US20200056157A1 - Liver organoid disease models and methods of making and using same - Google Patents

Liver organoid disease models and methods of making and using same Download PDF

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US20200056157A1
US20200056157A1 US16/346,190 US201716346190A US2020056157A1 US 20200056157 A1 US20200056157 A1 US 20200056157A1 US 201716346190 A US201716346190 A US 201716346190A US 2020056157 A1 US2020056157 A1 US 2020056157A1
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liver
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Takanori Takebe
Rie Ouchi
Masaki Kimura
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Cincinnati Childrens Hospital Medical Center
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Definitions

  • Irreversible epithelial organ remodeling is a major contributing factor to worldwide death and disease, costing healthcare systems billions of dollars every year (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).
  • COPD chronic obstructive pulmonary disease
  • inflammatory bowel disease Hynds and Giangreco, 2013.
  • most epithelial organ research performed primarily on animals fails to produce new therapies for these diseases and mortality rates remain unacceptably high. This is partly due to a lack of predictive human systems to test the efficacy of a vast growing number of compound libraries in pharmaceutical industries, imposing a fundamental challenge to develop a high-fidelity system for modeling inflammation and fibrosis in humans towards clinically relevant therapy development.
  • Non-alcoholic Fatty Liver Disease is one of the major challenges to be overcome in developed countries due to the increased chance of developing lethal liver disorders, yet effective treatment is lacking.
  • iatrogenic parenteral nutrition associated liver disease is a disorder that arises from parenteral nutrition and which currently has no effective treatment.
  • Models having steatohepatitis and/or clinical histopathological features of liver disease such as lipid droplet accumulation and cystoskeleton filament disorganization, steatosis, and hepatocellular ballooning, and which can be used to address these and other liver disorders or disease states, are needed.
  • current approaches are limited in their applications to unicellular, monogenic and relatively simple pathologies, failing to capture more prevailing and complex disease pathology such as epithelial organ fibrosis.
  • the human liver is a vital organ that provides many essential metabolic functions for life such as lipid metabolism, ammonium and bile production, coagulation, as well as the detoxification of exogenous compounds.
  • iPSC induced pluripotent stem cell
  • in vitro reconstitution of patients' liver reaction is attractive to the pharmaceutical industry, due to a number of promising applications including regenerative therapy, drug discovery, and drug toxicity studies.
  • conventional in vitro approaches study two-dimensional (2-D) and 3-D differentiation platforms to generate liver cells.
  • most of the reported methods predominantly differentiate cells into target epithelial cell type, completely lacking an essential supporting component such as pro-fibrotic and/or inflammatory cell types.
  • the methods may comprise the steps of contacting a liver organoid with a free fatty acid (FFA) composition.
  • FFA free fatty acid
  • the FFA composition may comprise oleic acid, linoleic acid, palmitic acid, or combinations thereof.
  • FIG. 1 Co-differentiation of pre-fibrotic and inflammatory lineages in human iPSC-liver organoid
  • A Schematic representation of retinoic acid (RA)-based liver organoid differentiation method and bright-field image of day 20 liver organoids. Scale bar, 100 ⁇ m.
  • B Bright-field image of liver organoids in Matrigel at day 20 after the culture in the presence and absence of 4 days RA.
  • C Organoid numbers were measured manually at day 20 after the culture in the presence (black bar) and absence (gray bar) of 4 days RA.
  • D Diameter of organoid was recognized and measured by Image J at day 20 after the culture in the presence (black bar) and absence (gray bar) of 4 days RA.
  • E is
  • FIG. 2 Generation of steatohepatitis organoids (sHLO) by fatty acid treatment
  • A Schematic representing the method for generating steatohepatitis HLO (sHLO)
  • B Live-cell imaging of lipid droplets (green), membrane (red), and nuclear (blue). The image was adopted from the overlay of 10-20 Z-stack images. Increase of lipid droplet accumulation and enlargement of lipid droplets and cells were observed in a dose depended manner.
  • C Representative total lipid volume normalized by each organoid size. Bar shows the mean of the total lipids volume.
  • Lipid droplets were increased in a dose depended manner 0 (black), 200 ⁇ M OA (red), 400 ⁇ M OA (green), and 800 ⁇ M OA (blue).
  • D Quantification of triglycerides in HLO.
  • HLOs were isolated from one Matrigel drop and divided into HCM media in the presence (blue bar) or absence (black bar) of oleic acid (800 ⁇ M) and cultured for 3 days.
  • E ELISA measurement of IL-6 with culture supernatants obtained from the wells that contain 20-30 HLO cultured in the presence or absence of oleic acid (800 ⁇ M) and cultured for 3 days. The final values were normalized by the number of organoids in each well.
  • IL-6 was 2.2-fold released in 800 ⁇ M OA treatment (blue bar), when compared with non-treatment (black bar).
  • F. Gene expression of pro-inflammatory cytokines TNF-alpha and IL-8. They were normalized by 18S. Both TNF-alpha and IL-8 gene expression were upregulated in 200 ⁇ M OA (red bar) and 800 ⁇ M OA (blue bar), compared with untreated (black bar).
  • G. 10-20 HLO were cultured in HCM media including 0, 400, 800 uM OA for 3 days. The cultured supernatants were collected from each well, and THP-1 migration was measured by using transmembrane with those supernatants.
  • FIG. 3 Cirrhotic transition of steatohepatitis HLO measured by AFM.
  • A Schematic representation for measuring the stiffness of HLO by AFM. The top region of each single HLO (14 ⁇ 14 matrix in a 25 ⁇ 25 ⁇ m square) was scanned with an AFM cantilever, which can provide a spatial mapping of topographical and mechanical information of HLO. Scale bar, 100 ⁇ m.
  • B The representative histogram of calculated Young's modulus (stiffness of HLO; E, kPa) of single HLO showed Gaussian-like distribution and its peak values and width were increased in a dose depended manner 0 (black), 200 (red), 400 (green) and 800 (blue) ⁇ M OA.
  • C The representative histogram of calculated Young's modulus (stiffness of HLO; E, kPa) of single HLO showed Gaussian-like distribution and its peak values and width were increased in a dose depended manner 0 (black), 200 (red),
  • Young's modulus (stiffness of HLO) was determined from 7-12 organoids and summarized by the dot plot with box-and-whisker plot. Increase of the median value was observed in a dose dependent manner 0 (black), 200 (red), 400 (green) and 800 (blue) ⁇ M OA.
  • FIG. 4 cHLO rigidity recapitulates clinical phenotype of Wolman disease
  • A Bright-field image of HLO and cHLO established from several iPSC lines including healthy person (317D6), NAFLD patients (NAFLD150, NAFLD77, and NAFLD27), and the Wolman disease patients (WD90, WD92, and WD91).
  • B Young's modulus (stiffness of HLO: Pa) of average of single HLO and cLO derived from several iPSC lines.
  • FIG. 5 Modeling human phenotypic variation of steatosis using iPSC-sHLO
  • A Representative allelic functions of PNPLA3, GCKR, and TM6SF2 responsible for hepatic TG content increase.
  • C The table summarizes the donor characteristic including polygenic score assigned in further study.
  • FIG. 6 Phagocyte activity on THP-1
  • FIG. 7 Lipids accumulation comparison of 800 ⁇ M of OA, PA, LA, and SA.
  • FIG. 8 Actual migration cell number of THP-1 by 400 and 800 ⁇ M of OA
  • FIG. 9 A. Photo of E-cad positive and negative sorted reconstituted spheroids from E-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.
  • FIG. 10 No effectiveness of resveratrol on ROS production of liver organoids
  • FIG. 12 Lipids accumulation in the organoid in the presence and absence of 400 ⁇ M Intralipid
  • FIG. 13 Bright-field image of liver organoids in the culture of non-treatment, 800 ⁇ M of OA alone, and 800 ⁇ M of OA with 40 ng/ml FGF19.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
  • totipotent stem cells are stem cells that can differentiate into embryonic and extra-embryonic cell types. Such cells can construct a complete, viable organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.
  • pluripotent stem cells encompasses any cells that can differentiate into nearly all cell types of the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissues and nervous system).
  • PSCs can be the descendants of inner cell mass cells of the preimplantation blastocyst or obtained through induction of a non-pluripotent cell, such as an adult somatic cell, 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.
  • iPSCs induced pluripotent stem cells
  • hiPSC human iPSCs.
  • embryonic stem cells also commonly abbreviated as ES cells, refers to cells that are pluripotent and derived from the inner cell mass of the blastocyst, an early-stage embryo.
  • ESCs is used broadly sometimes to encompass the embryonic germ cells as well.
  • a precursor cell encompasses any cells that can be used in methods described herein, through which one or more precursor cells acquire the ability to renew itself or differentiate into one or more specialized cell types.
  • a precursor cell is pluripotent or has the capacity to becoming pluripotent.
  • the precursor cells are subjected to the treatment of external factors (e.g., growth factors) to acquire pluripotency.
  • a precursor cell can be a totipotent (or omnipotent) stem cell; a pluripotent stem cell (induced or non-induced); a multipotent stem cell; an oligopotent stem cells and a unipotent stem cell.
  • a precursor cell can be from an embryo, an infant, a child, or an adult. In some embodiments, a precursor cell can be a somatic cell subject to treatment such that pluripotency is conferred via genetic manipulation or protein/peptide treatment.
  • one step is to obtain stem cells that are pluripotent or can be induced to become pluripotent.
  • pluripotent stem cells are derived from embryonic stem cells, which are in turn derived from totipotent cells of the 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 the blastocyst, an early-stage embryo. Methods for deriving embryonic stem cells from blastocytes are well known in the art. Human embryonic stem cells H9 (H9-hESCs) are used in the exemplary embodiments described in the present application, but it would be understood by one of skill in the art that the methods and systems described herein are applicable to any stem cells.
  • Additional stem cells that can be used in embodiments in accordance with the present invention include but are not limited to those provided by or described in the database hosted by 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, 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 hosted by Princeton University and the University of Pennsylvania.
  • NSCB National Stem Cell Bank
  • UW-SCRMC University of Wisconsin Stem Cell and Regenerative Medicine Center
  • UW-SCRMC University of Wisconsin Stem Cell and Regenerative Medicine Center
  • Novocell, Inc. San Diego, Calif.
  • Cellartis AB Goteborg, Sweden
  • Exemplary embryonic stem cells that can be used in embodiments in accordance with 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).
  • embryonic stem cells More 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,”.
  • ES Embryonic Stem Cell Lines Derived from Human Blastocysts
  • EC embryonal carcinoma
  • iPSCs Induced Pluripotent Stem Cells
  • iPSCs are derived by transfection of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection is typically achieved through viral vectors, such as retroviruses. Transfected genes include the master transcriptional regulators Oct-3/4 (Pouf51) and Sox2, although it is suggested that other genes enhance the efficiency of induction. After 3-4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection.
  • iPSCs include but are not limited to first generation iPSCs, second generation iPSCs in mice, and human induced pluripotent stem cells.
  • a retroviral system is used to transform human fibroblasts intopluripotent stem cells using four pivotal genes: Oct3/4, Sox2, Klf4, and c-Myc.
  • a lentiviral system is used to transform somatic cells with OCT4, SOX2, NANOG, and LIN28.
  • Genes whose expression are induced in iPSCs include but are not limited to Oct-3/4 (e.g., Pou5fl); certain members of the Sox gene family (e.g., Sox1, Sox2, Sox3, and Sox15); certain members of the Klf family (e.g., Klf1, Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, and LIN28.
  • Oct-3/4 e.g., Pou5fl
  • Sox gene family e.g., Sox1, Sox2, Sox3, and Sox15
  • Klf family e.g., Klf1, Klf2, Klf4, and Klf5
  • Myc family e.g., C-myc, L-myc, and N-myc
  • Nanog LIN28.
  • non-viral based technologies are employed to generate iPSCs.
  • an adenovirus can be used to transport the requisite four genes into the DNA of skin and liver cells of mice, resulting in cells identical to embryonic stem cells. Since the adenovirus does not combine any of its own genes with the targeted host, the danger of creating tumors is eliminated.
  • reprogramming can be accomplished via plasmid without any virus transfection system at all, although at very low efficiencies.
  • direct delivery of proteins is used to generate iPSCs, thus eliminating the need for viruses or genetic modification.
  • generation of mouse iPSCs is possible using a similar methodology: a repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency.
  • the expression of pluripotency induction genes can also be increased by treating somatic cells with FGF2 under low oxygen conditions.
  • embryonic stem cells More details on embryonic stem cells can be found in, for example, 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 hereby incorporated herein in its
  • exemplary iPS cell lines include but not limited to iPS-DF19-9; iPS-DF19-9; iPS-DF4-3; iPS-DF6-9; iPS(Foreskin); iPS(IMR90); and iPS(IMR90).
  • pluripotent cells e.g., iPSCs or ESCs
  • Methods for producing definitive endoderm from pluripotent cells are applicable to the methods described herein. Exemplary methods are disclosed in, for example, “Methods and systems for converting precursor cells into intestinal tissues through directed differentiation,” U.S. Pat. No. 9,719,068B2 to Wells et al., and “Methods and systems for converting precursor cells into gastric tissues through directed differentiation,” US20170240866A1, to Wells et al.
  • pluripotent cells may be derived from a morula.
  • pluripotent stem cells may be stem cells. Stem cells used in these methods can include, but are not limited to, embryonic stem cells.
  • Embryonic stem cells may be derived from the embryonic inner cell mass or from the embryonic gonadal ridges. Embryonic stem cells or germ cells can originate from a variety of 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 creating 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 to D'Amour et al.; U.S. Pat. No.
  • Non-alcoholic Fatty Liver Disease is one of the major challenges to be overcome in developed countries to do the increased chance of developing lethal liver disorders, yet effective treatment is lacking.
  • iatrogenic parental nutrition associated liver disease is a disorder that currently has no effective treatment.
  • Models having steatohepatitis and/or clinical histopathological features of liver disease such as lipid droplet accumulation and cystoskeleton filament disorganization, steatosis, and hepatocellular ballooning are needed.
  • current approaches are limited in their applications to unicellular, monogenic and relatively simple pathologies, failing to capture more prevailing and complex disease pathology such as epithelial organ fibrosis.
  • a method of making a lipotoxic organoid model may comprise the steps of contacting a liver organoid made according to the methods described herein, with a free fatty acid (FFA) composition.
  • FFA composition may comprise oleic acid, linoleic acid, palmitic acid, or combinations thereof, preferably oleic acid.
  • the amount of FFA may be determined by one of ordinary skill in the art.
  • the FFA preferably oleic acid
  • a liver organoid in an amount of from about 10 ⁇ M to about 10,000 ⁇ M, or from about 20 ⁇ M to about 5,000 ⁇ M, or from about 30 ⁇ M to about 2500 ⁇ M, or from about 40 ⁇ M to about 1250 ⁇ M or from about 50 ⁇ M to about 1000 ⁇ M, or from about 75 ⁇ M to about 900 ⁇ M or from about 80 ⁇ M to about 800 ⁇ M, or from about 90 ⁇ M to about 700 ⁇ M, or about 100 ⁇ M to about 500 ⁇ M, or from about 200 ⁇ M to about 400 ⁇ M.
  • the FFA may be contacted with the liver organoid for a period of time of from about 1 hour to about 10 days, or from about 2 hours to about 9 days, or from about 3 hours to about 8 days, or from about 4 hours to about 7 days, or from about 5 hours to about 6 days, or from about 6 hours to about 5 days, or from about 7 hours to about 4 days, or from about 8 hours to about 3 days, or from about 9 hours to about 2 days, or from about 10 hours to about 1 day.
  • the range is about 3 to about 5 days, +/ ⁇ 24 hours.
  • the lipotoxic organoid model is a model of fatty liver disease.
  • the lipotoxic organoid model is a model of steatohepatitis.
  • the lipotoxic organoid model is a model of cirrhosis.
  • the lipotoxic organoid model is a model of parenteral nutrition associated liver disease (PNALD).
  • PNALD parenteral nutrition associated liver disease
  • the lipotoxic organoid model is a model of NAFLD.
  • the lipotoxic organoid model may be characterized by cytoskeleton filament disorganization, ROS increase, mitochondrial swelling, trigycleride accumulation, fibrosis, hepatocyte ballooning, IL6 secretion, steatosis, inflammation, ballooning and Mallory's body-like, tissue stiffening, cell-death, and combinations thereof.
  • a method of screening for a drug for treatment of a liver disease including NAFLD and/or cholestasis is disclosed.
  • the method may comprise the step of contacting a candidate drug with a lipotoxic organoid model as disclosed herein.
  • a method of assaying the effectiveness of a nutritional supplement/TPN is disclosed.
  • the method may comprise the step of contacting the nutritional supplement/TPN with a lipotoxic organoid model as disclosed herein.
  • the three-dimensional (3D) liver organoid model of fatty liver disease is disclosed, wherein the organoid is characterized by steatosis, inflammation, ballooning and Mallory's bodies, ROS accumulation and mitochondrial overload; fibrosis and tissue stiffening, and cell death.
  • the three-dimensional (3D) liver organoid model is a model of drug induced hepatotoxicity and inflammation/fibrosis.
  • the three-dimensional (3D) liver organoid model is a model of parenteral nutrition associated liver disease (PNALD)
  • the three-dimensional (3D) liver organoid model does not comprise inflammatory cells, for example T-cells or other inflammatory secreted proteins.
  • the method may comprise the steps of
  • RA retinoic acid
  • Fibroblast growth factors are a family of growth factors involved in angiogenesis, wound healing, and embryonic development.
  • the FGFs are heparin-binding proteins and interactions with cell-surface associated heparan sulfate proteoglycans have been shown to be essential for FGF signal transduction. Suitable FGF pathway activators will be readily understood by one of ordinary skill 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.
  • FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23 may be used to activate these pathways.
  • DE culture is treated with the one or more molecules of a signaling pathway described herein at a concentration 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.
  • concentration of signaling molecule is maintained at a constant throughout the treatment. In other embodiments, concentration of the molecules of a signaling pathway is varied during the course of the treatment.
  • a signaling molecule in accordance with the present invention is suspended in media comprising DMEM and fetal bovine serine (FBS).
  • the FBS can be at a concentration of 2% and more; 5% and more; 10% or more; 15% or more; 20% or more; 30% or more; or 50% or more.
  • concentration of signaling molecule in accordance with the present invention is suspended in media comprising DMEM and fetal bovine serine (FBS).
  • the FBS can be at a concentration of 2% and more; 5% and more; 10% or more; 15% or more; 20% or more; 30% or more; or 50% or more.
  • the regiment described herein is applicable to any known molecules of the signaling pathways described herein, alone or in combination, including but not limited to any molecules in the FGF signaling pathway.
  • GSK3 inhibitors will be readily understood by one of ordinary skill in the art.
  • Exemplary GSK3 inhibitors include, but are not limited to: Chiron/CHIR99021, for example, which inhibits GSK30.
  • Chiron/CHIR99021 Chiron/CHIR99021
  • GSK3 inhibitors suitable for carrying out the disclosed methods may be administered in an amount of from about 1 uM to about 100 uM, or from about 2 uM to about 50 uM, or from about 3 uM to about 25 uM.
  • One of ordinary skill in the art will readily appreciate the appropriate amount and duration.
  • the stem cells may be mammalian, or human, iPSCs.
  • the foregut spheroids may be embedded in a basement membrane matrix, such as, for example, the commercially available basement membrane matrix sold under the tradename Matrigel.
  • the liver organoids may be characterized in that the liver organoids may express alpha-fetoprotein (AFP), albumin (ALB), retinol binding protein (RBP4), cytokeratin 19 (CK19), hepatocyte nuclear factor 6 (HNF6), and cytochrome P450 3A4 (CYP3A4), HNF4a, E-cadherin, DAPI, and Epcam. Such expression may occur, for example, at day 40 to day 50. The expression level may be similar to that observed in human liver cells, for example, that of an adult liver cell.
  • AFP alpha-fetoprotein
  • ALB albumin
  • RBP4 retinol binding protein
  • CK19 cytokeratin 19
  • HNF6 hepatocyte nuclear factor 6
  • CYP3A4 cytochrome P450 3A4
  • HNF4a E-cadherin
  • Epcam Epcam
  • the liver organoid may be characterized in that the liver organoid has bile transport activity.
  • the liver organoid may be derived from a stem cell and may comprise a luminal structure further containing internalized microvilli and mesenchymal cells.
  • the luminal structure may be surrounded by polarized hepatocytes and a basement membrane.
  • the liver organoid may comprise functional stellate cells and functional Kupffer cells.
  • the liver organoid may, in certain aspects, be characterized by having one or more of the following: bile production capacity, bile transport activity, Complement factor H expression of at least 50 ng/mL/1 ⁇ e 6 cells/24 hr, Complement factor B of at least 40 ng/mL/1 ⁇ e 6 cells/24 hr, C3 expression of at least 1000 ng/mL/1 ⁇ e 6 cells/24 hr; C4 expression of at least 1000 ng/mL/1 ⁇ e 6 cells/24 hr, fibrinogen production of at least 1,000 ng/mL/1 ⁇ e 6 cells/24 hr and albumin production of at least 1,000 ng/mL/1 ⁇ e 6 cells/24 hr.
  • the liver organoid may be characterized by having total hepatic protein expression of at least 10,000 ng/mL 1 ⁇ e 6 cells/24 hours.
  • the liver organoid may be characterized in that it may express one or more genes selected from PROX1, RBP4, CYP2C9, CYP3A4, ABCC11, CFH, C3, C5, ALB, FBG, MRP2, ALCAM, CD68, CD34, CD31.
  • the liver organoid may comprise cells comprising a drug metabolism cytochrome variant, such as, for example, a CY2C9*2 variant.
  • the liver organoid may comprise a vasculature, such as that described in US 20160177270.
  • the liver organoid may be characterized in that the liver organoid does not comprise inflammatory cells, for example T-cells or other inflammatory secreted proteins.
  • HLO human liver organoid
  • Basal fatty acid exposure enables persistent steatosis induction, followed by progressive activation of pro-inflammatory and fibrotic lineages that develops massive fibrosis in HLO.
  • expression of the steatohepatitis phenotype is strongly influenced by clinically reported heritable factors.
  • Atomic force microscopy measurement revealed that overall organoid stiffening correlates the severity of inflammation and fibrosis. The measurement fidelity to clinical phenotype was confirmed using three monogenic steatohepatitis-specific iPSC lines.
  • PNALD parental nutrition associated liver disease
  • Retinoic acid (RA) signaling is a well-known important specifier of thyroid, lung, and pancreas from foregut endoderm during the early organ specification phase, whereas it is clearly not essential for specification of the liver in model organisms (Kelly and Drysdale, 2015) with the exception of zebrafish (Negishi et al., 2010). Indeed, xenopus and chick are similar to mammals as liver specification occurs in embryos lacking RA (Chen et al., 2004; Stafford et al., 2004).
  • RA promotes hepatic stellate cell differentiation from mesenchyme, which is controlled in part by the zinc finger transcription factor WT1 expressed in the STM and stellate cells (Ijpenberg et al., 2007; Wang et al., 2013). Additionally, several studies suggest that the balanced RA regulation induces monocyte fate specification from human stem cells (Purton et al., 2000; Ronn et al., 2015). Also, later liver bud growth is promoted by RA signaling via unknown mechanisms (Zorn and Wells, 2009).
  • Applicant also established a screen platform to determine the severity of fibrosis by evaluating stiffness at single organoid level in the living state. This method serves as an invaluable application platform for the study of epithelial organ inflammation and fibrosis towards drug development and personalized therapy.
  • Applicant established a liver organoid model from human iPSCs by transient induction of RA.
  • Applicant initially differentiated iPSCs to foregut spheroids through definitive endoderm (DE) specification as previously described (Spence et al., 2011).
  • DE definitive endoderm
  • the foregut spheroids were mixed with the attached cells grown in the same well, and the mixture was embedded in Matrigel.
  • RA is a known specifier for diverse lineages through a context dependent process
  • Applicant set the duration of initial RA signaling before the hepatocyte maturation process which is cultured in hepatocyte culture media (HCM) and characterized the established organoids at day 20 as illustrated in FIG. 1 , A. Since the highest concentration of albumin in culture media was observed with 4 days of RA exposure among the various duration of RA from 0 to 5 days, the further characterization of the established organoids was compared between two conditions of 0 and 4 days-RA exposure. The number of organoids was increased 1.8-fold in RA treated wells compared to untreated and the size was increased 1.5-fold after RA treatment ( FIG. 1 , B, C, and D).
  • Albumin secretion was increased two-fold in the RA treated group (3.0-6.5 ⁇ g/ml) when compared with untreated (0.5-3.5 ⁇ g/ml; FIG. 1 , E).
  • albumin secretion was maintained more than 40 days after day 20 when albumin was first detected (data not shown).
  • the internal luminal structure appeared in 95% of RA treated organoids, whereas it was detected in only 12% of untreated organoids, indicating that the lumenization of the organoid is dependent on RA signaling ( FIG. 1 , F).
  • HLO human liver organoids
  • scRNA-seq single-cell RNA sequencing analysis of RA treated HLO showed the expression signatures specific to stellate cells, liver resident macrophage 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; FIG. 1 , H) (Bahar Halpern et al., 2017; El Taghdouini et al., 2015; van de Garde et al., 2016).
  • HLO RA-based liver organoid
  • FFA free fatty acids
  • HLO steatohepatitis
  • cHLO cirrhosis
  • OA oleic acid
  • LA linoleic acid
  • PA palmitic acid
  • SA stearic acid
  • Live-cell imaging and subsequent quantitation displayed that lipid accumulation was elevated in sHLO in a dose dependent fashion (up to 18-fold; FIG. 4 , B and C).
  • the size of lipid droplets was enlarged ( FIG. 4 , B).
  • Hepatocyte ballooning (enlargement) was one of the pathological grading indicators to determine the nonalcoholic steatohepatitis (NASH) activity (NAS scoring), which was confirmed in 800 ⁇ M OA treated sHLO by live imaging of cellular membrane (FIG., 4B).
  • NASH nonalcoholic steatohepatitis
  • Triglycerides a main constituent of lipids accumulated in the liver, were also detected in 800 ⁇ M treated sHLO, but not in untreated sHLO ( FIG. 4 , D).
  • ELISA of culture supernatant showed that IL-6 was secreted 2.2-fold in 800 ⁇ M OA treated sHLO culture media when compared to the untreated ( FIG. 4 , E).
  • IL-8 and TNF-alpha were also upregulated in a 200 ⁇ M or 800 ⁇ M OA treated condition (10- and 2-fold increase, respectively; FIG. 4 , F).
  • E-cad E-cad mRuby embryonic stem (ES) cells derived organoids, reconstructed the spheroids from either E-cad positive or negative cells, and applied for ELISA for IL6 and P3NP and RNA expression for IL8 and TNF-alpha.
  • FIG. 9 indicates, neither E-cad positive nor negative cells derived spheroids produced P3NP and IL-6 secreted production and overexpressed the gene expression of inflammatory markers.
  • Applicant also tested same experiments using human hepatocyte cell line HepG2, Macrophage cell line THP-1, and hepatic stellate cell LX-2, and had a similar result of E-cad positive and negative cells. The results demonstrated that liver organoids only response to FFA treatment on inflammation and fibrosis response.
  • liver stiffness is well correlated with the severity of liver fibrosis (Yoneda et al., 2008) and thereby measuring the stiffness of HLO has potential to evaluate the fibrosis severity of HLO.
  • Applicant's preliminary qualitative analysis of smooth muscle actin immunostaining indicates dose dependent fibrosis progression by OA exposure ( FIG. 2 , H, I, and J).
  • AFM Atomic Force Microscopy
  • each single HLO 14 ⁇ 14 matrix in a 25 ⁇ 25 ⁇ m square
  • AFM cantilever which can provide a spatial mapping of topographical and mechanical information of HLO.
  • the representative histogram of calculated Young's modulus (E, kPa) of single HLO clearly showed a Gaussian-like distribution and its peak values and width were increased in accordance with the OA concentration ( FIG. 4 , B; 0, 200, 400, 800 ⁇ M). Young's moduli determined from HLOs were summarized by dot plot with box-and-whisker plot ( FIG.
  • TNF-alpha and IL-8 were also upregulated by OA addition, known to be correlated with fibrosis severity in NAFLD patients (Ajmera et al., 2017). These results indicate that stiffness of HLO can be a quantifiable measurement for the severity of fibrosis, and thus can be potentially applied for a high-throughput screening of fibrosis.
  • Applicant established three Wolman disease patient-specific iPSC lines, which is a mono-genetic disorder with lethal steatohepatitis, and confirmed the significant rigidity increase relative to normal iPSC line-derived organoids, notably with remarkable correlation to enzymatic activity at the time of clinical diagnosis ( FIG. 5 , B).
  • Applicant therefore approached large scale cell bank, and mapped out 2504 cell lines with the polygenic score ( FIG. 6 , B) by a summation of reported effect size ⁇ (SNP) (Stender et al., 2017), per-allele change in standardized HTGC multiplying weighted dosage as follow: ⁇ (SNP) ⁇ (dosage of risk allele) ⁇ .
  • ⁇ (SNP) ⁇ (dosage of risk allele) ⁇ After the acquisition of iPSC lines with three different thresholds ( FIG. 6 , B and C), Applicant has generated seven iPSC organoids, and induced steatohepatitis to estimate polygenic impacts on the phenotype. Strikingly, Applicant has found the remarkable correlation of ‘steatosis’ (by live imaging, FIG.
  • liver measurement is a highly attractive readout due to its robustness, normalization and relative simplicity.
  • a functional swelling assay was established for cystic fibrosis patients' gut organoids, demonstrating an effective drug selection (Saini, 2016).
  • Live rigidity assessment on a single liver organoid from iPSC is an effective way to predict the severity of fibrosis.
  • a significant correlation between liver stiffness measurement and fibrosis stage is clinically reported in fatty liver disease patients by elastography (Yoneda et al., 2008).
  • liver stiffness increased in proportion to both LPS and FFAs, accompanied by inflammatory cytokine production and fibroblast expansion.
  • an organoid based rigidity detection assay could be used to analyze fibrosis using lung, kidney, cardiac and gut organoids.
  • organoid based assay platforms may be used to better understand the human specific mechanisms of fatty liver diseases and establishing high-content screening for drug discovery for these diseases. Combined with compound libraries and nutritional metabolites, organoid based mechanical screening would be highly attractive model system for implicating effective therapy in humans, otherwise not testable preclinically.
  • Patient-specific iPSC-derived organoids might be used to predict individualized drug efficacy and epithelial response.
  • Human iPSCs can be established from both healthy and diseased individuals.
  • the use of patients' iPSCs is expected to model inter-individual differences of lipotoxicity, drug efficacy and safety concerns (Warren et al., 2017a; Warren et al., 2017b).
  • phenotypic screening using a liver organoid-based platform will promote personalized selection of highly effective interventions for the disorders.
  • the disclosed system may be a highly compatible assay for reflecting nutrition associated conditions before administration.
  • patient-specific iPSC-derived organoids can be used to customize PN formulation to each patient with the aim of minimizing the possible progression to hepatic steatosis and fibrosis (PNALD).
  • PNALD hepatic steatosis and fibrosis
  • PN formulation is often customized (Mercaldi et al., 2012) as commercial solutions do not meet the caloric, amino acid, and electrolyte needs of critically ill patients, who are often obese and require fluid restriction, and display hepatic/renal dysfunction (Boullata et al., 2014). Beyond nutritional needs, there is an urgent need to gain insights for a reduction of possible adverse effects associated with PN products. The liver organoid would thus be useful to evaluate safety concerns especially for PNALD risk assessment, facilitating the customization strategy of PN formulations for each patient.
  • Applicant has demonstrated that enabling crosstalk between epithelial and stromal lineages in a 4-D organoid culture is useful for modeling clinically relevant pathology associated with steatosis and fibrosis of liver. This model will eventually lead to analysis of more prevailing pathology such as NAFLD or NASH, which is a growing concern with population aging. More broadly, Applicant has established a viable strategy for modeling humanistic complex pathology coupled with currently evolving organoid technology (Lancaster and Knooff, 2014), paving a new way for the discovery of effective treatments against unrecoverable diseases at single organoid level.
  • hPSCs 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 described previously (Takebe et al., 2015; Takebe et al., 2014). Undifferentiated hiPSCs were maintained on 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% CO 2 with 95% air.
  • 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) at day 1, 100 ng/mL Activin A and 0.2% fetal calf serum (FCS; Thermo Fisher Scientific Inc.) at day 2 and 100 ng/mL Activin A and 2% FCS at day 3.
  • FCS fetal calf serum
  • Day4-6 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 3 uM CHIR99021 (Stemgent, MA, USA). Cells were maintained at 37° C. in 5% CO 2 with 95% air and the medium was replaced every day. Spheroids appeared on the plate at day 7 of differentiation.
  • HLO induction At day 7, spheroids and attached cells are gently pipetted to be delaminated from dishes. They were centrifuged at 800 rpm for 3 minutes, embedded in a 100% Matrigel drop on the dishes in Advanced DMEM/F12 with B27, N2 and 2 uM retinoic acid (RA; Sigma, MO, USA) after removing supernatant, and cultured for 4 days.
  • RA retinoic acid
  • HCM Hepatocytes Culture Medium
  • HGF hepatocyte growth factor
  • OSM Oncostatin M
  • Albumin, IL-6, and P3NP ELISA To measure albumin secretion level of HLO, 200 ⁇ L of culture supernatant was collected from HLO embedded in Matrigel. For IL-6 and P3NP, 20-30 organoids were seeded and cultured on an ultra-low attachment multiwell plates 96 well plate (Corning). To define the exact number of organoids in each well and lastly normalize the secreted level for IL-6 and P3NP by the number, the organoids were captured on The KEYENCE BZ-X710 Fluorescence Microscope. The culture supernatants were collected at 24 hrs (for albumin), 96 hrs (for IL-6) and 120 hrs (P3NP) time points after the culture and stored at ⁇ 80° C.
  • Bile transport activity Fluorescein diacetate was used for evaluating bile transport activity in organoids. 10 mg/mL fluorescein diacetate (Sigma) was added into HCM media cultured with HLO and allowed to sit for 5 minutes and captured using fluorescent microscopy BZ-X710 (Keyence, Osaka, Japan).
  • Phagocyte, lipids, ROS live-cell imaging After being cultured in an ultra-low attachment 6 multi-well plate, 5-10 HLO were picked up and seeded in a Microslide 8 Well Glass Bottom plate (Ibidi, WI, USA) and subjected to live-cell staining.
  • the following antibodies were used: pHrodo® Red S. aureus Bioparticles® Conjugate for Phagocyte activity (Thermo Fisher Scientific Inc.), BODIPY® 493/503 for lipids (Thermo Fisher Scientific Inc.), Di-8-ANEPPS for membrane (Thermo Fisher Scientific Inc.), and CellROX green reagent for ROS detection (Thermo Fisher Scientific Inc.).
  • lipid droplet volume was calculated by IMARIS8 and normalized by each organoid size. Significance testing for lipid droplet volume and ROS production (%) was conducted by Student's t-test.
  • HLO HE staining and immunohistochemistry.
  • HLO were isolated from Matrigel and fixed in 4% paraformaldehyde and embedded in paraffin. Sections were subjected to HE 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).
  • HLO were isolated from 10 Matrigel droplets and washed by 1 ⁇ PBS. HLO were dissociated to single cells by the treatment of Trypsin-EDTA (0.05%), phenol red (Gibco) for 10 min. After PBS wash, the single cells were subjected to flow cytometry with BV421-conjugated Epcam antibody (BioLegend), PE-conjugated CD166 antibody (eBioscience, CA, USA), and PE/Cy7-CD68 (eBioscience). DNA was measured by propidium iodide staining.
  • HLO LPS and FFA exposure and OCA and FGF19 treatment.
  • 20-30 HLO which had been isolated from Matrigel and washed by 1 ⁇ PBS, were divided into each condition and cultured on an ultra-low attachment 6 multi-well plates (Corning). HLO were cultured with LPS (Sigma), OA (Sigma), LA (Sigma), SA (Sigma), or PA (Sigma) and collected at day 1 and 3 (for LPS HLO) and at day 3 and 5 (for OA) after the culture.
  • HLO human FGF19 recombinant
  • 20-30 HLO were cultured in HCM media in the presence or absence of oleic acid (800 ⁇ M), and 1 ⁇ M OCA and 40 ng/ml FGF19 were added into 800 ⁇ M OA condition.
  • HLO were collected at day 3 for lipids live-cell imaging and at day 5 for stiffness measurement.
  • HLO Whole mount immunofluorescence. HLO were fixed for 30 min in 4% paraformaldehyde and permeabilized for 15 min with 0.5% Nonidet P-40. HLO were washed by 1 ⁇ PBS three times and incubated with blocking buffer for 1 h at room temperature. HLO were then incubated with primary antibody; anti-alpha smooth muscle actin antibody (1:50 dilution; abcam) overnight at 4° C. HLO were washed by 1 ⁇ PBS and incubated in secondary antibody in blocking buffer for 30 min at room temperature. HLO were washed and mounted using Fluoroshield mounting medium with DAPI (abcam). The stained HLO were visualized and scanned on a Nikon A1 Inverted Confocal Microscope (Japan) using 60 ⁇ water immersion objectives.
  • RNA isolation, RT-qPCR RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany). Reverse transcription was carried out using the SuperScriptIII First-Strand Synthesis System for RT-PCR (Invitrogen, CA, USA) according to manufacturer's protocol. qPCR was carried out using TaqMan gene expression master mix (Applied Biosystems) on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific Inc.). All primers 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 conducted by Student's t-test.
  • HLO stiffness measurement by AFM HLOs treated with 0, 50200, 1400, 2800 ng/mL ⁇ M LPSOA were used for stiffness measurement with a AFM (NanoWizard IV, JPK Instruments, Germany).
  • M205 FA fluorescence stereo microscope
  • JPK CellHesion module JPK Instruments, Germany
  • E, Pa Young's moduli
  • THP-1 cell migration assay THP-1 cell, which was gifted from T. Suzuki, was maintained in Advanced DMEM/F12 (Thermo Fisher Scientific Inc.) containing 10% FBS. THP-1 floating cells were collected, and 200,000 cells were added with serum-free Advanced DMEM/F12 to the membrane chamber of the CytoSelectTM 96-Well Cell Migration Assay (5 ⁇ m, Fluorometric Format; Cell Biolabs, CA, USA). 10-20 HLO were cultured in HCM media including 0, 400, 800 uM OA with an ultra-low attachment 96 multi-well plate (Corning) for three days.
  • organoids were captured on The KEYENCE BZ-X710 Fluorescence Microscope. 150 ⁇ L of culture supernatant of HLO was collected and added to the feeder tray of the kit. The kit was incubated at 37° C. for 24 h in a 5% CO 2 cell culture incubator. Cells that had migrated were counted using Countess II FL Automated Cell Counter (Thermo Fisher Scientific Inc.). Significance testing was conducted by Student's t-test.
  • Triglyceride assay For quantitative determination of triglycerides, HLO were isolated from one Matrigel drop and divided into HCM media in the presence or absence of oleic acid (800 ⁇ M). They were cultured on an ultra-Low attachment 6 multi-well plate for three days. Quantitative estimation of hepatic triglyceride accumulation was performed by an enzymatic assay of triglyceride mass using the EnzyChrom Triglyceride assay kit (Bioassay Systems, CA, USA).
  • HLO survival assay HLO were collected from Matrigel and washed by 1 ⁇ PBS. 30-40 organoids were cultured on an ultra-low attachment 6 multi-well plate (Corning). HLO were captured on The KEYENCE BZ-X710 Fluorescence Microscope every day. The surviving and dead organoids were counted manually from the photo. HLO with a rounded configuration was counted as the surviving while the organoids with out of shape is counted as dead. To assess the survival rate of OA treated HLO at the same time point, 3D cell titer glo assay was used (Promega, Wi, USA).

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11584916B2 (en) 2014-10-17 2023-02-21 Children's Hospital Medical Center Method of making in vivo human small intestine organoids from pluripotent stem cells
US11767515B2 (en) 2016-12-05 2023-09-26 Children's Hospital Medical Center Colonic organoids and methods of making and using same

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011140441A2 (en) 2010-05-06 2011-11-10 Children's Hospital Medical Center Methods and systems for converting precursor cells into intestinal tissues through directed differentiation
WO2015183920A2 (en) 2014-05-28 2015-12-03 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
CN116790476A (zh) 2016-05-05 2023-09-22 儿童医院医疗中心 用于体外制造胃底组织的方法和与其相关的组合物
EP3635095A4 (en) * 2017-06-09 2021-12-01 Children's Hospital Medical Center LIVER ORGANOID COMPOSITIONS AND METHOD FOR MANUFACTURING AND USING THEREOF
US20210254012A1 (en) * 2018-06-09 2021-08-19 Arizona Board Of Regents On Behalf Of Arizona State University Next generation designer liver organoids and their methods of preparation and use
SG11202102431YA (en) * 2018-09-12 2021-04-29 Childrens Hospital Med Ct Organoid compositions for the production of hematopoietic stem cells and derivatives thereof
AU2019351022A1 (en) * 2018-09-27 2021-04-15 Children's Hospital Medical Center Liver support system comprising liver organoids and methods of making and using same
EP3950931A4 (en) 2019-03-29 2022-11-30 Nagasaki University CULTIVATED TISSUE AND PROCESS OF PRODUCTION
WO2020243613A1 (en) * 2019-05-31 2020-12-03 Children's Hospital Medical Center Methods of generating and expanding hematopoietic stem cells
JP7039045B2 (ja) * 2019-08-26 2022-03-22 株式会社フェニックスバイオ ヒト脂肪肝モデル細胞
JP2023516484A (ja) 2020-03-11 2023-04-19 ビット バイオ リミテッド 肝細胞作製方法
CN113717850A (zh) * 2020-05-26 2021-11-30 中国科学院大连化学物理研究所 一种基于三维类肝芯片的非酒精性脂肪肝体外模型建立方法
WO2021243526A1 (zh) * 2020-06-01 2021-12-09 苏州大学附属儿童医院 新生儿肠外营养相关性肝病特异性标志物lncRNA的应用
WO2021262676A1 (en) * 2020-06-23 2021-12-30 Children's Hospital Medical Center Model for insulin resistance
EP4332215A1 (en) * 2021-04-28 2024-03-06 National University Corporation Tokyo Medical and Dental University Method for producing cells
WO2022236144A1 (en) 2021-05-06 2022-11-10 Massachusetts Institute Of Technology Ex vivo tissue explant and graft platform and uses thereof
CN113430890A (zh) * 2021-07-01 2021-09-24 驻马店市华中公路设计有限公司 用于旧沥青路面改造的永久性复合式路面结构及施工装置
CN115125187B (zh) * 2022-06-14 2023-04-07 创芯国际生物科技(广州)有限公司 一种子宫内膜炎症的类器官体外模型及其建立方法和应用
CN115386535B (zh) * 2022-10-26 2023-02-03 天津外泌体科技有限公司 多谱系肝类器官模型及基于该模型的药物肝毒评价方法
CN115418386B (zh) * 2022-11-03 2023-03-24 成都诺医德医学检验实验室有限公司 一种基于皮肤类器官的损伤愈合评价方法及模型和应用
WO2024185901A1 (ja) * 2023-03-09 2024-09-12 国立大学法人 東京医科歯科大学 脂肪性肝疾患を処置することに用いるための組成物
WO2024206911A2 (en) 2023-03-30 2024-10-03 Children's Hospital Medical Center Clinical-grade organoids

Family Cites Families (44)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003050249A2 (en) 2001-12-07 2003-06-19 Geron Corporation Islet cells from human embryonic stem cells
CN103898045B (zh) 2003-12-23 2019-02-01 维亚希特公司 定形内胚层
ES2523591T3 (es) 2006-06-27 2014-11-27 Intercept Pharmaceuticals Inc. Derivados de ácidos biliares como ligandos de FXR para la prevención o el tratamiento de enfermedades o estados mediados por FXR
DE602007012293D1 (ja) * 2006-12-08 2011-03-10 Du Pont
EP2412800A1 (en) * 2010-07-29 2012-02-01 Koninklijke Nederlandse Akademie van Wetenschappen Liver organoid, uses thereof and culture method for obtaining them
EP2393917B1 (en) 2009-02-03 2016-04-06 Koninklijke Nederlandse Akademie van Wetenschappen Culture medium for epithelial stem cells and organoids comprising said stem cells.
WO2010094694A1 (en) 2009-02-23 2010-08-26 F. Hoffmann-La Roche Ag Assays to predict cardiotoxicity
RU2011139643A (ru) 2009-03-17 2013-04-27 Акскан Фарма, Инк. Способ лечения неалкогольного стеатогепатита повышенными дозами урсодезоксихолевой кислоты
EP2443230B1 (en) * 2009-06-18 2017-07-26 Takara Bio Europe AB 3D CULTURING SYSTEMS FOR GROWTH AND DIFFERENTIATION OF HUMAN PLURIPOTENT STEM (hPS) CELLS
CN101696396A (zh) * 2009-10-27 2010-04-21 中国人民解放军军事医学科学院卫生学环境医学研究所 乙型肝炎病毒体外感染模型的构建方法及应用
WO2011066369A2 (en) * 2009-11-24 2011-06-03 Alder Biopharmaceuticals, Inc. Antagonists of il-6 to raise albumin and/or lower crp
JP5897543B2 (ja) * 2010-03-22 2016-03-30 セルアーティス アーベー Ｗｎｔシグナル経路の調節による多能性細胞から肝細胞様細胞への分化誘導と成熟
WO2011140441A2 (en) 2010-05-06 2011-11-10 Children's Hospital Medical Center Methods and systems for converting precursor cells into intestinal tissues through directed differentiation
US20120291096A1 (en) * 2011-05-12 2012-11-15 Nokia Corporation Method and apparatus for secure signing and utilization of distributed computations
SI3336097T1 (sl) 2012-06-19 2021-07-30 Intercept Pharmaceuticals, Inc. Priprava nekristalinične oblike obetiholne kisline
US20160237400A1 (en) * 2013-03-15 2016-08-18 The Jackson Laboratory Isolation of non-embryonic stem cells and uses thereof
US20160245653A1 (en) 2013-04-30 2016-08-25 Sangtae Park Cylindrical resonator gyroscope
JP6455934B2 (ja) 2013-07-23 2019-01-23 公立大学法人横浜市立大学 生物学的組織に血管系を付与する方法
RU2020119611A (ru) 2014-03-21 2020-06-29 Тобира Терапьютикс, Инк. Сенекривирок для лечения фиброза
MX369921B (es) 2014-04-11 2019-11-26 Cymabay Therapeutics Inc Tratamiento de la hgna y ehna.
WO2015183920A2 (en) * 2014-05-28 2015-12-03 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
CA2950470A1 (en) 2014-06-06 2015-12-10 Vrije Universiteit Brussel Human hepatic 3d co-culture model and uses thereof
IL282156B (en) 2014-07-29 2022-07-01 Shenzhen Hightide Biopharmaceutical Ltd Salts of berberic acid, salts of ursodeoxycholic acid and their combinations, methods for their preparation and their uses
AU2015314830B2 (en) 2014-09-12 2021-01-07 Tobira Therapeutics, Inc. Cenicriviroc combination therapy for the treatment of fibrosis
SG11201701844YA (en) * 2014-10-08 2017-04-27 Agency Science Tech & Res Methods of differentiating stem cells into liver cell lineages
CN104387451B (zh) * 2014-11-07 2020-12-18 浙江海洋学院 一种对非酒精性脂肪肝病细胞模型具有修复作用的文蛤酶解寡肽及其制备方法
CN107208054B (zh) * 2014-11-26 2021-09-17 艾克塞利瑞提德生物技术公司 诱导的肝细胞及其用途
EA201892625A1 (ru) 2015-01-09 2019-07-31 Джилид Аполло, Ллс КОМБИНИРОВАННАЯ ТЕРАПИЯ С ПРИМЕНЕНИЕМ ИНГИБИТОРА АЦЕТИЛ-КоА-КАРБОКСИЛАЗЫ (ACC) ДЛЯ ЛЕЧЕНИЯ НЕАЛКОГОЛЬНОЙ ЖИРОВОЙ БОЛЕЗНИ ПЕЧЕНИ
US20160256672A1 (en) * 2015-02-10 2016-09-08 Cedars-Sinai Medical Center Induced pluripotent stem cell-derived hepatocyte based bioartificial liver device
CN105985395A (zh) 2015-02-13 2016-10-05 江苏奥赛康药业股份有限公司 一种奥贝胆酸化合物及含有该化合物的药物组合物
SG10202103552XA (en) 2015-03-09 2021-05-28 Intekrin Therapeutics Inc Methods for the treatment of nonalcoholic fatty liver disease and/or lipodystrophy
KR20170132879A (ko) * 2015-04-07 2017-12-04 인터셉트 파마슈티컬즈, 인크. 조합 요법을 위한 약제학적 조성물
US10557124B2 (en) * 2015-04-22 2020-02-11 The Regents Of The University Of Michigan Compositions and methods for obtaining stem cell derived lung tissue, and related uses thereof
PE20180690A1 (es) 2015-04-27 2018-04-23 Intercept Pharmaceuticals Inc Composiciones de acido obeticolico y metodos de uso
BR112017028414B1 (pt) 2015-06-30 2021-09-08 Basf Agro B.V. Arnhem Método de controle de pragas
CN110582564A (zh) * 2015-09-15 2019-12-17 新加坡科技研究局 从人类多能干细胞衍生肝类器官
MX2018003179A (es) 2015-09-16 2018-08-21 Tobira Therapeutics Inc Terapia de combinación con cenicriviroc para el tratamiento de fibrosis.
US10801068B2 (en) 2015-10-16 2020-10-13 The Trustees Of Columbia University In The City Of New York JAG1 expression predicts therapeutic response in NASH
CA3000016A1 (en) 2015-11-06 2017-05-11 Gemphire Therapeutics Inc. Gemcabene combinations for the treatment of cardiovascular disease
EP3426766B1 (en) * 2016-03-08 2022-12-28 Yissum Research and Development Company of the Hebrew University of Jerusalem Ltd. Method for continuous biosensing
EP3228306A1 (en) 2016-04-04 2017-10-11 ratiopharm GmbH Complex compound comprising obeticholic acid and cyclodextrin and pharmaceutical formulation comprising the complex compound
WO2017184586A1 (en) 2016-04-18 2017-10-26 The Trustees Of Columbia University In The City Of New York Therapeutic targets involved in the progression of nonalcoholic steatohepatitis (nash)
AU2017253228B2 (en) 2016-04-22 2020-04-09 Taiwanj Pharmaceuticals Co., Ltd. Nalmefene, naltrexone or derivatives thereof for use in treating (non)-alcoholic steatohepatitis or non-alcoholic fatty liver disease
EP3635095A4 (en) * 2017-06-09 2021-12-01 Children's Hospital Medical Center LIVER ORGANOID COMPOSITIONS AND METHOD FOR MANUFACTURING AND USING THEREOF

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11584916B2 (en) 2014-10-17 2023-02-21 Children's Hospital Medical Center Method of making in vivo human small intestine organoids from pluripotent stem cells
US11767515B2 (en) 2016-12-05 2023-09-26 Children's Hospital Medical Center Colonic organoids and methods of making and using same

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