL / 1xe 6 Complement factor H expression at least 40ng / mL / 1xe per cell / 24 hours 6 Complement factor B, at least 1000ng / mL / 1xe per cell / 24 hours 6 C3 expression at least 1000ng / mL / 1xe per cell / 24h 6 C4 expression at least 1000ng / mL / 1xe per cell / 24 hours 6 Fibrinogen production of at least 1000ng / mL / 1xe 6In one embodiment, the liver organoids have at least one of the following characteristics: albumin production in at least 10,000 ng / mL 1xe cells / 24 hours. 6 The liver organoid may be characterized by having total liver protein expression of cells / 24 hours.The liver organoid may be characterized by expressing one or more genes selected from PROX1, RBP4, CYP2C9, CYP3A4, ABCC11, CFH, C3, C5, ALB, FBG, MRP2, ALCAM, CD68, CD34, CD31.In one embodiment, the liver organoid may comprise cells that comprise drug-metabolizing cytochrome mutants, such as CY2C9*2 mutants.The liver organoid may comprise vasculature, such as those described in US2016 / 0177270.

[0050] In one embodiment, the liver organoids can be characterized in that the liver organoids do not contain inflammatory cells, such as T cells or other inflammatory secretory proteins. EXAMPLES

[0051] Human organoid systems that have achieved high-order functions of 3D tissues closely resemble in vivo organ architecture in health and disease, but have failed to capture more common and complex pathologies such as inflammation and fibrosis. Here, applicants developed a multicellular human liver organoid (HLO) model that exhibits essential features of steatohepatitis. Fatty acid exposure on culture allows induction of persistent steatosis, followed by progressive activation of proinflammatory and fibrotic lineages that develop massive fibrosis in HLO. Interestingly, the expression of steatohepatitis phenotype is strongly influenced by clinically reported genetic factors. Atomic force microscopy measurements revealed that overall organoid stiffness correlated with the severity of inflammation and fibrosis. The fidelity of measurements to clinical phenotype was confirmed using three monogenic steatohepatitis-specific iPSC lines. Furthermore, applicants established an iatrogenic parental nutrition-associated liver disease (PNALD) model in organoids.

[0052] Retinoic acid (RA) signaling is a well-known key specifier of thyroid, lung, and pancreas from foregut endoderm during the early organ specification phase, but is apparently not essential for liver specification in model organisms (Kelly and Drysdale, 2015), except for zebrafish (Negishi et al., 2010). Indeed, Xenopus and chicken resemble mammals in that liver specification occurs in embryos lacking RA (Chen et al., 2004; Stafford et al., 2004). Conversely, some animal studies suggest that RA promotes hepatic stellate cell differentiation from mesenchyme, which is in part controlled by the zinc finger transcription factor WT1 expressed in STMs and stellate cells (Ijpenberg et al., 2007; Wang et al., 2013). Moreover, some studies suggest that balanced RA regulation induces monocyte fate specification from human stem cells (Purton et al., 2013). (Ronn et al., 2000; Ronn et al., 2015). Subsequent liver bud outgrowth is also promoted by RA signaling through unknown mechanisms (Zorn and Wells, 2009). Given the broader role of RA on the specification of both parenchymal and non-parenchymal cells, applicants hypothesized that timed RA exposure could affect lineage diversification of hepatic stromal cells, including proinflammatory lineages, thereby facilitating the creation of human liver organoids to model inflammation and fibrosis.

[0053] Recently, directed differentiation into intestinal organoids by foregut spheroid generation from PSCs has been reported (McCracken et al., 2017; Spence et al., 2011). These organoids not only generate epithelial cells but also co-generate mesenchymal cell components. Here, by utilizing this foregut generation method, applicants tested the hypothesis that a timed RA pulse into liver organoids preferentially co-differentiates supporting lineages after hepatic specification from human iPSCs. By promoting close interactions between epithelial and supporting lineages in a 4-D condition where they can co-generate, applicants demonstrated the applicability of liver organoids to model inflammation and fibrosis. Applicants also established a screening platform to determine the severity of fibrosis by assessing stiffness at the single organoid level in a living condition. The present method serves as an invaluable application platform for the study of inflammation and fibrosis in epithelial organs toward drug development and personalized treatment.

[0054] result Generation of RA-based liver organoid model from human iPSCs To clarify whether RA signaling affects lineage differentiation decisions towards stromal cells, applicants established a liver organoid model from human iPSCs by transient induction of RA. As previously described (Spence et al., 2011), applicants first differentiated iPSCs into foregut spheroids via specification of definitive endoderm (DE). Foregut spheroids were mixed with adherent cells grown in the same well, and the mixture was embedded in Matrigel. Since RA is a known specifier for diverse lineages via an environment-dependent process, applicants set the duration of early RA signaling before the hepatocyte maturation process, which was characterized in organoids established at day 20, cultured in hepatocyte culture medium (HCM) as shown in Figure 1, A. Since the highest concentration of albumin in the culture medium was observed at 4 days of RA exposure among various durations of RA from 0 to 5 days, further characterization of established organoids was compared between two conditions of 0 to 4 days of RA exposure. In RA-treated wells, the number of organoids increased 1.8-fold compared to untreated ones, and the size increased 1.5-fold after RA treatment (Figure 1, B, C, and D). Albumin secretion increased 2-fold in the RA-treated group (3.0–6.5 μg / ml) when compared to untreated (0.5–3.5 μg / ml, Figure 1, E). Notably, albumin secretion was maintained for more than 40 days after 20 days when albumin was first detected (data not shown). Interestingly, internal luminal structures appeared in 95% of RA-treated organoids, but were only detected in 12% of untreated organoids, indicating that organoid lumenization is dependent on RA signaling (Figure 1, F).

[0055] Next, applicants tested bile transport activity by adding fluorescein diacetate, a marker of efflux transport in hepatocytes that turns to fluorescein after esterase hydrolysis in hepatocytes, into the culture medium and followed the flux of fluorescence. The fluorescent substance was absorbed by the cells of the organoids within minutes after the addition of fluorescein diacetate and was then excreted from the cells into the lumen (Figure 1, G). In contrast, fluorescence was not detected in the spheroids. Since bile transport activity is a key function of the liver, human liver organoids, hereafter defined as HLOs, can be used as a model representing multiple human hepatocyte functions, including albumin secretion and bile transport function.

[0056] Simultaneous differentiation of profibrotic and inflammatory lineages in human iPSC liver organoids Interestingly, single-cell RNA sequencing (scRNA-seq) analysis of RA-treated HLOs showed specific expression signatures for stellate cells, hepatic resident macrophages, Kupffer cells, and endothelial cells, as evidenced by stellate cell markers (ACTA2, DES, PDGFRB), Kupffer cell markers (CD68, IRF7), and endothelial cell markers (OIT3, DPP4, C1QTNF1; Figure 1, H) (Bahar Halpern et al., 2017; El Taghdouini et al., 2015; van de Garde et al., 2016). To confirm the presence of stellate cells and Kupffer cells, applicants performed quantitative analysis by FACS using the epithelial cell marker EpCAM, the stellate cell marker CD166 / ALCAM, and the Kupffer cell markers CD68 and F4 / 80 (Yanagimachi et al., 2013). The frequency of EpCAM+ cells was 78.85±7.35% in HLO, whereas EpCAM-CD166+, EpCAM-CD68+, and EpCAM-F4 / 80+ expressing cells were 32.4±1.2%, 1.69±0.3%, and 1.68±0.2%, respectively (Figure 1, I). By immunohistochemistry, expression of CD68 cells was also detected in HLO, and CD68 expression was localized to the luminal side of cells (Figure 1, J). Vimentin and GFAP, which are novel markers of human hepatic stellate cells and have been reported to be necessary for the process of stellate cell transdifferentiation in rodents (Geerts et al., 2001; Kordes et al., 2014), were both detected in HLO and co-expressed in the same cells (Figure 1, J), indicating that stellate cells are present in HLO.

[0057] To investigate whether CD68-expressing cells are functionally active in HLOs similar to Kupffer cells, applicants monitored phagocytic activity in HLOs using a pH-sensitive rhodamine-based live cell dye that undergoes a dramatic increase in fluorescence (red) in response to the environmental shift from high to low pH that occurs in phagocytosis, as observed in THP1 human macrophage cells, which have been reported to be active in liver-resident Kupffer cells (Figure 8). In HLOs, activity was detected and localized similarly to CD68-expressing cells, but was rarely detected in spheroids (Figure 1, K). These findings indicate that RA-based liver organoids (HLOs) are a unique liver model that naturally generates multilineage liver stromal cells, including stellate cells and Kupffer cells, from human iPSCs.

[0058] Inflammatory response and fibrosis induction of HLO by free fatty acids Currently, free fatty acids (FFAs) are widely used as initiating factors to establish in vitro lipotoxicity hepatocyte models; however, fat accumulation is the only phenotype (Kanuri and Bergheim, 2013). An alternative co-culture model of hepatocytes and Kupffer cells showed inflammatory responses such as increased expression of IL family cytokines, macrophage-related cytokines, and MMP-related cytokines, but did not show subsequent fibrosis (Hassan et al., 2014). Given the presence of hepatocytes, stellate cells, and Kupffer-like cells with lipid metabolism function-related genes shown by comprehensive transcriptome analysis, applicants hypothesized that FFA exposure naturally triggers an inflammatory response and a resulting fibrotic response in HLOs. To test this hypothesis, applicants treated HLOs with FFAs for 3–5 days. Applicants denoted 3- and 5-day OA-treated HLOs as steatohepatitis (s) HLOs and cirrhosis (c) HLOs (Figure 4, A). sHLOs are HLOs treated with oleic acid for 3 days, showing lipid accumulation and inflammation. cHLOs are HLOs treated with oleic acid for 5 days, which show fibrosis (HLOs are stiff) in addition to lipid accumulation and inflammation. First, to compare the effects of multiple FFAs, including oleic acid (OA, 18:1 n9), linoleic acid (LA, 18:2), palmitic acid (PA, 16:0), and stearic acid (SA, 18:0), on lipid accumulation, live-cell imaging was performed using the lipid dye BODIPY 3 days after FFA exposure. As Figure 7 shows, OA was the most effective at inducing lipid accumulation in hepatocyte-like cells, while SA was the least effective. Considering that OA causes a large amount of lipid accumulation, the concentration of OA was varied (0, 200, 400, and 800 μM) to induce an inflammatory response in sHLOs. Live-cell imaging and subsequent quantification showed that lipid accumulation was elevated in sHLOs in a dose-dependent manner (up to 18-fold; Figure 4, B and C). In addition to increased lipid accumulation, the size of lipid droplets expanded ( Fig. 4, B ).Hepatocyte ballooning (enlargement), one of the pathological grading indicators for determining nonalcoholic steatohepatitis (NASH) activity (NAS scoring), was confirmed in 800 μM OA-treated sHLO by live imaging of cell membranes (Figure 4B). Triglycerides, the main component of lipids accumulated in the liver, were also detected in 800 μM treated sHLO but not in untreated sHLO (Figure 4, D). More importantly, ELISA of culture supernatants showed that IL-6 was secreted 2.2-fold in 800 μM OA-treated sHLO medium when compared with untreated one (Figure 4, E). IL-8 and TNF-alpha were also upregulated under 200 μM or 800 μM OA treatment conditions (10-fold and 2-fold increase, respectively; Figure 4, F). Furthermore, OA-treated sHLO conditioned medium was used to evaluate THP1 migration 24 hours after culturing in transwells, and THP-1 migratory cells were elevated in OA-treated sHLO, suggesting that FFA treatment naturally induced an inflammatory response in sHLO, possibly by causing stress to hepatocyte-like cells (up to 2-fold: Figure 4, G and Figure 8). To confirm whether these inflammatory and fibrotic features were specific to HLO, applicant isolated the epithelial marker E-cad from E-cad mRuby embryonic stem (ES) cell-derived organoids, reconstructed spheroids from either E-cad positive or negative cells, and applied to ELISA for IL6 and P3NP and RNA expression for IL8 and TNF-α. As Figure 9 shows, neither E-cad positive nor negative cell-derived spheroids caused P3NP and IL-6 secretion production, nor overexpressed gene expression of inflammatory markers. Applicants also tested the same experiment with human hepatocyte cell line HepG2, macrophage cell line THP-1, and hepatic stellate cells LX-2, and had similar results for E-cad positive and negative cells. The results showed that liver organoids only responded to FFA treatment against inflammation and fibrosis responses.

[0059] To further determine whether HLOs progress toward a fibrosis-like state, applicants performed Masson's trichrome staining on sHLOs and cHLOs. Trichrome-positive HLOs were not observed in sHLOs, but were significantly increased in cHLOs (Figure 2, H). Furthermore, IF staining for epithelial marker Epcam and fibrosis marker Vimentin showed an increase in Epcam-positive HLOs and a decrease in Vimentin-positive HLOs in cHLOs (Figure 2, I), suggesting that OA treatment induced a selective increase in fibrotic populations as well as matrix deposition in cHLOs. Furthermore, ELISA for P3NP was also measured on the fifth day of OA exposure. When compared to untreated, P3NP secretion was increased 2.8-fold in the medium of 800 μM OA-treated HLOs (Figure 4, J). Taken together, these observations indicated that FFA exposure did not simply cause triglyceride accumulation but also induced an inflammatory response and fibrosis in HLOs.

[0060] cHLO profile of inflammation and fibrosis by FFA treatment at the single cell level High-throughput quantification of fibrosis by AFM Accumulating evidence indicates that liver stiffening correlates well with the severity of liver fibrosis (Yoneda et al., 2008), so measuring stiffening of HLOs may assess the severity of fibrosis in HLOs. Applicant's preliminary qualitative analysis of smooth muscle actin immunostaining indicates a dose-dependent fibrosis progression with OA exposure (Figure 2, H, I, and J). To gain more quantitative insight in a screenable format, Applicant thus assessed in live conditions whether HLO fibrosis can increase stiffening in a dose-dependent manner using micro-indentation with atomic force microscopy (AFM). As shown in Figure 4A, the top area of ​​each single HLO (14 × 14 matrix of 25 × 25 μm squares) was scanned with an AFM cantilever, which can provide spatial mapping of topographical and mechanical information of HLOs. Representative histograms of the calculated Young's modulus (E, kPa) of single HLOs clearly showed a Gaussian-like distribution, whose peak value and width increased according to the OA concentration (Figure 4,B; 0, 200, 400, 800 µM). The Young's modulus determined from HLOs was summarized by a boxplot with dots (Figure 4,C), which shows that the stiffness of HLOs gradually increased from 0 to 800 µM OA, i.e., a gradual shift in the median and an expansion of the stiffness range were observed according to the increasing OA concentration. The emedient values ​​were 1.2 kPa, 1.6 kPa, 2.4 kPa, and 2.8 kPa for 0, 200, 400, and 800 µM OA-treated organoids, respectively. Moreover, the difference between the 90th and 10th percentiles (ΔP90-10) was 3.2 kPa for untreated organoids and 7.0 kPa for 800 µM OA-treated organoids. As shown in Figure 3, F, TNF-alpha and IL-8 were also upregulated by OA addition, which are known to correlate with the severity of fibrosis in NAFLD patients (Ajmera et al., 2017). These results indicate that HLO stiffening can be a quantifiable measurement of fibrosis severity and therefore potentially applicable to high-throughput screening of fibrosis.

[0061] cHLO stiffness recapitulates the clinical phenotype of Wolman disease The clinical relevance of the pluripotent stem cell-derived model for fibrosis and cirrhosis is unclear due to the undetermined nature of genetic influences in patients. Predisposition to fibrosis will likely not be captured in this model. Therefore, applicants investigated the fidelity of the assay to clinical phenotypes by evaluating iPSC-derived congenital steatohepatitis patients, including healthy subjects (317D6), NAFLD / NASH patients (NAFLD150, 77, and 27), and Wolman disease patients (WD90, 91, and 92) (Figure 5, A). Specifically, applicants established three Wolman disease patient-specific iPSC lines, a monogenetic disease with fatal steatohepatitis, and confirmed significant stiffness increase compared to normal iPSC line-derived organoids, especially with a remarkable correlation with enzyme activity at the time of clinical diagnosis (Figure 5, B).

[0062] Modeling human phenotypic variation in adiposity using iPSC-sHLOs Second, Applicants extended this novel organoid model to evaluate polygenic effects on steatohepatitis progression. Three protein coding sequence variants PNPLA3 (patatin-like phospholipase 3) p.I148M, TM6SF2 (transmembrane 6 superfamily member 2) p.E167K, and GCKR (glucokinase regulatory protein) p.P446L (Xu et al., 2015) have been repeatedly shown by large-scale GWAS to be independent determinants of hepatic triglyceride content (HTGC) (Zain et al., 2015) (Figure 6, A). Therefore, applicants approached a large cell bank and mapped 2504 cell lines with a polygenic score (Figure 6, B) by the sum of reported effect sizes β(SNP) (Stender et al., 2017), the per-allele change in normalized HTGC multiplied by weighted dosage, as follows: Σ{β(SNP) × (dosage of risk allele)}. After obtaining iPSC lines with three different thresholds (Figure 6, B and C), applicants generated seven iPSC organoids and induced steatohepatitis to estimate the impact of polygenicity on the phenotype. Surprisingly, applicants found a striking correlation between "steatosis" (by live imaging, Figure 6, D and E) and "inflammation" (qRT-PCR, Figure 6, F), but no correlation with the polygenic score for "fibrosis" (AFM-based stiffness measurement, Figure 6, G). Applicants also found that OCA response differed between cell lines and that the response to OCA was dependent on the number of SNPs (Figure 6H).

[0063] discussion Multicellular liver organoids from human PSCs A series of recent studies reported successful integration of supporting lineages into endoderm-derived organoids by experimentally combining endothelial cells (Takebe et al., 2013), mesenchymal cells (Takebe et al., 2015), and neural crest cells (Workman et al., 2017). The method based on RA pulsation naturally engages cells to diversify with sustained cell polarity and is remarkably reproducible and scalable at a reasonable cost. Regardless of the exact nature of the supporting lineages, whether developmentally relevant or liver-specific, the stromal populations are fully responsive to known fibrosis inducers, including LPS and fatty acids, opening new methods to model multicellular and complex pathologies.

[0064] The Promise of Mechanical Organoid Screening to Analyze Inflammation and Fibrosis From a future screening perspective, single organoid-based liver measurements are a very attractive readout due to their robustness, normalization, and relative simplicity. For example, a functional swelling assay was established for intestinal organoids of cystic fibrosis patients based on live fluorescent imaging analysis of organoids, and effective drug selection was demonstrated (Saini, 2016). Live stiffness assessment of single liver organoids from iPSCs is an effective method to predict fibrosis severity. In patients with fatty liver disease, a significant correlation between measurements of liver stiffness and fibrosis stage has been reported clinically by elastography (Yoneda et al., 2008). Furthermore, a strong association between increased liver stiffness and the presence of diabetes mellitus (DM) and / or higher insulin resistance was observed in a subgroup of subjects with sonographically defined NAFLD (Koehler et al., 2016). Interestingly, human liver organoid (HLO) stiffness was increased proportionately by both LPS and FFA, accompanied by inflammatory cytokine production and fibroblast proliferation. Considering that numerous epithelial organ fibroses share similar phenotypes through diverse pathological mechanisms, the organoid-based stiffness detection assay can be used to analyze fibrosis using lung, kidney, heart and intestinal organoids.

[0065] In liver organoids, multiple measurements can be assessed in vivo using high-content imaging systems. In fact, lipid droplet number and size, bile transport activity, increased phagocytes, and deterioration of cell morphology can be effectively imaged in fat-treated liver organoids. Therefore, organoid-based assay platforms can be used to better understand the human-specific mechanisms of fatty liver disease and establish high-content screening for drug discovery against these diseases. Combined with compound libraries and nutritional metabolites, organoid-based mechanistic screening would be a very attractive model system for implicating effective treatments in humans that otherwise cannot be tested preclinically.

[0066] Nutritional precision medicine (individualized treatment) Patient-specific iPSC-derived organoids may be used to predict personalized drug efficacy and epithelial responses. Human iPSCs can be established from both healthy and diseased individuals. In parallel with the establishment of population iPSC panels, the use of patient iPSCs is expected to model inter-individual differences regarding lipotoxicity, drug efficacy and safety (Warren et al., 2017a, Warren et al., 2017b). In doing so, phenotypic screening with liver organoid-based platforms would facilitate personalized selection of highly effective interventions for disorders. For example, since nutritional supplementation can be modified according to the individual, the disclosed system could be a highly adaptable assay to reflect nutrition-related status before administration. Specifically, patient-specific iPSC-derived organoids could be used to customize PN formulations for each patient with the aim of minimizing possible progression to hepatic steatosis and fibrosis (PNALD). Indeed, in the clinic, PN formulations are often customized (Mercaldi et al., 2012), because commercially available solutions do not meet the caloric, amino acid, and electrolyte needs of critically ill patients, who are often obese, require fluid restriction, and exhibit hepatic / renal dysfunction (Boullata et al., 2014). Beyond nutritional needs, there is an urgent need to gain insight into the reduction of possible adverse effects associated with PN products. Thus, liver organoids are useful to evaluate safety concerns, especially for PNALD risk assessment, facilitating customization strategies of PN formulations for each patient.

[0067] Overall, applicants have demonstrated that allowing crosstalk between epithelial and stromal lineages in 4-D organoid cultures is useful for modeling clinically relevant pathologies associated with hepatic steatosis and fibrosis. This model will ultimately lead to the analysis of more common pathologies such as NAFLD and NASH, which are of increasing concern in an aging population. More broadly, applicants have established a viable strategy for modeling human and complex pathologies, coupled with currently evolving organoid technologies (Lancaster and Knoblich, 2014), paving the way for new ways to discover effective therapies for irreversible diseases at the single organoid level.

[0068] method hPSC Maintenance The TkDA3 human iPSC clone used in this study was kindly provided by K. Eto and H. Nakauchi. Human iPSC lines were maintained as previously described (Takebe et al., 2015; Takebe et al., 2014). Undifferentiated hiPSCs were maintained under feeder-free conditions in mTeSR1 medium (StemCell technologies, Vancouver, Canada) on plates coated with Matrigel (Corning Inc., NY, USA) at 1 / 30 dilution at 37°C in 5% CO2, 95% air.

[0069] Definitive endoderm induction Human iPSCs were differentiated into definitive endoderm using a previously described method with minor modifications (Spence et al., 2011). Briefly, colonies of human iPSCs were isolated in Accutase (Thermo Fisher Scientific Inc., MA, USA) and plated at 150,000 cells / mL onto Matrigel-coated tissue culture plates (VWR Scientific Products, West Chester, PA). The medium was changed to RPMI 1640 medium (Life Technologies) containing 100 ng / mL activin A (R&D Systems, MN, USA) and 50 ng / mL bone morphogenetic protein 4 (BMP4; R&D Systems) on day 1, to RPMI 1640 medium containing 100 ng / mL activin A and 0.2% fetal calf serum (FCS; Thermo Fisher Scientific Inc.) on day 2, and to RPMI 1640 medium containing 100 ng / mL activin A and 2% FCS on day 3. On days 4–6, cells were cultured in Advanced DMEM / F12 (Thermo Fisher Scientific Inc.) with B27 (Life Technologies) and N2 (Gibco, CA, USA) containing 500 ng / ml fibroblast growth factor (FGF4; R&D Systems) and uM CHIR99021 (Stemgent, MA, USA). Cells were maintained at 37°C in 5% CO2 with 95% air and medium was changed daily. Spheroids appeared on the plates on day 7 of differentiation.

[0070] On day 7 of HLO induction, spheroids and attached cells are gently pipetted to detach them from the dish. They are centrifuged at 800 rpm for 3 min, and after removing the supernatant, they are embedded in 100% Matrigel droplets on the dish in Advanced DMEM / F12 containing B27, N2 and 2 μM retinoic acid (RA; Sigma, MO, USA) and cultured for 4 days. After RA treatment, spheroids embedded in Matrigel droplets were cultured in hepatocyte culture medium (HCM; Lonza, MD, USA) containing 10 ng / mL hepatocyte growth factor (HGF; PeproTech, NJ, USA), 0.1 μM dexamethasone (Dex; Sigma) and 20 ng / mL oncostatin M (OSM; R&D Systems). Cultures for HLO induction were maintained at 37 °C in 5% CO2 with 95% air, and the medium was changed every 2–3 days. To analyze HLO (days 20–30), organoids were isolated from the Matrigel by scratching and pipetting.

[0071] Albumin, IL-6, and P3NP ELISA. To measure the albumin secretion levels of HLOs, 200 μL of culture supernatant was collected from HLOs embedded in Matrigel. For IL-6 and P3NP, 20–30 organoids were seeded and cultured on ultra-low attachment multi-well plates (Corning) in 96-well plates. To define the exact number of organoids in each well and finally normalize the secretion levels of IL-6 and P3NP by number, the organoids were captured under a KEYENCE BZ-X710 fluorescent microscope. Culture supernatants were collected at 24 h (for albumin), 96 h (for IL-6), and 120 h (for P3NP) after culture and stored at -80°C until use. The supernatant was centrifuged at 1,500 rpm for 3 min to pellet debris, and the resulting supernatant was assayed using the Human Albumin ELISA Quantitation Set (Bethyl Laboratories, Inc., TX, USA), Human IL-6 ELISA Kit (Biolegend, CA, USA), and Human N-terminal procollagen III propeptide, PIIINP ELISA Kit (My BioSource, CA, USA) according to the manufacturer's instructions. Significance tests were performed by Student's t test.

[0072] Bile transport activity Fluorescein diacetate was used to evaluate bile transport activity in organoids. 10 mg / mL fluorescein diacetate (Sigma) was added to HCM medium cultured with HLO, left to stand for 5 min, and captured using a fluorescence microscope BZ-X710 (Keyence, Osaka, Japan).

[0073] Phagocyte, lipid, and ROS live cell imaging. After culturing in ultra-low attachment 6 multi-well plates, 5–10 HLOs were picked up and seeded onto Microslide 8-well glass bottom plates (Ibidi, WI, USA) for live cell staining. The following antibodies were used: pHrodo® Red S. aureus Bioparticles® Conjugate (Thermo Fisher Scientific Inc.) for phagocytic activity, BODIPY® 493 / 503 (Thermo Fisher Scientific Inc.) for lipids, Di-8-ANEPPS (Thermo Fisher Scientific Inc.) for membranes, and CellROX green reagent (Thermo Fisher Scientific Inc.) for ROS detection. Nuclear staining was marked by NucBlue Live ReadyProbes Reagent (Thermo Fisher Scientific Inc.). hLOHLOs were visualized and scanned with a Nikon A1 inverted confocal microscope (Japan) using a 60x water immersion objective. The final lipid droplet volume was calculated by IMARIS8 and normalized by each organoid size. Significance tests for lipid droplet volume and ROS production (%) were performed by Student's t-test.

[0074] HE staining and immunohistochemistry HLOs were isolated from Matrigel, fixed in 4% paraformaldehyde, and embedded in paraffin. Sections were subjected to HE staining and immunohistochemistry staining. The following primary antibodies were used: anti-alpha smooth muscle actin antibody (1:200 dilution; Abcam, Cambridge, UK), Desmin antibody (prediluted; Roche, Basel, Switzerland), and CD68 antibody (1:200 dilution, Abcam).

[0075] Flow cytometry HLOs were isolated from 10 Matrigel droplets and washed with 1× PBS. HLOs were dissociated into single cells by treatment with trypsin-EDTA (0.05%), phenol red (Gibco) for 10 min. After PBS washing, single cells were subjected to flow cytometry using BV421-conjugated Epcam antibody (BioLegend), PE-conjugated CD166 antibody (eBioscience, CA, USA), and PE / Cy7-CD68 (eBioscience). DNA was measured by propidium iodide staining.

[0076] Exposure to LPS and FFA and treatment with OCA and FGF19. Twenty to thirty HLOs isolated from Matrigel and washed with 1x PBS were divided into each condition and cultured on ultra-low attachment six-multiwell plates (Corning). HLOs were cultured with LPS (Sigma), OA (Sigma), LA (Sigma), SA (Sigma), or PA (Sigma) and collected on days 1 and 3 (for LPS HLOs) and days 3 and 5 (for OA) after culture. To test the inhibitory effect of OCA (INT-747, MedChem Express, NJ, USA) and human FGF19 recombinant (Sigma) on HLOs, 20 to 30 HLOs were cultured in HCM medium in the presence or absence of oleic acid (800 μM), and 1 μM OCA and 40 ng / ml FGF19 were added to the OA condition at 800 μM. HLOs were collected on day 3 for lipid live cell imaging and on day 5 for stiffening measurements.

[0077] Whole-mount immunofluorescence. HLOs were fixed in 4% paraformaldehyde for 30 min and permeabilized with 0.5% Nonidet P-40 for 15 min. HLOs were washed three times with 1× PBS and incubated with blocking buffer for 1 h at room temperature. HLOs were then incubated with primary antibody, anti-α-smooth muscle actin antibody (1:50 dilution; abcam) at 4°C overnight. HLOs were washed with 1× PBS and incubated in secondary antibody in blocking buffer for 30 min at room temperature. HLOs were washed and mounted with Fluoroshield mounting medium with DAPI (abcam). Stained HLOs were visualized and scanned with a Nikon A1 inverted confocal microscope (Japan) using a 60x water-immersion objective.

[0078] RNA isolation, RT-qPCR. RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany). Reverse transcription was performed using the SuperScriptIII First-Strand Synthesis System for RT-PCR (Invitrogen, CA, USA) according to the manufacturer's protocol. qPCR was performed using TaqMan Gene Expression Master Mix (Applied Biosystems) on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific Inc.). All primer and probe information for each target gene was obtained from the Universal ProbeLibrary Assay Design Center (https: / / qpcr.probefinder.com / organism.jsp). Significance testing was performed by Student's t-test.

[0079] HLO cure measurements by AFMHLO treated with 0, 50200, 1400 and 2800 ng / mL μM LPSOA were used for cure measurements with AFM (NanoWizard IV, JPK Instruments, Germany). An AFM head with silicon nitride cantilevers (CSC37, k=0.3 N / m, MikroMasch, Bulgaria) was mounted on a fluorescence stereo microscope (M205 FA, Leica, Germany) coupled with a Z-axis piezo stage (JPK CellHesion module, JPK Instruments, Germany), which allows indentation measurements to a depth of approximately 100 μm. As substrate for organoids, fibronectin-coated dishes were used. Tissue culture dishes (φ=34 mm, TPP Techno Plastic Products, Switzerland) were first incubated overnight at 4 °C with 1 μg / mL fibronectin solution (Sigma). Afterwards, the tissue culture dishes were washed twice with distilled water and dried for 1 h. Then, the HLOs incubated with OALPS for 51 days were placed into fibronectin-coated dishes and incubated at 37 °C for 1 h. The sample dishes were then placed on the AFM stage, and the force-distance curves of a 14 × 14 matrix of 25 × 25 μm squares were measured from each HLO. Finally, the Young's modulus (E, Pa) of the HLOs was determined by fitting the obtained force-distance curves with a modified Hertz model (Sneddon, 1965). The Dunn-Holland-Wolfe test was performed for significance testing.

[0080] THP-1 cell migration assay. THP-1 cells, donated by T. Suzuki, were maintained in Advanced DMEM / F12 (Thermo Fisher Scientific Inc.) containing 10% FBS. THP-1 floating cells were collected, and 200,000 cells were added to the membrane chamber of a CytoSelect™ 96Well Cell Migration Assay (5μm, Fluorometric Format; Cell Biolabs, CA, USA) with serum-free Advanced DMEM / F12. 10–20 HLOs were cultured in HCM medium containing 0, 400, and 800μM OA for 3 days using ultra-low attachment 96 multiwell plates (Corning). To define the exact number of organoids in each well and finally normalize the number of cells that had migrated, the organoids were captured with a Keyence BZ-X710 fluorescent microscope. 150μL of the culture supernatant of the HLOs was collected and added to the feeder tray of the kit. The kit was incubated for 24 hours at 37°C in a 5% CO2 cell culture incubator. Migrated cells were counted using a Countess II FL Automated Cell Counter (Thermo Fisher Scientific Inc.). Significance testing was performed by Student's t-test.

[0081] Triglyceride assay For quantitative measurement of triglycerides, HLOs were isolated from one Matrigel droplet and split into HCM medium in the presence or absence of oleic acid (800 μM). They were cultured for 3 days on ultra-low attachment 6-multiwell plates. Quantitative estimation of hepatic triglyceride accumulation was performed by enzymatic assay of triglyceride mass using the EnzyChrom Triglyceride assay kit (Bioassay Systems, CA, USA).

[0082] HLO survival assay HLOs were harvested from Matrigel and washed with 1x PBS. Thirty to 40 organoids were cultured on ultra-low attachment 6 multiwell plates (Corning). HLOs were captured daily with a Keyence BZ-X710 fluorescent microscope. Viable and dead organoids were counted manually from the photographs. HLOs with a rounded shape were counted as viable, whereas organoids with different shapes were counted as dead. To assess the viability of OA-treated HLOs at the same time points, a 3D cell titer glo assay was used (Promega, Wisconsin, USA).

[0083] Statistics and replicates. Statistical analysis was performed using unpaired two-tailed Student's t-test, Dunn-Holland-Wolfe test, or Welch's t-test. Results are presented as mean ± SEM. P values ​​less than 0.05 were considered statistically significant. N values ​​refer to biologically independent replicates unless otherwise stated.

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Claims

1. A method for producing a lipotoxic organoid model, comprising contacting a liver organoid with a free fatty acid (FFA) composition, wherein the FFA composition comprises oleic acid, linoleic acid, palmitic acid, or a combination thereof, preferably oleic acid.

2. The method of claim 1, wherein the lipotoxic organoid model is a model of fatty liver disease.

3. The method of claim 1, wherein the lipotoxic organoid model is a model of steatohepatitis.

4. The method of claim 1, wherein the lipotoxic organoid model is a liver cirrhosis model.

5. The method of claim 1, wherein the lipotoxic organoid model is a model of parenteral nutrition-associated liver disease (PNALD).

6. The method of claim 1, wherein the lipotoxic organoid model is a model of NAFLD.

7. The method according to any of claims 1 to 6, wherein the lipotoxic organoid model is characterized by cytoskeletal filament disorganization, increased ROS, mitochondrial swelling, triglyceride accumulation, fibrosis, hepatocyte ballooning, IL6 secretion, steatosis, inflammation, ballooning and Mallory's body-like, tissue stiffening, cell death, and combinations thereof.

8. A method for screening drugs for the treatment of liver disease, including NAFLD and / or cholestasis, comprising contacting a candidate drug with the lipotoxic organoid model of claim 1.

9. A method for assaying the effectiveness of a nutritional supplement / TPN comprising contacting the nutritional supplement / TPN with a lipotoxic organoid model described in claim 1.

10. A three-dimensional (3D) liver organoid model of fatty liver disease, wherein the organoid is characterized by steatosis, inflammation, ballooning and Mallory bodies, ROS accumulation and mitochondrial overload, fibrosis and tissue stiffening, and cell death.

11. Three-dimensional (3D) liver organoid model of drug-induced hepatotoxicity and inflammation / fibrosis.

12. Three-dimensional (3D) liver organoid model of parenteral nutrition-associated liver disease (PNALD).

13. The liver organoid model according to any one of claims 1 to 12, wherein the model does not contain inflammatory cells, such as T cells or other inflammatory secretory proteins.

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