Liver organoid disease models and methods for their creation

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 the benefit and priority of U.S. Provisional Patent Application No. 62 / 471,371, filed on November 4, 2016, and No. 62 / 517,414, filed on June 9, 2016, the entire contents of each of which are incorporated herein by reference for all purposes.

Background Art

[0002] Irreversible epithelial organ remodeling is a major cause of death and disease worldwide and costs 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 cirrhosis, chronic obstructive pulmonary disease (COPD), and inflammatory bowel disease (Hynds and Giangreco, 2013). Unfortunately, most epithelial organ research, mainly conducted in animals, has been unable to produce new therapies for these diseases, and mortality rates remain unacceptably high. This is due to the lack of predictive human systems for testing the efficacy of the vast number of compound libraries in the pharmaceutical industry, and it poses a fundamental challenge to develop high - fidelity systems for modeling human inflammation and fibrosis towards the development of clinically appropriate therapies.

[0003] Non-alcoholic 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 currently there is no effective treatment. Similarly, iatrogenic parenteral nutrition-related liver disease (PNALD), a disorder resulting from parenteral nutrition, currently has no effective treatment. There is a need for models that possess the clinicopathological features of liver diseases such as steatohepatitis and / or fat droplet accumulation and bladder skeletal filamentous tissue breakdown, steatosis, and hepatocyte swelling, as well as models that can be used to address these and other liver disorders or conditions. Despite the promise of disease models using patient stem cells, current approaches are limited in their application to single-celled, single-genetic, and relatively simple conditions, and fail to capture more common and complex disease conditions such as epithelial organ fibrosis.

[0004] The human liver is a vital organ that provides many metabolic functions essential to life, including lipid metabolism, ammonium and bile production, coagulation, and detoxification of exogenous compounds. In vitro reconstruction of patient liver responses using induced pluripotent stem cell (iPSC) technology is attractive to the pharmaceutical industry due to numerous promising applications, including regenerative medicine, drug discovery, and pharmacotoxicology research. For this purpose, conventional in vitro approaches currently study two-dimensional (2-D) and 3-D differentiation platforms to generate hepatocytes. However, most reported methods differentiate cells primarily into target epithelial cell types, completely lacking essential supporting components such as profibrotic and / or inflammatory cell types. Alternatively, the applicants have proposed a co-culture-based approach by mixing epithelial and supporting lineages, but these assays are highly variable and often confuse by many artificial variations, such as the difficulty in selecting epithelial cell medium (ECM) and media that can maintain them simultaneously. Therefore, there is a need to establish a novel and robust assay system that co-develops supporting lineages for disease modeling and further screening applications. 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 / 185714 (Non-patent literature) (Non-patent document 1) Gori et al. "Investigating Non alcoholic Fatty Liver Disease in a Liver -on-a-Chip Microfluidic Device",PLoS One 11(7):e0159729. Doi10.1371 / journal.pone.0159729,July 20,2016. (Non-patent document 2) Park et al. "Lipotoxicity of Palmitic Acid on Neural Progenitor Cells and Hippocampal Neurogenesis", Toxicological Research, 27(2), 103:110, June 2011. (Non-patent document 3) Nandivada et al. "Treatment of Parenteral Nutrition-Associated Liver Disease: The Role of Lipid Emulsions", Nutr. Vol. 4: 711-717, November 2013. (Non-patent document 4) Cabezas et al. "Nonalcoholic Fatty Liver Disease: A Pathological View", Nobumi Tagaya, ISBN 978-953-51-0853-5, November 21, 2012. (Non-Patent Document 5) Kruitwagen et al. "Long-Term Adult Feline Liver Organoid Cultures for Disease Modeling of Hepatic Steatosis" Stem Cell Reports, vol. 8, no. 4, April 1, 2017. (Non-patent document 6) Kruitwagen et al. "Research Communications of the 26th ECVIM-CA Congress-20160908 to 2016091-SCH-O-5 LONG-TERM ADULT FELINE LIVER ORGANOID CULTURES FOR DISEASE MODELLING OF HEPATIC LIPIDOSIS", Journal of Veterinary Internal Medicine, vol.31,no.1,January 1,2017 (Non-patent document 7) Mori et al. "Micropatterned organoid culture of rat hepatocytes and HepG2 cells" Journal of Bioscience and Bioengineering. Vol. 106, no. 3, September 1, 2008. (Non-patent document 8) Nantasanti et al. "Concise Review: Organoids Are a Powerful Tool for the Study of Liver Disease and Personalized Treatment Design in Humans and Animals: Organoids for Disease Modeling and Therapy", Stem Cells Translational Medicine, vol.5, no.3.January 21,2016. (Non-Patent Document 9) Ricchi et al. "Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes" Journal of Gastroenterology and Hepatology, vol. 24, no. 5, May 1, 2009. [Overview of the project]

[0005] Disclosed herein are methods for preparing and using lipotoxic organoid models. In certain embodiments, the method may include the step of contacting liver organoids with a free fatty acid (FFA) composition. In one embodiment, the FFA composition may include oleic acid, linoleic acid, palmitic acid, or a combination thereof. [Brief explanation of the drawing]

[0006] 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.

[0007] A patent or application file must include at least one drawing made in color. A copy of the published patent or patent application containing the color drawing will be provided by the Office upon request and payment of the necessary fees.

[0008] [Figure 1]Co-differentiation of pre-fibrotic and inflammatory lineages in human iPSC liver organoids. A. Schematic diagram of a retinoic acid (RA)-based liver organoid differentiation method and bright-field images of liver organoids at day 20. Scale bar, 100 μm. B. Bright-field images of liver organoids in Matrigel at day 20 after 4 days of culture in and without RA. C. Organoid count was manually measured at day 20 after 4 days of culture in and without RA (black bars). D. Organoid diameter was recognized and measured by Image J at day 20 after 4 days of culture in and without RA (gray bars). E. Albumin production was measured from the culture supernatant collected 24 hours after 22-25 days of culture of liver organoids in Matrigel with and without 4 days of RA (black bars and gray bars). F. Organoids with internal lumen structures were manually counted on day 25 after 4 days of culture in and without RA. Red and gray bars indicate the percentage of organoids with / without internal lumen (HLO) / without internal lumen (spheroid), respectively. G. Bile transport activity was monitored by fluorescein diacetate (FD), which turns to fluorescein (green) after esterase hydrolysis of hepatocytes in culture medium, and the fluorescence flow was tracked. Results are expressed as mean ± standard deviation, n=3. The percentage of Epcam, CD166, and CD68-positive populations was determined by flow cytometry. Results are expressed as mean ± standard deviation, n=2-3. H. FPKM(log2) values ​​for hepatocytes, astrocytes, Kupffer cells, and endothelium-related genes expressed by unicellular RNA sequencing of human iPSCs, human iPSC-derived endoderm, foregut spheroids, HLOs, human adult liver tissue, and human fetal liver tissue. I. The proportion of Epcam, CD166, CD68, and F4 / 80-positive populations was determined by flow cytometry. Results are expressed as mean ± standard deviation, n=3. J. Immunofluorescence (IF) staining of HLO at day 25 for albumin, CD68, vimentin, GFAP, and Epcam. White arrows indicate the localization of cells positive for CD68, GFAP, and vimentin. Results are expressed as mean ± standard deviation, n=3.K. Phagocytic cell activity was analyzed by monitoring intracellular pH using a pHrodo indicator that reflects phagocytosis (red). Fluorescence expression was captured under a confocal microscope. Results are expressed as mean ± standard deviation, n=6. [Figure 2]Fatty Acid Treatment for Fatty Hepatitis Organoids (sHLOs) A. Schematic diagram of the method for generating fatty hepatitis HLOs (sHLOs) B. Live cell imaging of lipid droplets (green), membranes (red), and nuclei (blue). Images were obtained by superimposing 10-20 Z-stack images. Dose-dependent increases in lipid droplet accumulation and expansion of lipid droplets and cells were observed. C. Representative total lipid volume normalized by each organoid size. Bars indicate the average total lipid volume. 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 a single Matrigel droplet, separated into HCM medium in the presence or absence of oleic acid (800 μM) (blue bar) or (black bar), and cultured for 3 days. E. ELISA measurement of IL-6 using culture supernatant obtained from wells containing 20-30 HLOs cultured for 3 days in or without oleic acid (800 μM). Final values ​​were normalized by the number of organoids in each well. Compared to untreated (black bar), IL-6 was released 2.2 times more in the 800 μM OA-treated (blue bar). F. Gene expression of the pro-inflammatory cytokines TNF-alpha and IL-8. They were normalized by 18S. Both TNF-alpha and IL-8 gene expression were upregulated in the 200 μM OA (red bar) and 800 μM OA (blue bar) compared to untreated (black bar). G. 10-20 HLOs were cultured for 3 days in HCM medium containing 0, 400, and 800 μM OA. Culture supernatant was collected from each well, and THP-1 migration was measured by transmembrane use with these supernatants. Migratory 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 the HLO (black bar), sHLO (red bar), and cHLO (blue bar) populations. Results are expressed as mean ± standard deviation, for n=8-20 organoids. I. IF staining of HLOs on day 25 for Epcam and vimentin: Percentage of Epcam and vimentin-positive HLOs in the HLO (black bar), sHLO (red bar), and cHLO (blue bar) populations.Results are expressed as mean ± standard deviation, with n=8 to 20 organoids. J. 20 to 30 HLOs were cultured for 5 days in the presence or absence of oleic acid (800 μM) (blue bar). P3NPs were measured by ELISA using these supernatants. Final values ​​were normalized by the exact number of organoids in each well. P3NPs increased 2.8 times in the 800 μM OA treatment (blue bar) compared to the untreated group (black bar). [Figure 3] A. Schematic diagram for measuring the hardening of HLOs in fatty liver disease by AFM. The upper region of each single HLO (14 × 14 matrix in 25 × 25 sq μm) was scanned with an AFM cantilever, which can provide a spatial mapping of the topographic and mechanical information of the HLO. Scale bar, 100 μm. B. Representative histograms of the calculated Young's modulus (hardening of HLO; E, kPa) of single HLOs showed a Gaussian distribution, with its peak value and width increasing dose-dependently with respect to 0 (black), 200 (red), 400 (green), and 800 (blue) μM OA. C. Young's modulus (hardening of HLO) was determined from 7–12 organoids and summarized by dot plots with box plots. Increases in median values ​​were observed dose-dependently with respect to 0 (black), 200 (red), 400 (green), and 800 (blue) μM OA. [Figure 4] The stiffness of cHLO summarizes the clinical phenotype of Wolmann disease. A. Bright-field images of HLO and cHLO established from several iPSC lines, including healthy individuals (317D6), NAFLD patients (NAFLD150, NAFLD77, and NAFLD27), and Wolmann disease patients (WD90, WD92, and WD91). B. Mean Young's modulus (hardness of HLO: Pa) of single HLO and cLO derived from several iPSC lines. [Figure 5A] Modeling of human phenotypic mutations in steatosis using iPSC-sHLO: A. Representative allele functions of PNPLA3, GCKR, and TM6SF2 involved in increased 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 represent sHLO treated with 800 μM OA and 200 μM OA, respectively. [Figure 5F] F. Representative gene expression of pro-inflammatory cytokines in the total polygenic scoring of three groups of three alleles: 0.6 < x (black bar), 0.3 < x < 0.6 (red bar), x <= 0.3 (blue bar). [Figure 5G] G. Average Young's modulus of a single cLO in the total polygenic scoring of three groups of three alleles (HLO stiffening: 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- by 400 and 800 μM OA [Figure 9]A. Photographs of E-cad-positive and E-cad-negative sorted reconstituted spheroids from AE-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] Resveratrol has no effect on ROS production in liver organoids. [Figure 11] The percentage of BODIPY-positive cells (lipids) was measured by flow cytometry. The results are expressed as mean ± standard deviation, with n=5. [Figure 12] Lipid accumulation in organoids in the presence and absence of 400 μM intralipid. [Figure 13] Bright-field images of liver organoids in untreated cultures, cultures with 800 μM OA alone, and cultures with 800 μM OA and 40 ng / ml FGF19. [Modes for carrying out the invention]

[0009] Unless otherwise specified, terms should be understood in accordance with their conventional usage by those skilled in the art.

[0010] 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.

[0011] 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.

[0012] 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 (endoderm, gastrointestinal 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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 of 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).

[0018] 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; and Klimanskaya et al., 2005, "Human embryonic stem cells derived without feeder This can be found in "cells", Lancet 365(9471):1636-1641, and each of those descriptions is incorporated herein by reference in its entirety.

[0019] Induced pluripotent stem cells (iPSC) 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., Pou5fl); 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.

[0020] In some embodiments, non-viral techniques are used to produce iPSCs. In some embodiments, adenoviruses are used to deliver four required genes into 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 systems, 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 similar methodologies, and repeated treatment of cells with specific proteins guided into the cells 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.

[0021] 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 This can be found in "Proteins", Cell Stem Cell 4(5):381-384, and each of those descriptions 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-DF4-3;iPS-DF6-9;iPS(precipitous);iPS(IMR90); and iPS(IMR90).

[0023] 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.

[0024] 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 may be derived from a morula. In some embodiments, pluripotent stem cells may be 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 in embryos.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.

[0025] Non-alcoholic fatty liver disease (NAFLD) is one of the major challenges that developed countries must overcome due to its increased likelihood of developing fatal liver damage, but currently there is no effective treatment. Similarly, iatrogenic parental nutrition-related liver disease (PNALD) is a disease for which there is currently no effective treatment. There is a need for models that possess the clinicopathological features of liver disease, such as steatohepatitis and / or accumulation of lipid droplets and breakdown of bladder skeletal filamentous tissue, steatosis, and hepatocyte swelling. Despite the promise of disease models using patient stem cells, current approaches are limited in their application to single-celled, single-genetic, and relatively simple pathological conditions, and fail to capture more common and complex pathological conditions such as epithelial organ fibrosis.

[0026] In one embodiment, a method for preparing a lipotoxic organoid model is disclosed. This method may include the step of contacting liver organoids prepared according to the method herein with a free fatty acid (FFA) composition. The FFA composition may include oleic acid, linoleic acid, palmitic acid, or a combination thereof, preferably oleic acid. The amount of FFA can be determined by those skilled in the art. In one embodiment, FFA, preferably oleic acid, can be brought into contact with liver organoids in amounts of about 10 μM to about 10,000 μM, or about 20 μM to about 5,000 μM, or about 30 μM to about 2,500 μM, or about 40 μM to about 1,250 μM, or about 50 μM to about 1,000 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. FFA can be exposed to liver organoids for approximately 1 hour to 10 days, or approximately 2 hours to 9 days, or approximately 3 hours to 8 days, or approximately 4 hours to 7 days, or approximately 5 hours to 6 days, or approximately 6 hours to 5 days, or approximately 7 hours to 4 days, or approximately 8 hours to 3 days, or approximately 9 hours to 2 days, or approximately 10 hours to 1 day. In one embodiment, the range is approximately 3 to 5 days, ±24 hours.

[0027] In one embodiment, lipotoxic organoid models are used as models for fatty liver disease.

[0028] In one embodiment, lipotoxic organoid models are used as models for fatty liver disease.

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

[0030] In one embodiment, lipotoxic organoid models are models of parenteral nutrition-related liver disease (PNALD).

[0031] In one embodiment, lipotoxic organoid models are models of NAFLD.

[0032] In one embodiment, lipotoxic organoid models may be characterized by cytoskeletal filamentous tissue breakdown, increased ROS, mitochondrial swelling, triglyceride accumulation, fibrosis, hepatocyte ballooning, IL-6 secretion, steatosis, inflammation, ballooning and Mallory's body-like formation, tissue sclerosis, cell death, and combinations thereof.

[0033] In one embodiment, a method for screening drugs for the treatment of liver diseases, including NAFLD and / or cholestasis, is disclosed. This method may include the step of contacting a candidate drug with a lipotoxic organoid model, as disclosed herein.

[0034] In one embodiment, nutritional supplements , or high-calorie intravenous fluids ( TPN ) A method for assaying the effectiveness of nutritional supplements is disclosed. ,or, The procedure may include the step of contacting TPN with a lipotoxic organoid model such as those disclosed herein.

[0035] In one embodiment, 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 histosclerosis, and cell death.

[0036] In one embodiment, a three-dimensional (3D) liver organoid model is used to study drug-induced hepatotoxicity and inflammation. ,or, This is a model of fibrosis.

[0037] In one embodiment, a three-dimensional (3D) liver organoid model is used as a model for parenteral nutrition-related liver disease (PNALD).

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

[0039] Also disclosed is a method for inducing the formation of liver organoids from iPSC cells, which may be used in the aforementioned method and / or for obtaining the aforementioned composition. The method may include the following steps:

[0040] The process 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.

[0041] 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.

[0042] In some embodiments, the DE culture is treated with one or more molecules of the 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 molecule according to the present invention is suspended in a medium containing DMEM and fetal bovine serum (FBS). FBS may be present in concentrations of 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, or 50% or more. Those skilled in the art will understand that the regimens described herein are applicable, alone or in combination, to any known molecule in the signaling pathway described herein, including, but not limited to, any molecule in the FGF signaling pathway.

[0043] Appropriate GSK3 inhibitors will be readily apparent to those skilled in the art. Examples of GSK3 inhibitors include, but are not limited to, 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.

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

[0045] 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.

[0046] 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, on days 40 to 50. The expression levels may be similar to those observed in human hepatocytes, for example, adult hepatocytes.

[0047] In one embodiment, liver organoids may be characterized by having bile transport activity.

[0048] In one embodiment, liver organoids may be derived from stem cells, and internalized microvilli Hair Furthermore, the tubular structure , and mesenchymal cells It may include the following. The luminal structure may be surrounded by polar hepatocytes and a basement membrane. Liver organoids may contain functional stellate cells and functional Kupffer cells.

[0049] In a particular 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, liver organoids have at least 10,000 ng / mL 1xe 6 Liver organoids may be characterized by having total liver protein expression per cell / 24 hours. 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, liver organoids may include cells containing drug-metabolizing cytochrome mutants, such as CY2C9*2 mutants. Liver organoids may include vascular systems, such as those described in US2016 / 0177270.

[0050] In one embodiment, liver organoids may be characterized by the absence of inflammatory cells, such as T cells or other inflammatory secretory proteins. [Examples]

[0051] Human organoid systems that achieve higher-order functions of 3D tissue closely resemble in vivo organ structures in both healthy and diseased states, but they have failed to capture more common and complex pathological conditions such as inflammation and fibrosis. Here, the applicant developed a multicellular human liver organoid (HLO) model exhibiting essential features of steatohepatitis. Fatty acid exposure in culture allows for the induction of persistent steatosis, followed by the progressive activation of pro-inflammatory and fibrotic lineages that develop extensive fibrosis in the HLO. Interestingly, the expression of the steatohepatitis phenotype is strongly influenced by clinically reported genetic factors. Atomic force microscopy measurements revealed that overall organoid sclerosis correlates with the severity of inflammation and fibrosis. The fidelity of the measurements to the clinical phenotype was confirmed using three monogenic steatohepatitis-specific iPSC lines. Furthermore, the applicant established an iatrogenic parental nutrition-related liver disease (PNALD) model in organoids.

[0052] Retinoic acid (RA) signaling is a well-known and important specifier for the thyroid, lung, and pancreas from the foregut endoderm during the early organ specification phase, but it is clearly not essential for liver specification in model organisms (Kelly and Drysdale, 2015, with the exception of zebrafish (Negishi et al., 2010)). In fact, African clawed frogs and chickens are similar to mammals in that liver specification occurs in embryos lacking RA (Chen et al., 2004, Stafford et al., 2004). Conversely, several animal studies suggest that RA promotes hepatic astrocytocyte differentiation from mesenchyme, which is partially regulated by the zinc finger transcription factor WT1 expressed in STM and astrocytes (Ijpenberg et al., 2007, Wang et al., 2013). Furthermore, several studies suggest that balanced RA regulation induces monocyte fate specification from human stem cells (Purton (et al., 2000, Ronn et al., 2015). Furthermore, subsequent hepatomegaly growth is promoted by RA signaling via an unknown mechanism (Zorn and Wells, 2009). Given the broader role of RA in the identification of both parenchymal and non-parenchymal cells, the applicant hypothesized that timed RA exposure could influence the phylogenetic diversification of hepatic stromal cells, including pro-inflammatory lineages, thereby facilitating the creation of human liver organoids to model inflammation and fibrosis.

[0053] Recently, targeted differentiation of PSCs into intestinal organoids via foregut spheroid generation 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. Using this foregut generation method, the applicant tested the hypothesis that timely RA pulses to liver organoids preferentially co-differentiate the supporting lineage after liver specificization from human iPSCs. By promoting close interaction between epithelial and supporting lineages in a 4-D state where they can co-generate, the applicant demonstrated the applicability of liver organoids to model inflammation and fibrosis. The applicant also established a screening platform for determining fibrosis severity by evaluating sclerosis at the single organoid level in a living state. This method serves as a valuable application platform for the study of inflammation and fibrosis in epithelial organs toward drug development and personalized treatment.

[0054] result Generation of liver organoid models based on rheumatoid arthritis (RA) from human iPSCs To clarify whether RA signaling influences the determination of phylogenetic differentiation into stromal cells, the applicant established a liver organoid model from human iPSCs via transient induction of RA. As previously described (Spence et al., 2011), the applicant first differentiated iPSCs into foregut spheroids via identification of endoderm (DE) of the embryo. The foregut spheroids were mixed with adherent cells grown in the same well, and the mixture was embedded in Matrigel. Since RA is a known designator for diverse phylogenetic lines via an environment-dependent process, the applicant set the duration of early RA signaling prior to the hepatocyte maturation process, characterized by organoids established on day 20 as shown in Figure 1, A, cultured in hepatocyte culture medium (HCM). The highest concentration of albumin in the culture medium was observed with 4 days of RA exposure compared with various periods of RA from 0 to 5 days, so further characterization of the established organoids was compared between the two conditions of RA exposure from 0 to 4 days. In RA-treated wells, the number of organoids increased 1.8 times compared to untreated wells, and their size increased 1.5 times 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) compared to the untreated group (0.5–3.5 μg / ml, Figure 1, E). Notably, albumin secretion was maintained for more than 40 days from 20 days after albumin was first detected (data not shown). Interestingly, internal lumen structure appeared in 95% of RA-treated organoids, but was detected in only 12% of untreated organoids, indicating that organoid lumen formation is dependent on RA signaling (Figure 1, F).

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

[0056] Simultaneous differentiation of pre-fibrotic and inflammatory lineages in human iPSC liver organoids Interestingly, single-cell RNA sequencing (scRNA-seq) analysis of RA-treated HLO cells showed specific expression traces in astrocytes, hepatic endogenous macrophages, Kupffer cells, and endothelial cells, as evidenced by astrocyte 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 astrocytes and Kupffer cells, the applicant performed quantitative analysis by FACS using the epithelial cell marker EpCAM, the astrocyte 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% of HLO cells, while 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). Immunohistochemistry detected CD68 expression in HLO cells, and CD68 expression was localized to cells on the luminal side (Figure 1, J). Vimentin and GFAP (Geerts et al., 2001, Kordes et al., 2014), novel markers of human hepatic astrocytes reported to be necessary for the process of astrocyte differentiation in rodents, were both detected in HLO cells and co-expressed in the same cells (Figure 1, J), indicating the presence of astrocytes in HLO cells.

[0057] To investigate whether CD68-expressing cells are functionally active in HLOs similar to Kupffer cells, the applicant monitored phagocytic activity in HLOs, which has been reported to be active in Kupffer cells present in the liver, using a pH-sensitive rhodamine-based live cell dye that undergoes a dramatic increase in fluorescence (red) in response to the high-to-low pH environmental shift that occurs during phagocytosis, as observed in THP1 human macrophage cells (Figure 8). Activity was detected and localized in HLOs similar to that in 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 spontaneously generates multilineage hepatic stromal cells, including astrocytes 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 initiators to establish in vitro lipotoxic hepatocyte models, however, lipid accumulation is the only phenotype (Kanuri and Bergheim, 2013). Alternative co-culture models of hepatocytes and Kupffer cells showed inflammatory responses, including increased expression of IL family cytokines, macrophage-associated cytokines, and MMP-associated cytokines, but not subsequent fibrosis (Hassan et al., 2014). Given the presence of hepatocytes, astrocytes, and Kupffer-like cells with lipid metabolism function-related genes, as shown by comprehensive transcriptome analysis, the applicant hypothesized that FFA exposure spontaneously induces an inflammatory response and the resulting fibrous response in HLOs. To test this hypothesis, the applicant treated HLOs with FFAs for 3–5 days. The applicant presented 3-day and 5-day OA-treated HLOs as steatohepatitis (s)HLOs and cirrhotic (c)HLOs (Figure 4, A). sHLOs are HLOs treated with oleic acid for 3 days and show lipid accumulation and inflammation. cHLOs are HLOs treated with oleic acid for 5 days and exhibit fibrosis (HLOs become sclerotic) 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), with respect to lipid accumulation, live-cell imaging was performed using the lipid dye BODIPY 3 days after FFA exposure. As shown in Figure 7, OA was most effective in inducing lipid accumulation in hepatocyte-like cells, while SA was the least effective. Considering that OA causes massive lipid accumulation, the concentration of OA was varied to induce an inflammatory response in sHLOs (0, 200, 400, and 800 μM). Live-cell imaging and subsequent quantification showed that lipid accumulation increased 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 enlarged (Figure 4, B).Hepatocyte ballooning (magnification) is one of the pathological grading indicators for determining non-alcoholic steatohepatitis (NASH) activity (NAS scoring), and this was confirmed by live imaging of the cell membrane in 800 μM OA-treated sHLO (Figure 4B). Triglycerides, the main components 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 the culture supernatant showed that IL-6 was secreted 2.2 times more in 800 μM OA-treated sHLO medium compared to untreated medium (Figure 4, E). IL-8 and TNF-alpha were also upregulated under 200 μM or 800 μM OA-treated conditions (10-fold and 2-fold increases, respectively; Figure 4, F). Furthermore, THP1 migration was evaluated 24 hours after culturing in Transwell using OA-treated sHLO acclimatized medium, and THP-1 migrating cells were elevated in OA-treated sHLO, suggesting that FFA treatment spontaneously induced an inflammatory response in sHLO, possibly by stressing hepatocyte-like cells (up to 2x: Figure 4, G and Figure 8). To confirm whether these inflammatory and fibrotic features are specific to HLO, the applicant isolated the epithelial marker E-cad from organoids derived from E-cad mRuby embryonic stem (ES) cells, reconstituted spheroids from either E-cad-positive or E-cad-negative cells, and subjected them to ELISA for IL6 and P3NP, as well as RNA expression for IL8 and TNF-α. As shown in Figure 9, neither E-cad-positive nor E-cad-negative cell-derived spheroids induced P3NP and IL-6 secretion production, nor did they overexpress the genes for inflammatory markers. The applicant also tested the same experiment using the human hepatocyte cell line HepG2, the macrophage cell line THP-1, and the hepatic astrocytocyte LX-2, and obtained similar results for E-cad-positive and E-cad-negative cells. The results showed that liver organoids responded only to FFA treatment for inflammatory and fibrotic reactions.

[0059] To further determine whether HLOs progress toward a fibrosis-like state, the applicant 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 the epithelial marker Epcam and the 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 fibrous populations and matrix deposition in cHLOs. In addition, P3NP ELISA was also measured on day 5 of OA exposure. Compared to untreated HLOs, P3NP secretion increased 2.8-fold in the medium of 800 μM OA-treated HLOs (Figure 4, J). In summary, these observations indicate that FFA exposure not only causes triglyceride accumulation but also induces inflammatory responses and fibrosis in HLOs.

[0060] cHLO profiles of inflammation and fibrosis induced by FFA treatment at the single-cell level High-throughput quantification of fibrosis using AFM Accumulated evidence shows that liver sclerosis correlates well with the severity of hepatic fibrosis (Yoneda et al., 2008), thus suggesting that measuring HLO sclerosis may be an assessment of the severity of HLO fibrosis. The applicant's preliminary qualitative analysis of smooth muscle actin immunostaining shows dose-dependent fibrosis progression with OA exposure (Figure 2, H, I, and J). To gain more quantitative insights in a screenable format, the applicant thus assessed in living HLO fibrosis whether it could increase sclerosis in a dose-dependent manner using micro-indentation with atomic force microscopy (AFM). As shown in Figure 4A, the upper region of each single HLO (14×14 matrix of 25×25 μm squares) was scanned with an AFM cantilever, which can provide a spatial mapping of topographic and mechanical information of the HLO. Representative histograms of the calculated Young's modulus (E, kPa) of a single HLO clearly showed a Gaussian distribution, with its peak value and width increasing with increasing OA concentration (Figure 4, B; 0, 200, 400, 800 μM). The Young's moduli determined from HLO were summarized by a dot plot using box plots (Figure 4, C), which showed a gradual increase in the hardness of HLO from 0 to 800 μM OA, i.e., a gradual shift in the median and an expansion of the hardening range, observed with increasing OA concentration. The emmedian 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. Furthermore, 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 is known to correlate with fibrosis severity in NAFLD patients (Ajmera et al., 2017). These results suggest that HLO sclerosis can be a quantifiable measure of fibrosis severity and therefore potentially applicable to high-throughput screening for fibrosis.

[0061] cHLO stiffness replicates the clinical phenotype of Wolmann disease. The clinical relevance of pluripotent stem cell-derived models for fibrosis and cirrhosis is unclear due to the uncertain nature of genetic influences in patients. Predisposition to fibrosis is likely not captured by this model. Therefore, the applicant investigated the fidelity of the assay to clinical phenotypes by evaluating iPSC-derived congenital steatohepatitis patients, including healthy individuals (317D6), NAFLD / NASH patients (NAFLD150, 77, and 27), and Wolmann disease patients (WD90, 91, and 92) (Figure 5, A). Specifically, the applicant established three Wolmann disease patient-specific iPSC strains, which are monogenetic disorders with fatal steatohepatitis, and confirmed a significant increase in stiffness compared to organoids derived from normal iPSC strains, particularly with a notable correlation with enzyme activity at the time of clinical diagnosis (Figure 5, B).

[0062] Modeling of human phenotypic variations in steatosis using iPSC-sHLO Secondly, the applicant extended this novel organoid model to evaluate the polygenetic effects on the progression of steatohepatitis. 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) (Zain et al., 2015) (Figure 6, A), have been repeatedly shown by large-scale GWAS to be independent determinants of liver triglyceride content (HTGC). Therefore, the applicant approached a large cell bank and mapped 2504 cell lines with a polygenetic score (Figure 6, B) by summing the reported effect magnitude β(SNP) (Stender et al., 2017) and the allele-specific change in standardized HTGC multiplied by the weighted dose, as shown below: Σ{β(SNP) × (dose of risk allele)}. After acquiring iPSC lines with three different thresholds (Figure 6, B and C), the applicant generated seven iPSC organoids and induced steatohepatitis to estimate the effect of polygeneity on phenotype. Surprisingly, the applicant found a significant correlation between "steatosis" (by live imaging, Figure 6, D and E) and "inflammation" (qRT-PCR, Figure 6, F), but no correlation was found between "fibrosis" (sclerosis measurement based on AFM, Figure 6, G) and the polygenetic score. The applicant also found that OCA responses differed among cell lines, and that the response to OCA depended on the number of SNPs (Figure 6H).

[0063] discussion Multicellular liver organoids from human PSCs A series of recent studies have reported the 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 RA pulsation-based method naturally involves cells in diversifying with sustained cell polarity and is remarkably reproducible and scalable at a reasonable cost. Regardless of whether the precise nature of the supporting lineages is developmentally related or liver-specific, the stromal population is fully responsive to known fibrosis-inducing substances, including LPS and fatty acids, opening up new methods for modeling multicellular and complex pathologies.

[0064] Prospects for mechanical organoid screening to analyze inflammation and fibrosis From a future screening perspective, single organoid-based liver measurements are highly attractive readouts due to their robustness, normalization, and relatively simple nature. For example, a functional swelling assay has been established for intestinal organoids in cystic fibrosis patients based on live fluorescence imaging analysis of organoids, demonstrating effective drug selection (Saini, 2016). Live stiffness assessment of single liver organoids from iPSCs is an effective method for predicting the severity of fibrosis. In patients with fatty liver disease, a significant correlation between liver stiffness measurements and the stage of fibrosis has been clinically reported 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 has been observed in a subgroup of subjects with NAFLD as defined by ultrasound (Koehler et al., 2016). Interestingly, human liver organoid (HLO) sclerosis increased proportionally with both LPS and FFA, accompanied by inflammatory cytokine production and fibroblast proliferation. Given that numerous epithelial organ fibrosis species share similar phenotypes through diverse pathological mechanisms, organoid-based stiffness detection assays can be used to analyze fibrosis using lung, kidney, cardiac, and intestinal organoids.

[0065] Liver organoids allow for the in vivo evaluation of multiple parameters using high-content imaging systems. In fact, fat-treated liver organoids effectively visualize the number and size of lipid droplets, bile transport activity, increased phagocytic cells, and deterioration of cell morphology. 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 nutrient metabolites, organoid-based mechanical screening would be a very attractive model system for determining effective therapies in humans that would otherwise be difficult to test preclinically.

[0066] Nutritional precision medicine (personalized treatment) Patient-specific iPSC-derived organoids may be used to predict individualized 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 iPS cells is expected to model inter-individual differences in lipotoxicity, drug efficacy, and safety (Warren et al., 2017a, Warren et al., 2017b). In doing so, phenotypic screening using a liver organoid-based platform would facilitate the individualized selection of highly effective interventions for the disorder. For example, since nutritional supplementation may be modified on an individual basis, the disclosed system may be a highly suitable assay for reflecting nutrition-related status before administration. Specifically, patient-specific iPSC-derived organoids can be used to customize PN formulations for each patient with the aim of minimizing the possible progression to hepatic steatosis and fibrosis (PNALD). In fact, PN preparations are often customized in clinics (Mercaldi et al., 2012) because commercially available solutions do not meet the calorie, amino acid, and electrolyte needs of critically ill patients who are obese, require fluid restriction, and often exhibit hepatic / renal dysfunction (Boullata et al., 2014). Beyond nutritional needs, there is an urgent need to gain insights into mitigating potential adverse effects associated with PN products. Therefore, liver organoids are useful for assessing safety concerns, particularly regarding PNALD risk assessment, and facilitate customized PN preparation strategies for each patient.

[0067] Overall, the applicant demonstrated that enabling 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 concern with the increasing aging of the population. More broadly, the applicant has established a viable strategy for modeling complex human pathologies in conjunction with currently evolving organoid technologies (Lancaster and Knoblich, 2014) and opened up a new way to discover effective therapies for irreversible diseases at the single-organoid level.

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

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

[0070] On day 7 of HLO induction, spheroids and adherent cells were gently pipetted and detached from the dish. They were centrifuged at 800 rpm for 3 minutes, the supernatant was removed, and then embedded in 100% Matrigel droplets on a 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, the 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). The 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 Matrigel by scratching and pipetting.

[0071] Albumin, IL-6, and P3NP ELISA. To measure albumin secretion levels from 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 adhesion 96-well plates (Corning). To specify the exact number of organoids in each well and finally normalize the secretion levels of IL-6 and P3NP by number, organoids were captured using a KEYENCE BZ-X710 fluorescence microscope. Culture supernatant was collected at 24 hours (for albumin), 96 hours (for IL-6), and 120 hours (for P3NP) after incubation and stored at -80°C until use. The supernatant was centrifuged at 1,500 rpm for 3 minutes to form a pellet of fragments. 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 testing was performed by Student's t-test.

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

[0073] Phagocyte, lipid, and ROS live cell imaging. After culturing in ultra-low adhesion 6-multiwell plates, 5-10 HLO cells were picked and seeded onto Microslide 8-well glass-bottom plates (Ibidi, WI, USA) and stained for live cell action. The following antibodies were used: pHrodo® Red S. aureus Bioparticles® Conjugate (Thermo Fisher Scientific Inc.) for phagocyte 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 performed using NucBlue Live ReadyProbes Reagent (Thermo Fisher Scientific Inc.). HLO cells were visualized and scanned using a Nikon A1 inverted confocal microscope (Japan) with a 60x water immersion objective lens. The final lipid droplet volume was calculated using IMARIS8 and normalized by the size of each organoid. Significance testing for lipid droplet volume and ROS production (%) was performed by Student's t-test.

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

[0075] Flow cytometry was performed to isolate HLO cells from 10 Matrigel droplets and wash them with 1×PBS. HLO cells were dissociated into single cells by treatment with trypsin-EDTA (0.05%) and phenol red (Gibco) for 10 minutes. After washing with PBS, the 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, as well as treatment with OCA and FGF19, resulted in the isolation of 20-30 HLOs from Matrigel, washed with 1×PBS, and cultured under each condition on ultra-low adhesion 6-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). To test the inhibitory effects of OCA (INT-747, MedChem Express, NJ, USA) and recombinant human FGF19 (Sigma) on HLOs, 20-30 HLOs were cultured in HCM medium in or without oleic acid (800 μM), and 1 μM OCA and 40 ng / ml FGF19 were added to the 800 μM OA condition. HLOs were collected on day 3 for lipid living cell imaging and on day 5 for sclerosis measurements.

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

[0078] RNA isolation and RT-qPCR. RNA was isolated using the RNeasy mini-kit (Qiagen, Hilden, Germany). Reverse transcription was performed using the SuperScript III First-Strand Synthesis System (Invitrogen, CA, USA) for RT-PCR according to the manufacturer's protocol. qPCR was performed on a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific Inc.) 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). Significance testing was performed by Student's t-test.

[0079] AFM-based HLO curing measurement: HLO treated with 0, 50, 200, 1400, and 2800 ng / mL μM LPSOA was used for curing measurement using an AFM (NanoWizard IV, JPK Instruments, Germany). An AFM head with a silicon nitride cantilever (CSC37, k=0.3N / m, MikroMasch, Bulgaria) was attached to a fluorescence stereomicroscope (M205 FA, Leica, Germany) connected to a Z-axis piezo stage (JPK CellHesion module, JPK Instruments, Germany), allowing for indentation measurements to a depth of approximately 100 μm. Fibronectin-coated dishes were used as organoid substrates. Tissue culture dishes (φ=34 mm, TPP Techno Plastic Products, Switzerland) were first incubated overnight at 4°C with a 1 μg / mL fibronectin solution (Sigma). Subsequently, the tissue culture dish was washed twice with distilled water and dried for 1 hour. Then, the HLOs incubated with OALPS for 51 days were placed in a fibronectin-coated dish and incubated at 37°C for 1 hour. Next, the sample dish was placed on an AFM stage, and force-distance curves were measured from each HLO in a 25 × 25 μm square 14 × 14 matrix. Finally, the Young's modulus (E, Pa) of the HLOs was determined by fitting the obtained force-distance curves to a modified Hertz model (Sneddon, 1965). A 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 suspension cells were collected, and 200,000 cells were added to the membrane chamber of a CytoSelect® 96-Well Cell Migration Assay (5 μm, Fluorometric Format; Cell Biolabs, CA, USA) along with serum-free Advanced DMEM / F12. 10–20 HLOs were cultured for 3 days in HCM medium containing 0, 400, and 800 μM OA using an ultra-low adhesion 96-multiwell plate (Corning). Organoids were captured using a Keyence BZ-X710 fluorescence microscope to determine the exact number of organoids in each well and to normalize the number of ultimately migrated cells. 150 μL of the HLO culture supernatant was collected and added to the kit's feeder tray. The kit was incubated in a 5% CO2 cell culture incubator at 37°C for 24 hours. Migratory cells were counted using a Countess II FL Automated Cell Counter (Thermo Fisher Scientific Inc.). Significance testing was performed using Student's t-test.

[0081] Triglyceride Assay: For quantitative measurement of triglycerides, HLO was isolated from a single Matrigel droplet and separated into HCM medium in or without oleic acid (800 μM). These were cultured for 3 days on ultra-low adhesion 6-multiwell plates. Quantitative estimation of liver triglyceride accumulation was performed by an enzymatic assay of triglyceride mass using the EnzyChrom Triglyceride assay kit (Bioassay Systems, CA, USA).

[0082] HLO Survival Assay: HLOs were collected from Matrigel and washed with 1×PBS. 30–40 organoids were cultured on ultra-low adhesion 6-multiwell plates (Corning). HLOs were captured daily using a Keyence BZ-X710 fluorescence microscope. Surviving and dead organoids were manually counted from photographs. HLOs with a rounded shape were counted as viable, while organoids with different shapes were counted as dead. A 3D cell titer glo assay was used to assess the viability of OA-treated HLOs at the same time point (Promega, Wi, USA).

[0083] 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 ± standard error. P-values ​​less than 0.05 were considered statistically significant. Unless otherwise specified, the N-value refers to biologically independent copies.

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Claims

1. A three-dimensional (3D) liver organoid model of fatty liver disease, comprising an in vitro organoid characterized by ballooning, fibrosis, or histiosclerosis, wherein the in vitro organoid includes a tubular structure.

2. A three-dimensional (3D) liver organoid model of drug-induced hepatotoxicity and inflammation or fibrosis, comprising an in vitro organoid characterized by ballooning, fibrosis, or histiosclerosis, wherein the in vitro organoid includes tubular structures.

3. A three-dimensional (3D) liver organoid model of parenteral nutrition-related liver disease (PNALD), comprising an in vitro organoid characterized by ballooning, fibrosis, or histiosclerosis, wherein the in vitro organoid includes a tubular structure.

4. A 3D liver organoid model according to any one of claims 1 to 3, wherein the in vitro organoid is further characterized by one or more of steatosis, inflammation, ROS accumulation, or cell death.

5. A 3D liver organoid model according to claim 1 or 3, wherein the in vitro organoid does not contain T cells or inflammatory secreted proteins.

6. A 3D liver organoid model according to any one of claims 1 to 5, wherein the in vitro organoid comprises mesenchymal cells and the luminal structure comprises internalized microvilli.

7. A 3D liver organoid model according to any one of claims 1 to 6, wherein the in vitro organoid includes stellate cells and Kupffer cells.

8. A method for producing a 3D liver organoid model according to any one of claims 1 to 7, wherein the in vitro organoid is produced by activating the FGF pathway and inhibiting GSK3 in the endoderm (DE) of the embryo for a period of time sufficient to form the posterior foregut spheroid, and by incubating the posterior foregut spheroid for a period of time sufficient to form the in vitro organoid.

9. A method according to claim 8, wherein the period of time sufficient to form the posterior anorectal spheroid is 1 to 3 days.

10. A method according to claim 8 or 9, wherein the period of time sufficient to form the in vitro organoid is 1 to 5 days.

11. A method according to any one of claims 8 to 10, wherein the FGF pathway is activated by an FGF pathway activator 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.

12. The method according to claim 11, wherein the FGF pathway activator is FGF4.

13. A method according to any one of claims 8 to 12, wherein GSK3 is inhibited by CHIR99021.

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