WO2020030821A1 - Organoïdes biliaires - Google Patents

Organoïdes biliaires Download PDF

Info

Publication number
WO2020030821A1
WO2020030821A1 PCT/EP2019/071574 EP2019071574W WO2020030821A1 WO 2020030821 A1 WO2020030821 A1 WO 2020030821A1 EP 2019071574 W EP2019071574 W EP 2019071574W WO 2020030821 A1 WO2020030821 A1 WO 2020030821A1
Authority
WO
WIPO (PCT)
Prior art keywords
biliary
cells
population
progenitors
liver
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2019/071574
Other languages
English (en)
Inventor
Ludovic Vallier
Alexander Ross
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cambridge Enterprise Ltd
Original Assignee
Cambridge Enterprise Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cambridge Enterprise Ltd filed Critical Cambridge Enterprise Ltd
Publication of WO2020030821A1 publication Critical patent/WO2020030821A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the in vitro generation of organoids comprising biliary progenitor cells, for example for use in modelling liver disease or development, drug screening and regenerative medicine.
  • the liver is unique as an organ in the broad spectrum of its functions during adult life (Gille et al., 2010). These functions include iron, vitamin and mineral homeostasis, and the detoxification of alcohol, drugs and other chemicals circulating in the bloodstream. The liver also synthesises bile for the digestion of fat and secretes blood clotting factors and serum proteins such as albumin (Alb) that represents the most abundant protein in the plasma. Finally, the liver plays an essential metabolic activity by storing glycogen and lipids (Gille et al., 2010). Most of these activities are managed by hepatocytes which constitute over 80% of the liver mass (Blouin, Bolender and Weibel, 1977).
  • the cholangiocytes from the biliary tree also have an essential role in collecting waste, processing bile acids and possibly in hepatic regeneration after injury (Deng et al., 2018).
  • disorders affecting functions of these two cell types can be life-threatening and organ transplantation remains the only treatment for end stage liver diseases.
  • the number of organ donors has remained constant for the past 10 years while the demand for liver transplantation has more than doubled in the meantime (Hopkinson and Allen, 2017). This situation is likely to worsen in the foreseeable future due to the pandemic of liver disease associated with obesity and non-alcoholic fatty liver disease (Younossi et al., 2016).
  • understanding liver organogenesis especially the mechanisms by which hepatocytes and cholangiocytes are generated during development, could help to develop alternative regenerative approach such as cell-based therapies and also in vitro systems for disease modelling and drug screening.
  • hepatocytes and cholangiocytes originate from bi-potential progenitors, such as hepatoblasts (Yang et al., 2017)(Shiojiri et al., 2001 ). More precisely, hepatoblasts represent a proliferative population first detected in the liver bud at 5 weeks post conception in humans. These progenitors persist until 20 weeks of development playing a crucial role in liver
  • hepatoblasts are characterised by their capacity to express markers specific for both hepatocytes and cholangiocytes such as Epithelial Cell Adhesion Molecule (EPCAM), Albumin and Alpha-Fetoprotein (AFP).
  • EPCAM Epithelial Cell Adhesion Molecule
  • AFP Alpha-Fetoprotein
  • the signalling pathways involved in hepatoblast self-renewal and differentiation remain to be fully uncovered as contradicting reports have suggested that Wnt signalling could block or promote differentiation of hepatoblasts toward hepatocytes and cholangiocytes in the mouse embryo (Decaens et al., 2008; Tan et al., 2008).
  • TGF has been shown to direct differentiation of hepatoblasts toward the biliary lineage.
  • bi potent hepatic cells may also produce hepatocytes and cholangiocytes during foetal development (Simper-Ronan et al Development 2006 133: 4269-4279). These cells may be maintained in the adult liver and may play a key function in regeneration.
  • Organoid technology consists of growing stem cells, and more broadly a combination of progenitor and epithelial cells, in three-dimensional culture conditions. This technology was first developed using intestinal stem cells (Sato et al., 2011 ) and then was rapidly applied to a diversity of adult organs including the liver, pancreas, prostate, mammary gland, biliary three and lungs (Huch and Koo, 2015). In addition, similar culture conditions have been combined with differentiation of human Pluripotent Stem Cells (hPSCs) to generate brain, kidney, gut and liver organoids which mimic the cellular complexity and architecture of native organs.
  • hPSCs Pluripotent Stem Cells
  • organoids have been broadly demonstrated for basic studies, disease modelling (Schwank et al., 2013), drug screening (van de Wetering et al., 2015), and also regenerative medicine applications (Cruz-Acufia et al., 2017). Nonetheless, this technology has been more rarely applied to foetal tissues in the context of developmental studies. Indeed, primary organoids have only been derived so far from the foetal gut and lung tips. Interestingly, the resulting cells display capacity of differentiation in vitro (Kraiczy et al., 2017) and in vivo after co-culture with mesenchymal cells (Yui et al., 2018).
  • the present inventors have developed culture methods that allow the efficient long-term expansion of biliary progenitor cells, in the form of organoids. These biliary progenitor cells have not been previously described and may differentiate into cholangiocytes and hepatocytes. Populations of biliary progenitor cells expanded as described herein may be useful for example in regenerative medicine.
  • a first aspect of the invention provides a method for producing an expanded population of biliary progenitor cells in vitro comprising:
  • a biliary progenitor expansion medium comprising epidermal growth factor (EGF), a TGF inhibitor, a non-canonical Wnt signalling potentiator and a ROCK inhibitor, to produce an expanded population of biliary progenitor cells.
  • EGF epidermal growth factor
  • TGF tumor necrosis factor
  • ROCK ROCK inhibitor
  • the primary liver cells are human primary liver cells.
  • the population of primary liver cells is cultured in three-dimensional culture in the biliary progenitor expansion medium.
  • the biliary progenitor cells in the population may form organoids in the expansion medium during expansion.
  • the method may further comprise disrupting the organoids to produce a population of isolated biliary progenitor cells.
  • the isolated biliary progenitor cells may be further cultured in the expansion medium to expand or propagate the population.
  • the biliary progenitor cells may be further differentiated, for example into
  • a second aspect of the invention provides an isolated population of biliary progenitor cells produced by a method according to the first aspect or hepatocytes or cholangiocytes differentiated therefrom.
  • a third aspect of the invention provides a biocompatible scaffold comprising an isolated population of the second aspect.
  • a fourth aspect provides a method of treating a liver disease comprising administering an isolated population of the second aspect or a scaffold of the third aspect to an individual in need thereof.
  • a fifth aspect of the invention provides a method of screening comprising;
  • the population contacted with the test compound is in the form of organoids.
  • a sixth aspect of the invention provides a kit for the production of biliary progenitor cell organoids comprising a biliary progenitor expansion medium that comprises epidermal growth factor (EGF), a TQRb inhibitor, non- canonical Wnt signalling potentiator, and a ROCK inhibitor.
  • EGF epidermal growth factor
  • TQRb inhibitor non- canonical Wnt signalling potentiator
  • ROCK inhibitor a ROCK inhibitor
  • Figure 1 shows the derivation and characterisation of Fetal Biliary Organoids.
  • 1A shows a schematic representation of the process of derivation of organoid from human foetal liver.
  • F Feature plot derived from the tSNE in E, displaying levels of gene expression for each gene listed, with darker red points representing higher gene expression in that cell, and lighter points demonstrating lower gene expression.
  • G Violin plots of gene expression for the markers in F across the cells in each cluster; CoP or Co.
  • H Heatmap demonstrating the comparison of the top 20 differentially expressed genes within principal component 1 that separates the two clusters, CoP and Co.
  • FIG. 1 shows the basic characterisation of Hepatoblast Organoid (HO).
  • HO Hepatoblast Organoid
  • B) Representative brightfield images of HO (scale bar 400uM, top, 200uM, bottom).
  • Figure 3 shows the characterisation of Primary Fetal Liver (PFL) 3A Sections of an 8 post-conceptional week (pew) primary fetal liver, stained using haematoxylin and eosin (H&E, far left), immunohistochemistry for AFP (center left), KRT19 (center right), and hepatocyte marker Hep Par-1 (HEPPAR1 ) (far right).
  • PFL Primary Fetal Liver
  • B) tSNE plot derived from whole primary fetal liver scRNAseq data (n 2, 6pcw, 1406 cells) demonstrating independent clustering into six groups, labelled according to cellular identity (stellate, hepatoblast, haematopoietic stem/progenitor cells (HSPC), megakaryocytes, lymphocytes, and Kupffer cells).
  • Figure 4 shows a comparison of HO and FBO to primary foetal liver.
  • E Violin plots of scRNAseq data from PFL hepatoblasts, HO, CoP and Co, demonstrating the respective hepatoblast and cholangiocytic profiles.
  • This invention relates to the in vitro expansion of primary biliary progenitor cells using a biliary progenitor expansion medium comprising epidermal growth factor (EGF), a TGF inhibitor, a non-canonical Wnt signalling potentiator, and a ROCK inhibitor.
  • a biliary progenitor expansion medium comprising epidermal growth factor (EGF), a TGF inhibitor, a non-canonical Wnt signalling potentiator, and a ROCK inhibitor.
  • Biliary cells are cells from the epithelium of biliary tissue, which is a monolayer covering the luminal surface of the biliary tree, including the bile duct and gall bladder. Biliary cells play important roles in bile secretion and electrolyte transport in vivo and may include cells of the cholangiocyte lineage including cholangiocytes and progenitors thereof. Biliary progenitors are bipotent biliary cells that are distinct from foetal hepatoblasts and are capable of further differentiation into cholangiocytes and hepatocytes.
  • the biliary progenitors described herein may be expanded from a population of immature liver cells.
  • Immature liver cells may include neonatal and prenatal liver cells.
  • the population of immature liver cells may comprise immature biliary cells.
  • Immature liver cells may be primary cells isolated from a sample of immature liver tissue.
  • the sample of immature liver tissue may comprise or consist of immature biliary tissue, for example from the immature biliary epithelium.
  • a sample of immature liver tissue may be obtained from an individual of 2 years or less, 1 year or less 6 months or less or 1 month or less (e.g. a neonate (Tolosa et al (2014) Cell Transplant. 23 (10) 1229-1242)).
  • neonatal liver cells may be employed.
  • immature liver cells may be obtained from a sample of foetal liver tissue pre-birth, for example a sample of tissue having a gestational age of 5 weeks or more, 6 weeks or more, 8 weeks or more or 12 weeks or more, for example to 5 to 20 weeks, or 6 to 12 weeks. Suitable samples of foetal liver tissue may for example be obtained from patients following elective terminations. Immature liver cells may express hepatic markers, such as ALB, AFP and EpCAM. In other embodiments, a sample of immature liver tissue may be obtained from individual of more than 2 years old, for example an adult, who has undergone liver injury. Liver injury has been shown to induce ductal plate reactions in liver tissue that comprise immature liver cells.
  • Immature liver cells may be isolated from primary liver tissue derived from heathy individuals or from patients with known pathology to enable disease modelling.
  • Biliary progenitors derived from an individual with a liver disorder such as a biliary disorder, may be used to generate expanded populations of biliary progenitors which display a genotype or phenotype associated with the liver disorder.
  • the immature liver cells may be isolated from a sample of immature liver tissue using any convenient technique.
  • a sample of immature liver tissue may be dissociated into a single cell suspension by enzymatic treatment, for example with collagenase and hyaluronidase, and the suspension sorted for EpCAM positive cells using anti-EpCAM coated microbeads.
  • Immature liver cells for use as described herein may be mammalian cells, for example human, mouse or rat cells, preferably human.
  • biliary progenitors may be expanded from the immature liver cells, preferably immature biliary cells.
  • Immature biliary cells may correspond to biliary cells in immature liver tissue, for example foetal, prenatal or neonatal biliary cells, or cells mature liver tissue following injury .
  • the biliary progenitors are expanded in a biliary progenitor expansion medium. This is a cell culture medium that supports the proliferation of biliary progenitors in the form of organoids (referred to herein as“foetal biliary cell organoids” or FBOs).
  • the biliary progenitor expansion medium is a nutrient medium which comprises epidermal growth factor (EGF), a TGFp inhibitor, a non-canonical Wnt signalling potentiator, a canonical Wnt potentiator and a ROCK inhibitor.
  • EGF Epidermal Growth Factor
  • EGF epidermal growth factor receptor
  • EGFR epidermal growth factor receptor
  • EGF may be produced using routine recombinant techniques or obtained from commercial suppliers (e.g. R&D Systems, Minneapolis, MN; Stemgent Inc, USA). Suitable concentrations of EGF for expanding cholangiocyte organoids as described herein may be readily determined using standard techniques.
  • the expansion medium may comprise 2 to 500ng/ml EGF, preferably about 20ng/ml.
  • a TGF inhibitor is a compound that reduces, blocks or inhibits TGF signalling through the TGFpRI and TGF RII receptors.
  • Suitable TGF inhibitors include A83-01 3-(6-MethyI-2-pyridinyI)-N-phenyI-4-(4- quinolinyl)-1 H-pyrazole-1-carbothioamide), D4476 (4-[4-(2,3-Dihydro-1 ,4-benzodioxin-6-yl)-5-(2-pyridinyl)- 1 W-imidazol-2-yl]benzamide), GW788388 (4-[4-[3-(2-PyridinyI)-1 W-pyrazol-4-yl]-2-pyridinyl]-W-(tetrahydro- 2/+pyran-4-yI)-benzamide), IN1 130 (3-[[5-(6-Methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1
  • TGF inhibitors are available from commercial suppliers.
  • the TGF inhibitor may be A8301 , for example at 10 to 1000pM, preferably about 50mM.
  • a non-canonical Wnt signalling potentiator is a compound that stimulates, promotes or increases the activity of the non-canonical Wnt signalling pathway.
  • the non- canonical Wnt signalling pathway is a b-catenin- independent pathway involved in tissue polarity and morphogenetic processes in vertebrates (Komiya, Y. & Habas, R. Organogenesis 4, 68-75 (2008); Patel, V. et ai. Hum. Mol. Genet. 17, 1578-1590 (2008);
  • Wnt4a Wnt5a
  • Wnt1 1 LRP5/6
  • Dsh Dsh
  • Fz Daaml
  • Rho Rho
  • Rac Prickle
  • a non- canonical Wnt signalling potentiator may selectively potentiate non-canonical Wnt signalling or more preferably, may potentiate both the non-canonical Wnt signalling and the canonical Wnt signalling pathway (i.e. a Wnt signalling agonist).
  • Wnt signalling potentiators include the Wnt signalling agonist R-spondin.
  • R-spondin is a secreted activator protein with two cysteine-rich, furin-like domains and one thrombospondin type 1 domain that positively regulates Wnt signalling pathways.
  • R-spondin is human R-spondin.
  • R-spondin may include RSP01 (GenelD 284654 nucleic acid sequence reference NM__001038633.3, amino acid sequence reference NP_001033722.1 ), RSP02 (GenelD 340419 nucleic acid sequence reference NM_001282863.1 , amino acid sequence reference NP_001269792.1 ), RSP03 (GenelD 84870, nucleic acid sequence reference NM_032784.4, amino acid sequence reference NP_1 16173.2) or RSP04 (GenelD 343637, nucleic acid sequence reference NM_001029871.3, amino acid sequence reference
  • R-spondin is readily available from commercial sources (e.g. R&D Systems, Minneapolis, MN). Suitable concentrations of R-spondin for expanding cholangiocytes as described herein may be readily determined using standard techniques.
  • the expansion medium may comprise 50ng/ml to 5pg/ml R- spondin, preferably about 500ng/ml.
  • a ROCK inhibitor is a compound that reduces, blocks or inhibits Rho kinase (ROCK) (Liao et al (2007) J. Cardiovasc Pharmacol. 50 (1 ) 17-24).
  • Suitable ROCK inhibitors include fasudil, Y39983 (4-[(1 F?)-1- Aminoethyl]-/V-1 /-/-pyrrolo[2,3-£>]pyridin-4-ylbenzamide dihydrochloride), azabenzimidazole-aminofurazans and Y-27632 (trans-4-[(1 R)-1-Aminoethyl]-N-4-pyridinylcyclohexanecarboxamide) and are available from commercial suppliers.
  • the hepatoblast expansion medium may comprise 1 to 100mM Y-27632, preferably about 10 mM.
  • the biliary progenitor expansion medium may be devoid of growth factors other than the epidermal growth factor (EGF), TGF inhibitor, non-canonical Wnt signalling potentiator, and ROCK inhibitor.
  • the biliary progenitor expansion medium may consist of a basal medium supplemented with epidermal growth factor (EGF), TGFp inhibitor, non-canonical Wnt signalling potentiator, and ROCK inhibitor.
  • Suitable biliary cell expansion media may include CO-M (Table 1 ).
  • the immature liver cells are cultured in the hepatocyte expansion medium in three-dimensional culture in the methods described above.
  • the expansion medium further comprises a scaffold matrix which supports the growth and proliferation of cells in 3-dimensions and allows the biliary cells to assemble into organoids.
  • the expansion medium may comprise or consist of the scaffold matrix and the nutrient medium.
  • Suitable scaffold matrices are well-known in the art and include hydrogels, such as collagen,
  • the scaffold matrix may be chemically defined, for example a collagen or densified collagen hydrogel, or non-chemically defined, for example a complex protein hydrogel.
  • the scaffold matrix in the expansion medium is a complex protein hydrogel.
  • Suitable complex protein hydrogels may comprise extracellular matrix components, such as laminin, collagen IV, enactin and heparin sulphate proteoglycans.
  • Complex protein hydrogels may also include hydrogels of extracellular matrix proteins from Engelbreth- Holm-Swarm (EHS) mouse sarcoma cells.
  • EHS Engelbreth- Holm-Swarm
  • Suitable complex protein hydrogels are available from commercial sources and include MatrigelTM (Corning Life Sciences) or CultrexTM BME 2 RGF (AmsbioTM Inc).
  • the expansion medium may comprise 66% MatrigelTM.
  • the biliary progenitor expansion medium may comprise or consist of a scaffold matrix and a nutrient medium supplemented with epidermal growth factor (EGF), a TGF inhibitor, a non-canonical Wnt signalling potentiator, such as R-spondin, and a ROCK inhibitor.
  • EGF epidermal growth factor
  • TGF tumor necrosis factor
  • ROCK inhibitor non-canonical Wnt signalling potentiator, such as R-spondin
  • ROCK inhibitor ROCK inhibitor
  • a nutrient medium may comprise a basal medium.
  • Suitable basal media include Iscove’s Modified
  • IMDM IMDM
  • Ham Ham
  • F12 Ham
  • DMEM Advanced Dulbecco’s modified eagle medium
  • DMEM/F12 Price et al Focus (2003), 25 3-6
  • Williams E Woods L.K., (1976) Tissue Culture Association Manual. 3, 503-508.
  • DMEM/F12 medium may be preferred.
  • the basal medium may be supplemented with a media supplement, such as B27 (ThermoFisher Scientific) and/or one or more additional components, for example L-glutamine or substitutes, such as L-alanyl-L- glutamine (e.g. GlutamaxTM), nicotinamide, N-acetylcysteine, buffers, such as HEPES, and antibiotics such as penicillin and streptomycin.
  • a media supplement such as B27 (ThermoFisher Scientific) and/or one or more additional components, for example L-glutamine or substitutes, such as L-alanyl-L- glutamine (e.g. GlutamaxTM), nicotinamide, N-acetylcysteine, buffers, such as HEPES, and antibiotics such as penicillin and streptomycin.
  • B27 ThermoFisher Scientific
  • additional components for example L-glutamine or substitutes, such as L-alanyl-L- glutamine (e
  • the nutrient medium may be a chemically defined basal nutrient medium.
  • a chemically defined medium is a nutritive solution for culturing cells which contains only specified components, preferably components of known chemical structure.
  • a chemically defined medium is devoid of undefined components or constituents which include undefined components, such as feeder cells, stromal cells, serum, serum albumin and complex extracellular matrices, such as MatrigelTM.
  • a chemically defined medium may be humanised.
  • a humanised chemically defined medium is devoid of components or supplements derived or isolated from non-human animals, such as Foetal Bovine Serum (FBS) and Bovine Serum Albumin (BSA), and mouse feeder cells.
  • Conditioned medium includes undefined components from cultured cells and is not chemically defined.
  • Suitable chemically defined nutrient media include DMEM/F12 supplemented with B27, nicotinamide, N-acetylcysteine, L-alanyl-L-glutamine, HEPES, penicillin and streptomycin.
  • the biliary progenitors may form organoids in the expansion medium.
  • the biliary progenitor organoids may be disrupted as required, such that the expanded population comprises individual cells.
  • the biliary progenitors may be cultured in the expansion medium for multiple passages.
  • the biliary progenitors may be cultured for 25 or more, 30 or more, 40 or more or 50 or more passages.
  • a passage may take 7-14 days, preferably about 10 days.
  • the biliary progenitors may be passaged by digesting the scaffold matrix, harvesting organoids by centrifugation and disrupting the organoids into individual biliary cells.
  • the biliary progenitors may be resuspended and cultured as described above in the expansion medium where they reform into organoids.
  • Suitable techniques for cell culture are well-known in the art (see, for example, Basic Cell Culture Protocols, C. Helgason, Humana Press Inc. U.S. (15 Oct 2004) ISBN: 1588295451 ; Human Cell Culture Protocols (Methods in Molecular Medicine S.) Humana Press Inc., U.S. (9 Dec 2004) ISBN: 1588292223; Culture of Animal Cells: A Manual of Basic Technique, R. Freshney, John Wiley & Sons Inc (2 Aug 2005) ISBN:
  • Media and ingredients thereof may be obtained from commercial sources (e.g. Gibco, Roche, Sigma, Europa bioproducts, R&D Systems). Standard mammalian cell culture conditions may be employed for the above culture steps, for example 37°C, 21 % Oxygen, 5% Carbon Dioxide. Media is preferably changed every two days and cells allowed to settle by gravity
  • the population of biliary progenitors may be expanded 10 10 fold or more, 10 20 fold or more, 10 30 fold or more, 10 40 fold or more or 10 50 fold or more as organoids in the expansion medium as described herein.
  • Organoids are three-dimensional multicellular assemblies or cysts that comprise a layer of the biliary cells linked by tight junctions which surrounds an interior lumen and separates it from the external environment.
  • the morphology and physical characteristics of biliary progenitor organoids may be determined by standard microscopic procedures.
  • Biliary cell may express one or more of epithelial cellular adhesion molecule (EpCAM), gamma-glutamyl transferase (GGT), cystic fibrosis transmembrane conductance regulator (CFTR), and cyto keratin- 19 (KRT19).
  • EpCAM epithelial cellular adhesion molecule
  • GTT gamma-glutamyl transferase
  • CFTR cystic fibrosis transmembrane conductance regulator
  • KRT19 cyto keratin- 19
  • cell markers may be determined by any suitable technique, including
  • the biliary progenitors in the expanded population may display long term stability.
  • the biliary progenitors may be maintained in culture for at least 6 months without DNA copy number or other genetic abnormalities and with stable, preferably homogeneous, expression of biliary markers, such as EpCAM, GGT, CTFR and KRT19.
  • the presence of genetic abnormalities may be determined for example by comparative genomic hybridisation (CGH).
  • the biliary progenitors in the expanded population may display high proliferative potential.
  • the biliary cells may display a doubling time of 2-4 days, for example about 3 days.
  • a population of biliary progenitors expanded as described herein may comprise cholangiocyte progenitors and cholangiocytes.
  • Biliary progenitors may express one or more, preferably all of AFP, APOE, AMBP, SOX4, CD24, CLDN6, APOA2, DLK1 , LCN15, KRT19, SOX9, alpha-1 -antitrypsin (SERPINA1 ), APOB, and TTR.
  • the markers IGF2, AFP and DLK1 are specific to biliary progenitors and may be useful in separating biliary progenitors from other cell types such as cholangiocytes.
  • Other biliary progenitor markers may include LCN1 , FGB, APOA1 , CA4, VTN, DUSP5, LAPTM4B, AMBP, CLDN6, MDK, and BEX1.
  • biliary progenitors expanded as described herein may be further isolated and/or purified, for example from other cell types, including more differentiated cells, such as cholangiocytes. This may be performed, for example, using standard cell sorting techniques, such as flow cytometry. For example, the presence of biliary progenitor markers may be used to isolate cholangiocyte progenitors.
  • the population of biliary progenitors may be free or substantially free from other cell types i.e. the population of biliary progenitors may be homogeneous or substantially homogeneous.
  • the population may contain, 80% or more, 90% or more, 95% or more, 98% or more or 99% or more biliary cells, following culture in the medium.
  • the population of biliary progenitors is sufficiently free of other cell types that no purification is required.
  • Biliary progenitors expanded as described herein may be cultured or maintained using standard mammalian cell culture techniques or subjected to further manipulation or processing.
  • the cell populations produced as described herein may be stored, for example by lyophilisation and/or
  • the biliary progenitors may be stored as organoids, sub-organoid assemblies or individual cells. Suitable storage methods are well known in the art.
  • the biliary cells may be suspended in a cryopreservation medium (for example, CellbankerTM (AMS Biotechnology Ltd, UK) and frozen, for example at -70°C or below.
  • biliary progenitors expanded by the methods described above may be used directly, for example in regenerative medicine applications.
  • the biliary progenitors may be further differentiated in vitro.
  • the biliary progenitors may be differentiated into cholangiocytes by culturing in a cholangiocyte
  • cholangiocyte differentiation medium Culturing in the cholangiocyte differentiation medium may increase the expression of cholangiocytic markers, such as KRT19, and reduce the expression of biliary progenitor markers.
  • cholangiocytes produced as described herein may express cholangiocytic markers and not biliary progenitor markers.
  • Cholangiocytes may express one or more, preferably all of KRT7, KRT19, KRT20, MUC5B, CTFR,
  • the markers KRT7, MMP1 , CYSTM1 and REG4 are specific to cholangiocytes and may be useful in separating cholangiocytes from biliary progenitors.
  • Other cholangiocyte markers may include TFF1 , TFF2, TSPAN8, NEAT1 , LGALS4, ANXA10, S100A14, CTSE, TESC, GPX2, CEACAM6, LYZ, ADIRF, and S100A6.
  • the absence of biliary progenitor markers and/or the presence of cholangiocyte markers may be used to isolate cholangiocytes.
  • the biliary progenitors may be differentiated into hepatocytes by culturing in a hepatocyte differentiation medium. Culturing in the differentiation medium may increase the expression of hepatocytic markers, such as ASGR1 , CYP3A4, albumin (ALB), alpha-1 -antitrypsin (SERPINA1 ), APOH, APOM, APOC1 , CYP1A1 , RBP4, HP, AHSG and TTR, and reduce the expression of biliary progenitor markers.
  • hepatocytes produced as described herein may express hepatocytic markers and not biliary progenitor markers.
  • hepatocytic markers may be used to isolate hepatocytes.
  • the population of biliary progenitors produced as described herein, or cholangiocytes or hepatocytes differentiated therefrom, may be admixed with other reagents, such as buffers, carriers, diluents, preservatives, and/or pharmaceutically acceptable excipients. Suitable reagents are described in more detail below.
  • a method described herein may comprise admixing the population with a therapeutically acceptable excipient to produce a therapeutic composition.
  • the admixed hepatic cells may be in the form of organoids, sub-organoid assemblies or individual cells.
  • the biliary progenitors produced as described herein or cholangiocytes or hepatocytes differentiated therefrom may be useful in therapy.
  • the biliary progenitors, cholangiocytes or hepatocytes are preferably clinical grade cells.
  • Populations of biliary progenitors, cholangiocytes or hepatocytes, for use in treatment are preferably produced from immature liver cells, such as foetal or neonatal liver cells as described herein using chemically defined media.
  • the biliary progenitors, cholangiocytes or hepatocytes may be in the form of organoids, sub-organoid assemblies or individual cells, depending on the specific application.
  • the population of biliary progenitors, cholangiocytes or hepatocytes may be transplanted, infused or otherwise administered into the individual. Suitable techniques are well known in the art.
  • the biliary progenitors, cholangiocytes or hepatocytes, produced as described herein may be admixed with a biocompatible scaffold.
  • a biocompatible scaffold may be seeded with biliary progenitors, cholangiocytes or hepatocytes produced as described above.
  • individual biliary progenitors or sub-organoid assemblies of biliary progenitors may be injected on or into a scaffold or mixing into the scaffold during the manufacturing process.
  • the scaffold containing the biliary progenitors may then be cultured in expansion medium, such that the biliary progenitors populate the scaffold.
  • the biliary progenitors may proliferate within the scaffold and assemble into organoids.
  • Suitable biocompatible scaffolds may include hydrogels, such as fibrin, chitosan, glycosaminoglycans, silk, fibrin, fibronectin, elastin, collagen, glycoproteins such as fibronectin, or polysaccharides such as chitin, or cellulose collagen, collagen/laminin, densified collagen, alginate, agarose, complex protein hydrogels, such as Base Membrane Extracts, bio-organic gels, and synthetic polymer hydrogels, such as polylactic acid (PLA) polyglycol ic acid (PGA), polycapryolactone (PCL) hydrogels, crosslinked dextran and PVA hydrogels (e.g.
  • PLA polylactic acid
  • PGA polyglycol ic acid
  • PCL polycapryolactone
  • inert matrices such as porous polystyrene, polyester, soluble glass fibres porous polystyrene, and isolated natural ECM scaffolds, for example decellularized gall bladder and bile duct scaffolds (Engitix Ltd, London UK).
  • the scaffold may be biodegradable.
  • Suitable scaffold shapes may for example include patches, sheets and tubes, including straight and branched tubes, with diameters up to for example 10-12 mm.
  • Biliary progenitors cultured within a biocompatible scaffold organize into a functional hepatic tissue, for example functional hepatocytic or biliary tissue.
  • a functional hepatic tissue for example functional hepatocytic or biliary tissue.
  • biliary progenitors, or cholangiocytes or hepatocytes produced therefrom cultured within a biocompatible scaffold may organize into a functional biliary epithelium that displays one or more properties of the biliary epithelium.
  • An aspect of the invention provides an isolated population of biliary progenitors, produced by a method described above or cholangiocytes or hepatocytes produced therefrom.
  • the cells may be in the form of organoids, sub-organoid clusters or individual cells.
  • a population of cells generated as described herein may be substantially free from other cell types.
  • the population may contain 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more cells, following culture in the expansion medium.
  • the presence or proportion of biliary progenitors in the population may be determined through the expression of biliary progenitor markers as described above.
  • the population of biliary progenitors, or cholangiocytes or hepatocytes produced therefrom is sufficiently free of other cell types that no purification is required.
  • the population of cells or organoids may be purified by any convenient technique, including FACS.
  • the biliary progenitors, or cholangiocytes or hepatocytes produced therefrom may be engineered to express a heterologous protein, for example a marker protein, such as GFP, or an enzyme and/or to reduce or prevent expression of one or more endogenous protein, for example proteins associated with immunogenicity, such as HLA antigens.
  • a heterologous protein for example a marker protein, such as GFP, or an enzyme and/or to reduce or prevent expression of one or more endogenous protein, for example proteins associated with immunogenicity, such as HLA antigens.
  • the population of biliary progenitors, or cholangiocytes or hepatocytes produced therefrom may be within a biocompatible scaffold .
  • Another aspect of the invention provides a biocompatible scaffold comprising an isolated population of biliary progenitors, produced by a method described herein or cholangiocytes or hepatocytes produced therefrom. Suitable scaffolds are described above.
  • Another aspect of the invention provides a collection of isolated populations of biliary progenitors as described herein or cholangiocytes or hepatocytes produced therefrom, wherein each population in the collection comprise a different set of tissue markers. For example, each population in the collection may have a different antigenic profile or HLA type. This may be useful in matching a population in the collection to a recipient individual without generating a host immune response.
  • aspects of the invention also extend to a pharmaceutical composition, medicament, drug or other composition comprising biliary progenitors produced as described herein or cholangiocytes or hepatocytes produced therefrom in solution or in a biocompatible scaffold, and a method of making a pharmaceutical composition comprising admixing such cells with a pharmaceutically acceptable excipient, vehicle, carrier or biodegradable scaffold , and optionally one or more other ingredients.
  • a pharmaceutical composition containing biliary progenitors produced as described herein or cholangiocytes or hepatocytes produced therefrom may comprise one or more additional components.
  • Pharmaceutical compositions may comprise, in addition to the cells, a pharmaceutically acceptable excipient, carrier, buffer, preservative, stabiliser, anti-oxidant, or other material well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the activity of the cells. The precise nature of the carrier or other material will depend on the route of administration.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • Physiological saline solution, tissue or cell culture media, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the composition may be in the form of a pa rente rally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a pa rente rally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride, Ringer's Injection, or Lactated Ringer's Injection.
  • a composition may be prepared using artificial cerebrospinal fluid.
  • Another aspect of the invention provides a method of treatment of a liver disease comprising administering a population of biliary progenitors as described herein or cholangiocytes or hepatocytes produced therefrom to an individual in need thereof.
  • Another aspect of the invention provides a population of biliary progenitors produced as described herein or cholangiocytes or hepatocytes produced therefrom for use in a method of treatment of a liver disease in an individual in need thereof comprising administering the population to the individual.
  • Another aspect of the invention provides the use of a population of biliary progenitors, produced as described herein or cholangiocytes or hepatocytes produced therefrom in the manufacture of a medicament for use in the treatment of a liver disease.
  • the biliary progenitors, or cholangiocytes or hepatocytes produced therefrom may be in the form of organoids, sub-organoid assemblies or clusters or individual cells.
  • a liver disease is a condition in which liver tissue in an individual is damaged, defective or otherwise dysfunctional, for example, disorders characterised by damage to or destruction of liver tissue, or aberrant liver tissue.
  • Liver disease may include hepatitis (e.g. hepatitis A, B, C, D, E, G or K), cirrhosis, fibrosis, hepatocellular carcinoma, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), drug induced liver injury (DILI), alcoholic liver disease, autoimmune liver disease or an inherited metabolic disorder such as Alpha 1 Antitrypsin deficiency, a Glycogen Storage Disease, for example Glycogen Storage Disease Type 1a, Familial Hypercholesterolemia, Hereditary Tyrosinaemia, Crigler Najjar syndrome, ornithtine transcarbamylase deficiency, or factor IX deficiency or other haemophilia, haemochromatosis, Wilson's disease, Dubin
  • the liver disease may be a biliary disorder.
  • a biliary disorder is a condition in which the biliary tissue in an individual is damaged, defective or otherwise dysfunctional, for example, disorders characterised by damage to or destruction of bile ducts, aberrant bile ducts or the absence of bile ducts.
  • Biliary disorders may include biliary tissue injury, ischaemic strictures, traumatic bile duct injury and cholangiopathies, for example inherited, developmental, autoimmune and environment-induced cholangiopathies, such as Cystic Fibrosis associated cholangiopathy, drug induced cholangiopathy, Alagille Syndrome, polycystic liver disease, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), AIDS associated cholangiopathy, disappearing bile duct syndrome, biliary cancer, ductopenias such as adult idiopathic ductopenia, postoperative biliary complications, and biliary atresia.
  • PBC primary biliary cirrhosis
  • PSC primary sclerosing cholangitis
  • AIDS associated cholangiopathy disappearing bile duct syndrome
  • biliary cancer ductopenias such as adult idi
  • a population of biliary progenitors, or cholangiocytes or hepatocytes produced therefrom may be administered to the individual in solution.
  • the administration of a population of cells, in solution may be useful for example in the treatment of liver diseases, such as ductopenias, including ischaemic ductopenia, congenital ductopenia, such as alagille syndrome, metabolic ductopenia, complex diseases, such as intrahepatic PSC and PBC, drug induced ductopenia, vanishing bile duct syndrome and conditions affecting the intrahepatic biliary tree, as well as acute liver injury and chronic liver disease as described above.
  • liver diseases such as ductopenias, including ischaemic ductopenia, congenital ductopenia, such as alagille syndrome, metabolic ductopenia, complex diseases, such as intrahepatic PSC and PBC, drug induced ductopenia, vanishing bile duct syndrome and conditions
  • a population of biliary progenitors, or cholangiocytes or hepatocytes produced therefrom may be administered to the individual within a biocompatible scaffold.
  • a scaffold populated with cells may be administered to the individual.
  • the administration of a population of bile progenitors in a scaffold may be useful for example in the treatment of biliary atresia, biliary strictures, traumatic or iatrogenic biliary injury and conditions affecting the extrahepatic biliary tree
  • Biliary progenitors, or cholangiocytes or hepatocytes produced therefrom, in solution or in scaffolds may be implanted into a patient by any technique known in the art (e.g. Lindvall, O. (1998) Mov. Disord. 13 Suppl. 1 :83-7; Freed, C.R., et al. , (1997) Cell Transplant, 6 201-202; Kordower, et al., (1995) New England Journal of Medicine, 332 1118-1124; Freed, C.R.,(1992) New England Journal of Medicine, 327, 1549-1555, Le Blanc et al, Lancet 2004 May 1 ;363(9419):1439-41 ).
  • any technique known in the art e.g. Lindvall, O. (1998) Mov. Disord. 13 Suppl. 1 :83-7; Freed, C.R., et al. , (1997) Cell Transplant, 6 201-202; Kordower, et
  • cell suspensions may be injected or infused into the bile duct, gallbladder, portal vein, liver parenchyma, peritoneal cavity or spleen of a patient.
  • a hepatic cell suspension may be administered intravenously, intraperitoneally or via an endoscopic retrograde cholangio-pancreatography (ERCP) or percutaneous cholangiography (PTC).
  • ERCP endoscopic retrograde cholangio-pancreatography
  • PTC percutaneous cholangiography
  • a scaffold populated with hepatic cells may be administered to the individual by surgical implantation.
  • composition in accordance with the present invention is preferably in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.
  • a “prophylactically effective amount” or a “therapeutically effective amount” as the case may be, although prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors.
  • composition comprising biliary progenitors produced as described herein or cholangiocytes or hepatocytes produced therefrom may be administered alone or in combination with other treatments, either
  • Populations of isolated biliary progenitors, produced as described herein or cholangiocytes or hepatocytes produced therefrom, may be useful in modelling the interaction of test compounds with the cells, for example in toxicity screening, modelling biliary disorders or screening for compounds with potential therapeutic effects.
  • Another aspect of the invention provides the use of a population of biliary progenitors, produced as described herein or cholangiocytes or hepatocytes produced therefrom for disease modelling and study of pathogenesis of liver diseases, including biliary disorders.
  • Cells for use in modelling and screening may be in the form of organoids (biliary progenitor organoids), suborganoid clusters or individual cells (biliary progenitors) produced, for example by disruption of organoids.
  • organoids biliary progenitor organoids
  • suborganoid clusters or individual cells (biliary progenitors) produced, for example by disruption of organoids.
  • a method of screening a compound may comprise;
  • the proliferation, growth, apoptosis or viability of the biliary progenitors, cholangiocytes or hepatocytes , protein production, metabolic activity of key enzymes, expression of stress response genes, or the ability of the hepatoblasts to perform one or more cell or organoid functions may be determined in the presence relative to the absence of the test compound.
  • a decrease in proliferation, growth, viability or ability to perform one or more cell or organoid functions is indicative that the compound has a toxic effect and an increase in growth, viability or ability to perform one or more cell or organoid functions is indicative that the compound has an ameliorative effect on the biliary progenitors, cholangiocytes or hepatocytes.
  • Gene expression may be determined in the presence relative to the absence of the test compound. For example, the expression of one or more biliary marker genes may be determined, for example one or more marker genes listed above. Combined decrease in expression is indicative that the compound has a toxic effect or can modify the functional state of the hepatic cells.
  • Gene expression may be determined at the nucleic acid level, for example by RT-PCR, or at the protein level, for example, by immunological techniques, such as ELISA, or by activity assays.
  • Cytochrome p450 assays for example, luminescent, fluorescent or chromogenic assays are well known in the art and available from commercial suppliers.
  • the metabolism, degradation, or breakdown of the test compound by the biliary progenitors, hepatocytes, or cholangiocytes may be determined.
  • changes in the amount or concentration of test compound and/or a metabolite of said test compound may be determined or measured over time, either continuously or at one or more time points. For example, decreases in the amount or concentration of test compound and/or increases in the amount or concentration of a metabolite of said test compound may be determined or measured.
  • the rate of change in the amount or concentration of test compound and/or metabolite may be determined. Suitable techniques for measuring the amount of test compound or metabolite include mass spectrometry.
  • kits and their use for production of expanded populations as described herein relate to kits and their use for production of expanded populations as described herein.
  • a kit for the production of an expanded population of biliary progenitors may comprise a biliary progenitor expansion medium comprising epidermal growth factor (EGF), TGF inhibitor, a non-canonical Wnt signalling potentiator, and a ROCK inhibitor.
  • EGF epidermal growth factor
  • TGF inhibitor a non-canonical Wnt signalling potentiator
  • ROCK inhibitor a ROCK inhibitor
  • kits may further comprise a scaffold matrix, such as MatrigelTM.
  • the scaffold matrix may be provided as part of the expansion medium or may be provided separately.
  • the expansion medium may be formulated in deionized, distilled water.
  • the expansion medium will typically be sterilized prior to use to prevent contamination, e.g. by ultraviolet light, heating, irradiation or filtration.
  • the one or more media may be frozen (e.g. at -20°C or -80°C) for storage or transport.
  • the one or more media may contain one or more antibiotics to prevent contamination.
  • the kit may further comprise a dissociation buffer to dissociate immature liver cells from sample tissue.
  • Suitable buffers include Hanks Buffered Saline Solution (HBSS) supplemented with Liberase DH (Roche Applied Science) and Hyaluronidase (Sigma-Aldrich).
  • the kit may further comprise cryopreservation solution. Suitable cryopreservation media are described above.
  • the one or more media may be a 1x formulation or a more concentrated formulation, e.g. a 2x to 250x concentrated medium formulation.
  • each ingredient in the medium is at the concentration intended for cell culture, for example a concentration set out above.
  • a concentrated formulation one or more of the ingredients is present at a higher concentration than intended for cell culture.
  • Concentrated culture media are well known in the art. Culture media can be concentrated using known methods e.g. salt precipitation or selective filtration.
  • a concentrated medium may be diluted for use with water (preferably deionized and distilled) or any appropriate solution, e.g. an aqueous saline solution, an aqueous buffer or a culture medium.
  • the one or more media in the kit may be contained in hermetically-sealed vessels.
  • Hermetically-sealed vessels may be preferred for transport or storage of the culture media, to prevent contamination.
  • the vessel may be any suitable vessel, such as a flask, a plate, a bottle, a jar, a vial or a bag.
  • biliary progenitor expansion medium for the production of an expanded population of biliary progenitors.
  • HBSS Hanks Buffered Saline Solution
  • 70U/ml Hyaluronidase Sigma-Aldrich
  • the sample was subsequently washed three times in HBSS using centrifugation at 400g for five minutes each time.
  • the single cell suspension could then be sorted for EPCAM/CD326 positive cells using CD326 microbeads (Miltenyi), according to the manufacturer’s guidelines.
  • the single cell suspension was resuspended in cholangiocyte organoid media (CO-M) for FBO.
  • CO-M cholangiocyte organoid media
  • To the resuspended cells was added an equal volume of Growth Factor Reduced Phenol Free Matrigel (Corning), and the mixture pipetted into 48 well plates (20uL per well). The plates were placed at 37°C for fifteen minutes to allow the mixture to set, and subsequently 200uL of fresh HO-media applied to each well.
  • the respective culture medium was changed every 48-72 hours, and organoids mechanically passaged every 7-10 days. Organoids were passaged by scraping the gel away from the plate, pipetting the resulting solution into 1.5ml tubes, and pipetting the solution up and down to break individual organoids into pieces. If a precise cell number was required, the organoids can alternatively be processed to a single cell solution as described below, and then re-plated at the required dilution in the 55% Matrigel-medium solution.
  • Organoids established in culture were dissociated to single cell suspensions for splitting/ single cell sequencing/ cell counting by first removing the media from each well and replacing with Cell Recovery Solution (Corning). The organoids in the Matrigel could then be scraped off the plate and placed on ice for 30 minutes to remove the Matrigel. The organoids should then be washed with PBS, and placed in TrypLE (ThermoFisher Scientific) for 15 mins at 37°C. Cells should finally be washed three times in PBS or basal media.
  • hepatocytes Primary hepatocytes were purchased from Biopredic International (Rennes, France). Hepatocytes were isolated from human liver resects from three donors (two male and one female), and met the manufacturer’s quality control requirements in regards to viability, confluency and functionality, which was assessed by Phase I and II dependent enzymatic activity. Cells were purchased as monolayer cultures and maintained in William’s E (Gibco) supplemented with 1 % Glutamine (Gibco), 1 % Penicillin-streptomycin (Gibco), 700nM Insulin (Sigma) and 50mM Hydrocortisone (Sigma) for no longer than 48h upon receipt. qPCR analysis
  • Organoids were dissociated to single cell solution as described above.
  • the single cells were then fixed and permeabilised in FixPerm (ThermoFisher Scientific) for 20 minutes, then washed three times in PBS before resuspension in PermWash (ThermoFisher Scientific). Blocking was performed using 10% donkey serum (Biorad) in PBS for 30 minutes.
  • Primary antibodies were diluted 1 :100 in 1 % donkey serum in PBS and incubated with the cell suspension at room temperature for 1 hour. After three washes in PBS, cells were incubated with secondary antibodies diluted 1 :1000 in 1 % donkey serum in PBS. Data were collected using Cyan ADP flow cytometer, and analysed using FlowJo X. Assessment of cell proliferation rates
  • Organoids were extracted and dissociated to single cells as described above. The cells were then counted using the countess II automated cell counter (Bio-Rad), and the final number adjusted according to the number of wells used and the volume of resuspension.
  • Luciferin I PA CYP3A4 specific
  • Luciferin PFBE CYP3A7 & CYP3A5 > CYP3A4
  • BODIPY 493/503 (ThermoFisher Scientific) was diluted 1 :1000 in the organoid culture media, and applied to organoids for 30 minutes. After this time the organoids were washed with fresh media, and imaged in situ using a fluorescent microscope.
  • Organoids were extracted from Matrigel using cell recovery solution as described above. The organoids were then washed in PBS and resuspended in Cell Culture Freezing Medium (ThermoFisher Scientific), placed in cryogenic vials (ThermoFisher Scientific), and cooled to -80oC using a“Mr. Frosty” Freezing Container (ThermoFisher Scientific). Organoids could then be kept long term in liquid nitrogen. To thaw, cryovials were kept at 4oC until the freezing medium had melted. Organoids were then washed three times in PBS to remove any residual freezing medium, and then replated as above.
  • Organoids have been previously derived from the adult liver using two approaches.
  • the first method consists of deriving adult stem cells from the intrahepatic biliary epithelium using WNT, Noggin and Rock-inhibitor for the first three days of in vitro culture (Huch et at., 2015).
  • the resulting liver stem cell organoids (LSCO) mainly express biliary markers while displaying a limited capacity to differentiate into hepatocytes (Huch et ai, 2015).
  • the second method derived similar cells from the extra-hepatic biliary epithelium (Sampaziotis et ai, 2017).
  • the fetal biliary organoids express key biliary markers at both bulk and single cell RNA expression level; including Epithelial cellular adhesion molecule (EPCAM), gamma-glutamyl transferase (GGT), cystic fibrosis transmembrane conductance regulator (CFTR), and cytokeratin-19 (KRT19) (Fig 1 D- H).
  • EPCAM Epithelial cellular adhesion molecule
  • GTT gamma-glutamyl transferase
  • CFTR cystic fibrosis transmembrane conductance regulator
  • KRT19 cytokeratin-19
  • FBO cell line contains two distinct populations of cells expressing KRT19. Indeed, one population appears to be positive for traditional cholangiocytes markers such as KRT19 and SOX9, but also hepatoblast markers such as alpha-1 -antitrypsin (SERPINA1 ), AFP and TTR (although not albumin). The other group co-express high levels of KRT19 with more mature markers such as KRT7, CFTR, and CFTR. Thus, FBO organoids seem to contain a combination of cholangiocyte progenitors (CoP) and more differentiated cholangiocytes (Co).
  • CoP cholangiocyte progenitors
  • Co differentiated cholangiocytes
  • Heat map visualisation of genes differentially expressed between CoP and Co identify additional markers specific for the two populations (CoP: APOA2, APOE, DLK1 and LCN15; Co: KRT20, CEACAM6, CYSTM1 , and ADIRF).
  • CoP APOA2, APOE, DLK1 and LCN15
  • Co KRT20, CEACAM6, CYSTM1 , and ADIRF.
  • a number of CoP specific genes are expressed at the protein level in the liver and intrahepatic bile duct while most genes marking Co were expressed in biliary cells.
  • GO term analyses show that genes differentially expressed are involved in processes related to. Visualization of gene expression with cells ordered in pseudotime revealed temporal sequence of gene expression events between CoP and Co, and many cells at intermediate stages (Fig 6F).
  • genes known to be expressed in hepatoblast AFP, APOB,
  • tSNE analysis demonstrated independent clustering of non-parenchymal cell types including Kupffer Cells (CD68, FCGR3A, CD33, ITGAM, SPI1 ) Stellate Cells (COL1A1 , ACTA2, VIM, NES), hematopoietic stem/progenitor cells (PTPRC, SPN, MYB, KLF1 , TFRC), lymphocytes (LSP1 , CD52, CD37, CD48) and megakaryocytes (NRGN, PPBP, ITGA2B, GP1 BA, GP6), as well as a homogeneous population of hepatoblasts (ALB, AFP, KRT8, KRT18, EPCAM, SERPINA1 , TTR) ( Figure 3B).
  • Kupffer Cells CD68, FCGR3A, CD33, ITGAM, SPI1
  • Stellate Cells COL1A1 , ACTA2, VIM, NES
  • hepatoblasts showed great similarity between the biological replicates and showed a homogeneity in their gene expression signature, demonstrating the global ability of the liver parenchyma at this stage to expand the hepatoblast population.
  • this population of hepatoblasts did express a spectrum of known hepatic markers including albumin, alpha-fetoprotein, TTR and SERPINA1 and also new genes which were not identified previously such as RBP4, AHSG, APOH, APOM or FABP1.
  • RBP4 AHSG, APOH, APOM or FABP1.
  • a majority of these genes are expressed at the protein level in adult hepatocytes based on the protein atlas with the exception of SPINK1 and FGB (Table S1 ).
  • hepatoblast could be defined by the combined expression of ALB, SPINK1 , AFP, SERPINA1 , CYP3A7. Taken together, these data show that the foetal human liver contains a diversity of cell types including a homogeneous population of hepatoblast.
  • Hepatoblast organoids form a homogeneous population that closely resembles the in vivo counterpart
  • the Co and CoP populations maintain their distinct clustering, to each other and to the hepatoblasts, with the most differentially expressed genes confirming the different identities of the hepatoblasts (SERPINA1 , APOB, ALB, ASGR1 , TF), CoP (SOX4, CD24, CLDN6) and Co (KRT7, KRT20, MUC5B).
  • genes driving differences between the two populations included higher expression of genes from the Methallothionein sub-family of genes (MT 1 H, MT1A, MT1G, MT1 E, MT1 F, MT2A, MT1X) and also higher expression of some markers of immaturity such as AFP, whilst the HO had higher levels of expression of functional enzymes such as CYP1A1 and CYP2E1 , and metabolic proteins such as fatty acid synthase (FASN) and fatty acid desaturase (FADS1 ).
  • CYP1A1 and CYP2E1 CYP1A1 and CYP2E1
  • metabolic proteins such as fatty acid synthase (FASN) and fatty acid desaturase (FADS1 ).
  • FASN fatty acid synthase
  • FADS1 fatty acid desaturase
  • the ability to generate cells that could be expanded and transplanted into tissue matched recipients could represent an exciting opportunity to manage patients requiring liver transplant.
  • the efficiency of organoid generation alongside a ready supply of tissue offers the potential of rapidly generating a large-scale biobank of hepatoblast organoids that could cover the majority of variations in tissue markers such as HLA.
  • the ability of the organoids to produce serum albumin and differentiate to daughter cell types present an exciting prospect of developing a future bridge therapy for end stage biliary disease, restoring some level of hepatic function through tissue matched donor organoids.
  • this new organoid system represents an exciting system to model development, cell fate decisions, and opportunities for regenerative medicine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des procédés de culture qui permettent l'expansion à long terme efficace de cellules progénitrices biliaires, sous la forme d'organoïdes. Les procédés comprennent la culture d'une population de cellules hépatiques primaires dans un milieu d'expansion progénitrice biliaire comprenant Le Facteur de croissance épidermique (EGF), un inhibiteur de ΤGFβ, un potentialisateur de signalisation Wnt non canonique et un inhibiteur ROCK, pour produire une population expansée de cellules progénitrices biliaires. Les cellules progénitrices biliaires sont bipuissantes et peuvent être encore différenciées en hépatocytes et cholangiocytes. L'invention concerne des vecteurs de virus adéno-associés (AAV) et leurs utilisations.
PCT/EP2019/071574 2018-08-10 2019-08-12 Organoïdes biliaires Ceased WO2020030821A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1813128.4 2018-08-10
GBGB1813128.4A GB201813128D0 (en) 2018-08-10 2018-08-10 Biliary organoids

Publications (1)

Publication Number Publication Date
WO2020030821A1 true WO2020030821A1 (fr) 2020-02-13

Family

ID=63667215

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2019/071574 Ceased WO2020030821A1 (fr) 2018-08-10 2019-08-12 Organoïdes biliaires

Country Status (2)

Country Link
GB (1) GB201813128D0 (fr)
WO (1) WO2020030821A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114426946A (zh) * 2020-12-18 2022-05-03 成都中医药大学 基于干细胞/类器官技术的珍稀濒危动物中药材替代品的制备方法
CN115161261A (zh) * 2022-07-06 2022-10-11 南方医科大学南方医院 一种人胆管胆囊细胞3d培养的培养基与培养方法
WO2025166848A1 (fr) * 2024-02-05 2025-08-14 刘雅明 Utilisation de l'apolipoprotéine h pour préparer un médicament ou un produit de soins de santé pour prévenir et/ou traiter les maladies liées à l'alcool

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2412800A1 (fr) * 2010-07-29 2012-02-01 Koninklijke Nederlandse Akademie van Wetenschappen Organoïde du foie, ses utilisations et son procédé de culture pour l'obtenir
WO2016207621A1 (fr) * 2015-06-22 2016-12-29 Cambridge Enterprise Limited Production in vitro de cholangiocytes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2412800A1 (fr) * 2010-07-29 2012-02-01 Koninklijke Nederlandse Akademie van Wetenschappen Organoïde du foie, ses utilisations et son procédé de culture pour l'obtenir
WO2016207621A1 (fr) * 2015-06-22 2016-12-29 Cambridge Enterprise Limited Production in vitro de cholangiocytes

Non-Patent Citations (54)

* Cited by examiner, † Cited by third party
Title
"Human Cell Culture Protocols (Methods in Molecular Medicine S.", 9 December 2004, HUMANA PRESS INC.
"NCBI", Database accession no. NP_001171601.1
A. BONGSO, STEM CELLS: FROM BENCH TO BEDSIDE, 2005
A. CHIUM. RAO, HUMAN EMBRYONIC STEM CELLS, 2003
A. DOYLEJ. B. GRIFFITHS, MAMMALIAN CELL CULTURE: ESSENTIAL TECHNIQUES, 1997
BLOUIN, A.BOLENDER, R. P.WEIBEL, E. R., THE JOURNAL OF CELL BIOLOGY, vol. 72, no. 2, 1977, pages 441 LP - 455, Retrieved from the Internet <URL:http://jcb.rupress.org/content/72/2/441.abstract>
C. HELGASON: "Basic Cell Culture Protocols", 15 October 2004, HUMANA PRESS INC.
CLOTMAN, F. ET AL., GENES & DEVELOPMENT, vol. 19, no. 16, 2005, pages 1849 - 54
COLIN H. BECKWITT ET AL: "Liver 'organ on a chip'", EXPERIMENTAL CELL RESEARCH, vol. 363, no. 1, 1 February 2018 (2018-02-01), AMSTERDAM, NL, pages 15 - 25, XP055534749, ISSN: 0014-4827, DOI: 10.1016/j.yexcr.2017.12.023 *
DECAENS, T. ET AL., HEPATOLOGY (BALTIMORE, MD.), vol. 47, no. 5, 2008, pages 1667 - 1679
DENG, X. ET AL., CELL STEM CELL, vol. 23, no. 1, 2018, pages 114 - 122.e3, Retrieved from the Internet <URL:https://doi.org/10.1016/j.stem.2018.05.022>
FOTIOS SAMPAZIOTIS ET AL: "Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells", NATURE PROTOCOLS, vol. 12, no. 4, 23 March 2017 (2017-03-23), GB, pages 814 - 827, XP055534582, ISSN: 1754-2189, DOI: 10.1038/nprot.2017.011 *
FREED, C.R. ET AL., CELL TRANSPLANT, vol. 6, 1997, pages 201 - 202
FREED, C.R., NEW ENGLAND JOURNAL OF MEDICINE, vol. 327, 1992, pages 1549 - 1555
GILLE, C. ET AL., MOLECULAR SYSTEMS BIOLOGY, vol. 6, no. 1, 2010, Retrieved from the Internet <URL:http://msb.embopress.org/content/6/1/411.abstract>
GJOREVSKI ET AL., NATURE, vol. 539, 2016, pages 560 - 564
HARUNA, Y. ET AL.: "Hepatology", vol. 23, 1996, WILEY ONLINE LIBRARY, pages: 476 - 481
HEDWIG S. KRUITWAGEN ET AL: "Long-Term Adult Feline Liver Organoid Cultures for Disease Modeling of Hepatic Steatosis", STEM CELL REPORTS, vol. 8, no. 4, 1 April 2017 (2017-04-01), United States, pages 822 - 830, XP055534697, ISSN: 2213-6711, DOI: 10.1016/j.stemcr.2017.02.015 *
HO WY ET AL., J IMMUNOL METHODS, vol. 310, 2006, pages 40 - 52
HOPKINSON, C.ALLEN, E.: "Annual Report on Liver Transplantation 2016/2017", 2017, NHS BLOOD AND TRANSPLANT
HUCH, M.KOO, B.-K., DEVELOPMENT, vol. 142, no. 18, 2015, pages 3113 LP - 3125, Retrieved from the Internet <URL:http://dev.biologists.org/content/142/18/3113.abstract>
J. POLLARDJ. M. WALKER, BASIC CELL CULTURE PROTOCOLS, 1997
K. TURKSEN, HUMAN EMBRYONIC STEM CELL PROTOCOLS, 2006
KATSURA, N. ET AL., JOURNAL OF SURGICAL RESEARCH, vol. 106, no. 1, 2002, pages 115 - 123, Retrieved from the Internet <URL:https://doi.org/10.1006/jsre.2002.6446>
KAZUYA ANZAI ET AL: "Foetal hepatic progenitor cells assume a cholangiocytic cell phenotype during two-dimensional pre-culture", SCIENTIFIC REPORTS, vol. 6, no. 1, 23 June 2016 (2016-06-23), XP055534175, DOI: 10.1038/srep28283 *
KOMIYA, Y.HABAS, R., ORGANOGENESIS, vol. 4, 2008, pages 68 - 75
KORDOWER ET AL., NEW ENGLAND JOURNAL OF MEDICINE, vol. 332, 1995, pages 1118 - 1124
KRAICZY, J. ET AL., GUT, vol. 19, 2017, pages 1326, Retrieved from the Internet <URL:http://gut.bmj.com/content/early/2017/11/15/gutjnl-2017-314817.abstract>
LAURA BROUTIER ET AL: "Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation", NATURE PROTOCOLS, vol. 11, no. 9, 25 August 2016 (2016-08-25), GB, pages 1724 - 1743, XP055534706, ISSN: 1754-2189, DOI: 10.1038/nprot.2016.097 *
LAURA BROUTIER ET AL: "Human primary liver cancer-derived organoid cultures for disease modeling and drug screening", NATURE MEDICINE, vol. 23, no. 12, 13 November 2017 (2017-11-13), New York, pages 1424 - 1435, XP055534757, ISSN: 1078-8956, DOI: 10.1038/nm.4438 *
LE BLANC ET AL., LANCET, vol. 363, no. 9419, 1 May 2004 (2004-05-01), pages 1439 - 41
LIAO ET AL., J. CARDIOVASC PHARMACOL., vol. 50, no. 1, 2007, pages 17 - 24
LINDVALL, O., MOV. DISORD., vol. 13, no. 1, 1998, pages 83 - 7
MATHAPATI SANTOSH ET AL: "Small-Molecule-Directed Hepatocyte-Like Cell Differentiation of Human Pluripotent Stem Cells", CURRENT PROTOCOLS IN STEM CELL BIOLOGY 2012,, vol. 38, 17 August 2016 (2016-08-17), pages 1.6.1 - 1.6.18, XP009509972, ISSN: 1938-8969, DOI: 10.1002/CPSC.13 *
MERITXELL HUCH ET AL: "In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration", NATURE, vol. 494, no. 7436, 1 January 2013 (2013-01-01), pages 247 - 250, XP055058163, ISSN: 0028-0836, DOI: 10.1038/nature11826 *
MINA OGAWA ET AL: "Directed differentiation of cholangiocytes from human pluripotent stem cells. Supplementary Text and Figures.", NATURE BIOTECHNOLOGY, 13 July 2015 (2015-07-13), pages 853 - 861, XP055282317, Retrieved from the Internet <URL:http://www.nature.com/nbt/journal/v33/n8/extref/nbt.3294-S1.pdf> [retrieved on 20160621], DOI: 10.1038/nbt.3294 *
MOORE, G.E.WOODS L.K., TISSUE CULTURE ASSOCIATION MANUAL, vol. 3, 1976, pages 503 - 508
OERTEL, M. ET AL., HEPATOLOGY (BALTIMORE, MD.), vol. 37, no. 5, 2003, pages 994 - 1005
OHKAWARA ET AL., DEV DYN, vol. 240, no. 1, 2011, pages 188 - 194
PATEL, V. ET AL., HUM. MOL. GENET., vol. 17, 2008, pages 1578 - 1590
PETERSONLORING: "Human Stem Cell Manual: A Laboratory Guide", 2012, ACADEMIC PRESS
PRICE ET AL., FOCUS, vol. 25, 2003, pages 3 - 6
R. FRESHNEY: "Culture of Animal Cells: A Manual of Basic Technique", 2 August 2005, JOHN WILEY & SONS INC
SCHWANK, G. ET AL., CELL STEM CELL, vol. 13, no. 6, 2013, pages 653 - 658, Retrieved from the Internet <URL:https://doi.org/10.1016/j.stem.2013.11.002>
SHIOJIRI, N. ET AL.: "Laboratory Investigation", vol. 81, 2001, THE UNITED STATES AND CANADIAN ACADEMY OF PATHOLOGY, INC., pages: 17
SIMPER-RONAN ET AL., DEVELOPMENT, vol. 133, 2006, pages 4269 - 4279
STRAZZABOSCO, M.SOMLO, S., GASTROENTEROLOGY, vol. 140, 2011
TOLOSA ET AL., CELL TRANSPLANT., vol. 23, no. 10, 2014, pages 1229 - 1242
TOUBOUL THOMAS ET AL: "Stage-specific regulation of the WNT/[beta]-catenin pathway enhances differentiation of hESCs into hepatocytes", JOURNAL OF HEPATOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 64, no. 6, 26 February 2016 (2016-02-26), pages 1315 - 1326, XP029538552, ISSN: 0168-8278, DOI: 10.1016/J.JHEP.2016.02.028 *
VAN DE WETERING, M. ET AL., CELL, vol. 161, no. 4, 2015, pages 933 - 945, Retrieved from the Internet <URL:https://doi.org/10.1016/j.cell.2015.03.053>
WILLIAMS, G.M. ET AL., EXP. CELL RESEARCH, vol. 89, 1974, pages 139 - 142
XINPING TAN ET AL: "[beta]-Catenin deletion in hepatoblasts disrupts hepatic morphogenesis and survival during mouse development", HEPATOLOGY, vol. 47, no. 5, 7 April 2008 (2008-04-07), US, pages 1667 - 1679, XP055534724, ISSN: 0270-9139, DOI: 10.1002/hep.22225 *
YANG, L. ET AL., HEPATOLOGY (BALTIMORE, MD.), vol. 66, no. 5, 2017, pages 1387 - 1401
YOUNOSSI, Z. M. ET AL., HEPATOLOGY, vol. 64, no. 1, 2016, pages 73 - 84

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114426946A (zh) * 2020-12-18 2022-05-03 成都中医药大学 基于干细胞/类器官技术的珍稀濒危动物中药材替代品的制备方法
CN115161261A (zh) * 2022-07-06 2022-10-11 南方医科大学南方医院 一种人胆管胆囊细胞3d培养的培养基与培养方法
WO2025166848A1 (fr) * 2024-02-05 2025-08-14 刘雅明 Utilisation de l'apolipoprotéine h pour préparer un médicament ou un produit de soins de santé pour prévenir et/ou traiter les maladies liées à l'alcool

Also Published As

Publication number Publication date
GB201813128D0 (en) 2018-09-26

Similar Documents

Publication Publication Date Title
US20250057888A1 (en) Use of alveolar or airway organoids for the treatment of lung diseases and disorders
US12497596B2 (en) Hepatocyte expansion methods
EP3401392B1 (fr) Procédé de production de cellules souches/précurseurs hépatiques à partir de cellules hépatiques matures à l&#39;aide d&#39;un composé de faible poids moléculaire
Schmelzer et al. Effects of mesenchymal stem cell coculture on human lung small airway epithelial cells
WO2020030821A1 (fr) Organoïdes biliaires
AU2019360857B2 (en) Method for producing stem/precursor cells, by using low molecular weight compound, from cells derived from endodermal tissue or organ
US20230392122A1 (en) LONG-TERM AND FUNCTIONAL CULTURE OF HEPATIC ORGANOIDS (eHEPO) DERIVED FROM EPCAM+ ENDODERMAL PROGENITOR CELLS DIFFERENTIATED FROM INDUCED PLURIPOTENT STEM CELLS
US20250327033A1 (en) Methods of Expanding Cholangiocytes
WO2020030822A1 (fr) Organoïdes hépatoblastes
JP7148134B2 (ja) 肝芽細胞から胆管上皮前駆細胞への段階的誘導方法
Hayashi et al. A basic study on self-reconstitution of alveolar epithelium-like cells by tissue stem cells in mouse lung: Hayashi et al.
CN114317398B (zh) Gli1和EpCAM基因共同标记的肝祖细胞群及其应用
Rabata¹ et al. a Role for FGF and WNT Signaling in Regulation of Lung Epithelial Cell Fate and Morphogenesis
Tian et al. Lung Organoids: A New Pathway into Lung Regeneration and Repair
Wong et al. Directed Differentiation of Human Pluripotent Stem Cells to Functionally Mature Lung Alveolar Cells.
Turgut Endodermal Hepatic Organoid (eHEPO) Culture Development and Characterization with Single-Cell Sequencing
Turgut Endodermal Hepatik Organoid (eHEPO) Kültürünün Geliştirilmesi Ve Tek Hücre Bazında Karakterizasyonu
HK40058180A (en) Method for producing stem/precursor cells, by using low molecular weight compound, from cells derived from endodermal tissue or organ
HK40058180B (en) Method for producing stem/precursor cells, by using low molecular weight compound, from cells derived from endodermal tissue or organ
CN121653041A (zh) 一种小细胞肺癌类器官培养基及培养方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19758638

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19758638

Country of ref document: EP

Kind code of ref document: A1