WO2016148216A1 - 肝細胞及び肝非実質細胞、並びにそれらの調製方法 - Google Patents
肝細胞及び肝非実質細胞、並びにそれらの調製方法 Download PDFInfo
- Publication number
- WO2016148216A1 WO2016148216A1 PCT/JP2016/058411 JP2016058411W WO2016148216A1 WO 2016148216 A1 WO2016148216 A1 WO 2016148216A1 JP 2016058411 W JP2016058411 W JP 2016058411W WO 2016148216 A1 WO2016148216 A1 WO 2016148216A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- hepatic
- cell
- progenitor
- progenitor cells
- Prior art date
Links
Images
Classifications
-
- 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
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/37—Digestive system
- A61K35/407—Liver; Hepatocytes
-
- 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
- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
-
- 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
- C12N5/0672—Stem cells; Progenitor cells; Precursor cells; Oval 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
- C12N5/0679—Cells of the gastro-intestinal tract
-
- 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
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5067—Liver 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
- C12N2320/00—Applications; Uses
- C12N2320/10—Applications; Uses in screening processes
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- 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
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/12—Hepatocyte growth factor [HGF]
-
- 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
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- 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
- C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
-
- 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
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- 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
- C12N2501/999—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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/14—Coculture with; Conditioned medium produced by hepatocytes
-
- 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
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- 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
- C12N2503/00—Use of cells in diagnostics
- C12N2503/04—Screening or testing on artificial tissues
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/14—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from hepatocytes
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to hepatocytes, hepatic progenitor cells, bile duct epithelial cells, hepatic sinusoidal endothelial progenitor cells, hepatic sinusoidal endothelial cells, hepatic stellate progenitor cells, hepatic stellate cells, and hepatocyte tissue models, and methods for preparing them.
- the present invention also relates to a cell fraction comprising hepatic progenitor cells, hepatic sinusoidal progenitor cells, or hepatic stellate progenitor cells.
- the present invention also relates to a pharmaceutical composition or kit comprising the cell, hepatocyte tissue model or cell fraction.
- the present invention also provides a method for screening a liver disease therapeutic agent, a method for evaluating the hepatotoxicity of a drug, a hepatocyte of an infectious liver disease model and a preparation method thereof, an infectious liver disease model tissue and a preparation method thereof, and an infectious liver
- the present invention relates to a method for screening a disease therapeutic agent.
- liver is responsible for a variety of functions such as metabolism, detoxification, and serum protein synthesis, and is an essential organ for maintaining homeostasis.
- Liver cells which are the parenchymal cells of the liver, are responsible for these various liver functions.
- hepatocytes are used for drug discovery studies such as drug toxicity tests because they express many metabolic enzymes such as cytochrome P450 (CYP) enzymes.
- CYP cytochrome P450
- liver transplantation therapy is a basic treatment for severe liver diseases (fulminant hepatitis, cirrhosis, liver cancer, etc.)
- liver transplantation a basic treatment for severe liver diseases (fulminant hepatitis, cirrhosis, liver cancer, etc.)
- problems such as shortage of donors and the need for lifelong immunosuppressive therapy, which replaces liver transplantation.
- Development of new treatments is desired.
- One of them is cell transplantation therapy.
- hepatocytes isolated from living liver rapidly lose metabolic enzyme activity due to culture. Moreover, it is difficult to supply a large amount of human hepatocytes from a human body.
- hepatocytes are in close contact with hepatic sinusoidal endothelial cells (Liver Sinusoidal Endothelial Cell: LSEC) and hepatic stellate cells (Hepatic Stellate Cell: HSC) that constitute the sinusoid that is the capillary system of the liver.
- LSEC Liver Sinusoidal Endothelial Cell
- HSC Hepatic Stellate Cell
- Non-Patent Documents 4 and 5 There have been reported examples of successful regeneration of functional cells and tissues by three-dimensionally constructing different cells derived from stem cells by co-culture.
- the conventionally known methods for inducing human hepatocytes from human pluripotent stem cells need to undergo multi-step differentiation induction, and it is difficult to prepare uniform and large amounts of hepatocytes in a short period of time. Furthermore, in human hepatocytes obtained by this method, the expression level of CYP, an enzyme involved in drug metabolism, is lower than that of living hepatocytes, leading to induction of hepatocytes having physiological functions equivalent to those of living hepatocytes. There is no current situation.
- hepatocytes and liver tissues can be induced by faithfully reproducing cell-cell interactions during liver development in vitro.
- an object of the present invention is to provide a method for efficiently and efficiently preparing hepatocytes, hepatic non-parenchymal cells and their progenitor cells having high functionality.
- the present inventors have been able to selectively sort hepatic progenitor cells from a cell fraction containing hepatic progenitor cells using a carboxypeptidase M (CPM) positive phenotype as an index, and It was found that uniform and highly functional hepatocytes can be prepared by inducing differentiation of the obtained hepatic progenitor cells.
- CPM carboxypeptidase M
- the present inventors selectively separated uniform and highly functional hepatic sinusoidal progenitor cells from the cell fraction containing hepatic sinusoidal progenitor cells using FLK1-positive, CD34-positive and CD31-positive phenotypes as indices. It was found that uniform and highly functional hepatic sinusoidal endothelial cells can be prepared by inducing differentiation of the obtained hepatic sinusoidal progenitor cells.
- the present inventors have used homogeneous and highly functional hepatic stellate progenitor cells from the cell fraction containing hepatic stellate progenitor cells, using an activated leukocyte cell adhesion molecule (ALCAM) positive phenotype as an index. It was found that can be selectively sorted.
- ACAM activated leukocyte cell adhesion molecule
- hepatic stellate progenitor cells can be induced to differentiate into hepatic stellate cells using a Rho kinase (Rho-associated protein kinase: ROCK) inhibitor.
- Rho kinase Rho-associated protein kinase: ROCK
- the present inventors have found that the differentiation from hepatic progenitor cells to hepatocytes can be promoted by co-culturing the hepatic progenitor cells obtained above with hepatic non-parenchymal cells.
- hepatocytes of an infectious liver disease model can be prepared from the hepatic progenitor cells and hepatocytes obtained above.
- a method for preparing hepatic progenitor cells comprising a step of sorting hepatic progenitor cells from a cell fraction containing hepatic progenitor cells using a CPM positive phenotype as an index.
- the method according to [1] further comprising a step of preparing the cell fraction containing hepatic progenitor cells by inducing differentiation of definitive endoderm cells or hepatic endoderm cells.
- the method according to [1] further comprising the step of preparing the definitive endoderm cells or the hepatic endoderm cells by inducing differentiation of pluripotent stem cells.
- a cell fraction containing 90% or more hepatic progenitor cells with respect to all cells, the hepatic progenitor cells having a CPM positive phenotype, proliferating ability, and differentiation into hepatocytes or bile duct epithelial cells A cell fraction having a capacity.
- a hepatocyte having proliferative ability which can be prepared by the method according to [8].
- a method for preparing bile duct epithelial cells comprising a step of inducing differentiation of hepatic progenitor cells according to [5] or [6] into bile duct epithelial cells.
- a biliary epithelial cell having proliferative ability which can be prepared by the method according to [10].
- a method for preparing hepatic sinusoidal endothelial progenitor cells comprising a step of sorting hepatic sinusoidal endothelial progenitor cells from a cell fraction containing hepatic sinusoidal endothelial progenitor cells using FLK1-positive, CD34-positive and CD31-positive phenotypes as indices.
- [16] [12] to [15] can be prepared by the method according to any one of the above, having FLK1-positive, CD34-positive and CD31-positive phenotypes, proliferating ability, and differentiation into hepatic sinusoidal endothelial cells Hepatic sinusoidal endothelial progenitor cells having the ability.
- a cell fraction containing 90% or more hepatic sinusoidal endothelial progenitor cells with respect to all cells, wherein the hepatic sinusoidal endothelial progenitor cells have a phenotype of FLK1 positive, CD34 positive and CD31 positive, and have a proliferative ability A cell fraction that has and has the ability to differentiate into hepatic sinusoidal endothelial cells.
- [18] (1) a step of inducing differentiation of hepatic sinusoidal endothelial progenitor cells according to [16] using a TGF- ⁇ inhibitor to prepare a cell fraction containing hepatic sinusoidal endothelial cells; and (2) livers
- a method for preparing hepatic sinusoidal endothelial cells comprising a step of sorting hepatic sinusoidal endothelial cells from the cell fraction containing sinusoidal endothelial cells using CD31 positive and Fc ⁇ R II positive phenotypes as indices.
- a hepatic sinusoidal endothelial cell that can be prepared by the method according to [18], has a CD31 positive and Fc ⁇ R II positive phenotype, and has a proliferative ability.
- a method for preparing hepatic stellate progenitor cells comprising a step of sorting hepatic stellate progenitor cells from a cell fraction containing hepatic stellate progenitor cells using an ALCAM positive phenotype as an index.
- a method for preparing hepatic stellate progenitor cells further comprising the step of preparing the cell fraction containing hepatic stellate progenitor cells by inducing differentiation of pluripotent stem cells.
- hepatic stellate progenitor cells which can be prepared by the method according to any one of the above, have an ALCAM positive phenotype, have proliferative ability, and have the ability to differentiate into hepatic stellate cells. .
- the method according to [25] wherein the hepatic stellate progenitor cell is the hepatic stellate progenitor cell according to [23].
- a hepatic stellate cell that can be prepared by the method according to [25] or [26] and has a proliferative ability.
- a method for preparing a hepatocyte tissue model [29] The method according to [28], wherein the hepatic sinusoidal endothelial cell is the hepatic sinusoidal endothelial cell according to [19].
- hepatic stellate cell is the hepatic stellate cell according to [27].
- a hepatocyte tissue model that can be prepared by the method of [32] [5] or hepatic progenitor cell according to [6], cell fraction according to [7], hepatocyte according to [9], bile duct epithelial cell according to [11], liver according to [16] Sinusoidal endothelial progenitor cells, cell fraction according to [17], hepatic sinusoidal endothelial cells according to [19], hepatic stellate progenitor cells according to [23], cell fraction according to [24],
- a method for screening for a hepatic disease therapeutic agent comprising administering a hepatic cell therapeutic model according to [9] or a hepatocyte tissue model according to [31] to a candidate for a hepatic disease therapeutic agent.
- a method for evaluating the hepatotoxicity of a drug comprising administering the drug to the hepatocyte according to [9] or the hepatocyte tissue model according to [31].
- [35] Infecting a hepatic progenitor cell according to [5] or [6] or a hepatic progenitor cell in the cell fraction according to [7] with a pathogen, (2) A method for preparing hepatocytes of an infectious disease model, comprising preparing the hepatocytes of a pathogen-infected disease model by inducing differentiation of the hepatic progenitor cells infected with the pathogen. [36] [9] A method for preparing hepatocytes of an infectious disease model, comprising the step of infecting hepatocytes according to [9] with a pathogen.
- [37] The preparation method according to [35] or [36], wherein the pathogen is hepatitis virus or malaria parasite.
- [38] [35] A hepatocyte of an infectious disease model that can be prepared by the method according to [37]. [39] (1) Infecting a hepatic progenitor cell according to [5] or [6] or a hepatic progenitor cell in the cell fraction according to [7] with a pathogen, (2) infection comprising the step of co-culturing the hepatic progenitor cells infected with a pathogen with at least one hepatic non-parenchymal cell selected from the group consisting of hepatic sinusoidal endothelial cells, hepatic stellate cells, and hepatic mesothelial cells.
- uniform and highly functional hepatocytes, hepatic non-parenchymal cells and their progenitor cells can be prepared with high purity and efficiency.
- the obtained hepatocytes can be used, for example, for drug discovery screening.
- the obtained hepatocytes, hepatic non-parenchymal cells and their progenitor cells can be used for cell therapy, for example.
- the obtained hepatocytes, hepatic non-parenchymal cells and their progenitor cells can be used for the preparation of a disease model.
- FIG. 1 shows the results of flow cytometry analysis showing the transition of the CPM positive cell fraction over time when differentiation was induced from human iPS cells to hepatocytes.
- IPCs in the figure indicates human iPS cells.
- DE in the figure indicates a cell group including definitive endoderm cells.
- SH in the figure indicates a cell group including hepatic endoderm cells.
- IH in the figure indicates a cell group including hepatic progenitor cells.
- MH in the figure indicates a cell group including mature hepatocytes. The percentage in the figure indicates the proportion of CPM positive cells in the cell group.
- FIG. 2 shows the results of flow cytometry analysis when cells were separated from a cell group (IH) containing hepatic progenitor cells using CPM positive as an index. By this cell separation, 97.7% of hepatic progenitor cells could be collected.
- FIG. 3 shows the results of immunocytochemical staining of CPM positive cells.
- FIG. 4 shows a comparison result of expression levels of hepatic progenitor cell markers in CPM positive cells and CPM negative cells. * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001. Results are expressed as the mean ⁇ SEM of 8 experiments.
- FIG. 5 shows the ability of CPM positive cells to proliferate (top) and the relative number of cells (bottom) after multiple passages of CPM positive cells.
- FIG. 6 shows the results of observation with a bright-field microscope, albumin staining with immunocytochemical staining, and glycogen accumulation by PAS staining with respect to hepatic progenitor cells before differentiation induction and hepatocytes obtained by differentiation induction. Indicates.
- FIG. 7 shows human iPS cell-derived hepatocytes (iPSC Heps) obtained by inducing differentiation of hepatic progenitor cells without sorting using CPM positive as an index, and hepatic progenitors sorted using CPM positive as an index.
- iPSC Heps human iPS cell-derived hepatocytes
- FIG. 8 shows human iPS cell-derived hepatocytes (iPSC Heps) obtained by inducing differentiation of hepatic progenitor cells without sorting using CPM positive as an index, and hepatic progenitor cells sorted using CPM positive as an index.
- FIG. 9 shows the results of analysis of mRNA expression of AFP using quantitative RT-PCR in hepatic progenitor cells sorted using CPM positive as an index and human biliary epithelial cells induced to differentiate from the hepatic progenitor cells. .
- LPCs refer to hepatic progenitor cells prepared by the method of the invention
- Chol refers to human biliary epithelial cells induced to differentiate from the hepatic progenitor cells. Results are expressed as the mean ⁇ SEM of 4 independent experiments. ** p ⁇ 0.01
- FIG. 10 shows human biliary epithelial cells obtained by inducing differentiation of hepatic progenitor cells sorted using CPM positive as an index, and differentiation induction of hepatic progenitor cells without sorting using CPM positive as an index. The results of analyzing the mRNA expression of a bile duct epithelial cell-specific marker in the obtained human bile duct epithelial cells using quantitative RT-PCR are shown.
- CPM + Cholangiocytes in the figure refers to hepatic progenitor cells obtained by inducing differentiation of hepatic progenitor cells sorted using CPM positivity as an index
- iPSC Cholangiocytes in the figure refers to CPM positivity as an index. It is a human biliary epithelial cell obtained by inducing differentiation of hepatic progenitor cells without sorting. Results are expressed as the mean ⁇ SEM of 4 independent experiments. ND: not detected, ** p ⁇ 0.01, *** p ⁇ 0.001.
- FIG. 11 shows gene expression levels of marker molecules in human iPS cells (iPSC), human mesoderm cells (Meso), and human hepatic sinusoidal endothelial progenitor cells (LSEC pro) (before cell separation).
- iPSC human iPS cells
- Meso human mesoderm cells
- LSEC pro human hepatic sinusoidal endothelial progenitor cells
- n 3, 3, 5, ** p ⁇ 0.01, *** p ⁇ 0.001.
- FIG. 12 shows the results of flow cytometry analysis of a cell group containing human hepatic sinusoidal endothelial progenitor cells obtained by inducing differentiation of human mesoderm cells.
- CD31 + CD34 +/ ⁇ cells (right) are present in the FLK1 + cell fraction (left).
- FIG. 14 shows a phase contrast microscopic image of FLK1 + CD31 + CD34 + human hepatic sinusoidal endothelial progenitor cells.
- FIG. 17 shows an immunocytochemical staining image of FLK1 + CD31 + CD34 + cells after cryopreservation. Blue: nucleus, red: CD31. Scale bar, 100 ⁇ m.
- FIG. 18 shows the expression levels of hepatic sinusoidal endothelial cell markers in human hepatic sinusoidal endothelial progenitor cells of FLK1 + CD31 + CD34 + and human hepatic sinusoidal endothelial cells derived from the progenitor cells.
- Pre in the figure indicates human hepatic sinusoidal endothelial progenitor cells.
- + A83-01” in the figure indicates human hepatic sinusoidal endothelial cells.
- “F8” in the figure indicates Factor VIII.
- n 3, mean ⁇ SEM.
- FIG. 19 shows the results of flow cytometric analysis of a cell fraction containing human hepatic sinusoidal endothelial cells derived from human hepatic sinusoidal endothelial progenitor cells.
- FIG. 20 shows a phase-contrast microscope image of CD31 positive and FcR ⁇ II positive cells. Scale bar, 100 ⁇ m.
- FIG. 21 shows expression analysis of human hepatic sinusoidal endothelial cell marker molecules in CD31 + FcR ⁇ II ⁇ cells and CD31 + FcR ⁇ II + cells. Human cord blood vein endothelial cells (HUVEC) were used as controls.
- n 3. Mean ⁇ SEM. ND: not detected.
- FIG. 22 is an immunocytochemical staining image showing the uptake ability of acetylated LDL (Ac-LDL) and hyaluronic acid (HA) of human iPS cell-derived CD31-positive FcR ⁇ II-positive hepatic sinusoidal endothelial cells (iPS-LSEC). is there. HUVEC was used as a control. Blue: nucleus, red: acetylated LDL, green: hyaluronic acid scale bar, 100 ⁇ m.
- FIG. 22 is an immunocytochemical staining image showing the uptake ability of acetylated LDL (Ac-LDL) and hyaluronic acid (HA) of human iPS cell-derived CD31-positive FcR ⁇ II-positive hepatic sinusoidal endothelial cells (iPS-LSEC). is there. HUVEC was used as a control. Blue: nucleus, red: acetylated LDL, green: hyaluronic acid
- FIG. 24 shows an immunocytochemical staining image of human iPS cell-derived CD31-positive FcR ⁇ II-positive hepatic sinusoidal endothelial cells. Blue: Nuclear, Red: Factor VIII (F8) FIG.
- FIG. 25 shows the results of flow cytometric analysis of intracellular protein (factor VIII: F8) of CD31-positive FcR ⁇ II-positive hepatic sinusoidal endothelial cells derived from human iPS cells.
- FIG. 26 shows the results of flow cytometry analysis of human iPS cell-derived mesoderm cells.
- FIG. 27 shows the expression level of hepatic stellate progenitor cell marker molecules in ALCAM positive cells (ALCAM + ) and ALCAM negative cells (ALCAM ⁇ ) before separation of human iPS cell-derived mesoderm cells.
- n 3. Mean ⁇ SEM.
- FIG. 29 shows the results of flow cytometry analysis of human iPS cell-derived human hepatic stellate cells (iPS-HSC) and human bone marrow-derived mesenchymal stem cells (hMSC). Intracellular vitamin A was detected by autofluorescence.
- FIG. 29 shows the results of flow cytometry analysis of human iPS cell-derived human hepatic stellate cells (iPS-HSC) and human bone marrow-derived mesenchymal stem cells (hMSC). Intracellular vitamin A was detected by autofluorescence.
- FIG. 30 shows phase contrast microscopic images of human iPS cell-derived human hepatic stellate cells (iPS-HSC) and human bone marrow-derived mesenchymal stem cells (hMSC) after addition of vitamin A.
- iPS-HSC human iPS cell-derived human hepatic stellate cells
- hMSC human bone marrow-derived mesenchymal stem cells
- FIG. 31 shows expression of hepatic progenitor cell marker (LSEC HSC) when co-cultured with human iPS cell-derived CPM + hepatic progenitor cells using human iPS cell-derived hepatic sinusoidal endothelial cells and human iPS cell-derived hepatic stellate cells as feeder cells. ).
- HUVEC and hMSC were used as feeder cells (HUVEC hMSC).
- n 1 FIG.
- FIG. 32 shows the results of expression analysis of genes involved in hepatocyte differentiation when human iPS cell-derived hepatic sinusoidal endothelial cells and mouse-derived non-parenchymal cells are co-cultured.
- the left bar shows the result of single culture of iPS cell-derived hepatic progenitor cells
- the right bar shows the result of co-culturing human iPS cell-derived hepatic progenitor cells and mouse-derived non-parenchymal cells.
- FIG. 33 shows the results of expression analysis of genes involved in hepatocyte differentiation when human iPS cell-derived hepatic progenitor cells and mouse non-parenchymal cells are cultured in a plane.
- IPS hepatocytes in the figure indicate those obtained by culturing human iPS cell-derived hepatic progenitor cells alone.
- + Liver sinusoidal endothelial cell in the figure indicates a co-cultured human iPS cell-derived hepatic progenitor cell and mouse liver sinusoidal endothelial cell.
- + Stellate cell in the figure indicates a co-cultured human iPS cell-derived hepatic progenitor cell and mouse stellate cell.
- the “+ mesothelial cell” in the figure indicates a co-cultured human iPS cell-derived hepatic progenitor cell and mouse mesothelial cell.
- n 3. Mean ⁇ SEM.
- FIG. 34 shows a bright field image on the second day of the three-dimensional co-culture of human iPS cell-derived hepatic progenitor cells and mouse non-parenchymal cells.
- IPS hepatocytes in the figure indicate those obtained by culturing human iPS cell-derived hepatic progenitor cells alone.
- Liver sinusoidal endothelial cell in the figure indicates a co-cultured human iPS cell-derived hepatic progenitor cell and mouse liver sinusoidal endothelial cell.
- + Stellate cell indicates a co-cultured human iPS cell-derived hepatic progenitor cell and mouse stellate cell.
- the “+ mesothelial cell” in the figure indicates a co-cultured human iPS cell-derived hepatic progenitor cell and mouse mesothelial cell. (Scale bar: 50 ⁇ m)
- FIG. 35 shows the results of expression analysis of genes involved in hepatocyte differentiation when human iPS cell-derived hepatic progenitor cells and mouse non-parenchymal cells are three-dimensionally cultured.
- “IPS hepatocytes” in the figure indicate those obtained by culturing human iPS cell-derived hepatic progenitor cells alone.
- “+ Liver sinusoidal endothelial cell” in the figure indicates a co-cultured human iPS cell-derived hepatic progenitor cell and mouse liver sinusoidal endothelial cell.
- “+ Stellate cell” in the figure indicates a co-cultured human iPS cell-derived hepatic progenitor cell and mouse stellate cell.
- the “+ mesothelial cell” in the figure indicates a co-cultured human iPS cell-derived hepatic progenitor cell and mouse mesothelial cell.
- n 3. Mean ⁇ SEM. *** P ⁇ 0.005, * P ⁇ 0.05 FIG.
- FIG. 36 shows the expression analysis results of albumin when planar culture and three-dimensional culture of human iPS cell-derived hepatic progenitor cells and mouse non-parenchymal cells are performed.
- IPS hepatocytes in the figure indicate those obtained by culturing human iPS cell-derived hepatic progenitor cells alone.
- Liver sinusoidal endothelial cell in the figure indicates a co-cultured human iPS cell-derived hepatic progenitor cell and mouse liver sinusoidal endothelial cell.
- + Stellate cell indicates a co-cultured human iPS cell-derived hepatic progenitor cell and mouse stellate cell.
- the “+ mesothelial cell” in the figure indicates a co-cultured human iPS cell-derived hepatic progenitor cell and mouse mesothelial cell.
- FIG. 37 shows the expression analysis results of liver function markers when human iPS cell-derived hepatic progenitor cells and mouse non-parenchymal cells are planarly cultured (two-dimensional culture) and three-dimensionally cultured.
- “2D” in the figure indicates the result of planar culture
- “3D” in the figure indicates the result of three-dimensional culture.
- FIG. 38 shows the results of quantification of HBs antigen in the culture supernatant of hepatic progenitor cells (Hep-pro) infected with hepatitis B virus (HBV) and hepatocytes (Hep) induced to differentiate therefrom.
- FIG. 39 shows the amount of HBs antigen in the culture supernatant of hepatic progenitor cells infected with HBV (Hep-pro) and differentiation-induced hepatocytes (Hep).
- FIG. 40 shows the results of expression analysis in hepatocytes of molecules involved in HBV, HCV, and malaria infection.
- FIG. 41 shows the expression analysis results of CPM in mouse liver progenitor cells (LPC), mouse mature hepatocytes (Hep), and mouse bile duct epithelial cells (Chol).
- FIG. 42 shows the results of flow cytometric analysis of embryonic day 12.5 day mouse fetal liver. CD31 + CD34 +/ ⁇ cells are present in the CD45 ⁇ FLK1 + cell fraction (left).
- FIG. 43 shows the expression analysis results of hepatic sinusoidal progenitor cell marker molecules in mouse CD45 ⁇ FLK1 + CD31 + CD34 ⁇ cells (CD34 ⁇ ) and mouse CD45 ⁇ FLK1 + CD31 + CD34 + cells (CD34 + ).
- n 3. Mean ⁇ SEM. * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001.
- FIG. 42 shows the results of flow cytometric analysis of embryonic day 12.5 day mouse fetal liver. CD31 + CD34 +/ ⁇ cells are present in the CD45 ⁇ FLK1 + cell fraction (left).
- FIG. 43 shows the expression analysis results of hepatic sinusoidal progenitor cell marker molecules in mouse CD45 ⁇ FLK1 + CD31 + CD34 ⁇ cells (CD34
- FIG. 44 shows the proliferative ability of mouse CD45 ⁇ FLK1 + CD31 + CD34 + cells (hepatic sinusoidal progenitor cells).
- n 1
- hepatocytes are liver parenchymal cells that have major functions as a liver such as bile production and metabolism of various substances. Hepatocytes express the drug metabolizing enzyme CYP. Hepatocytes have albumin producing ability and glycogen storage ability.
- liver non-parenchymal cells refers to a group of cells other than hepatic parenchymal cells (hepatocytes) constituting the liver, hepatic sinusoidal endothelial cells, hepatic stellate cells, mesothelial cells, Bile duct epithelial cells, pit cells, Kupffer cells and the like are included.
- pluripotent stem cell refers to a cell having self-renewal ability and pluripotency, and having the ability to form all cells constituting a living body.
- Self-replicating ability refers to the ability to make the same undifferentiated cell from one cell.
- “Differentiation ability” refers to the ability of a cell to differentiate.
- Examples of pluripotent stem cells include embryonic stem cells (embryonic stem cells: ES cells), Muse cells (Multi-lineage differentiating Stress Enduring cells), sperm stem cells (germline stem cells: GS cells), embryonic germ cells (embryonics) Examples include, but are not limited to, germ cells (EG cells) and induced pluripotent cells (iPS cells).
- the origin of pluripotent stem cells may be any of mammals, birds, fishes, reptiles and amphibians, and is not particularly limited. Mammals include primates (human, monkey, etc.), rodents (mouse, rat, guinea pig, etc.), cats, dogs, rabbits, sheep, pigs, cows, horses, donkeys, goats, ferrets and the like.
- hepatic endoderm cells refers to the formation of organs derived from the intestinal tract such as the esophagus, stomach, small intestine and large intestine, and lungs, liver, thymus, parathyroid, thyroid, gallbladder, and pancreas. It is a germ layer cell.
- hepatic endoderm cells are cells differentiated from definitive endoderm cells, and are cells having the ability to differentiate into hepatic progenitor cells.
- definitive endoderm cells” and “hepatic progenitor cells” include not only those present in the living body but also those obtained by differentiation from pluripotent stem cells.
- mesoderm cells refers to mesothelial, muscle, skeletal, cutaneous dermis, connective tissue, heart / blood vessels (including vascular endothelium), blood (including blood cells), lymphatic vessels, spleen, kidney, Germ cells are responsible for the formation of ureters, gonads (testis, uterus, gonadal epithelium) and the like.
- mesoderm cells include not only those present in the living body but also those obtained by differentiation from pluripotent stem cells.
- hepatic progenitor cells are cells derived from definitive endoderm cells that have the ability to self-replicate and have the ability to differentiate into hepatocytes or bile duct epithelial cells.
- hepatic progenitor cells include not only those existing in the living body but also those obtained by differentiation from pluripotent stem cells.
- hepatic sinusoidal progenitor cells refers to cells derived from mesoderm cells that have the ability to self-renew and differentiate into hepatic sinusoidal endothelial cells.
- hepatic sinusoidal progenitor cells include not only those present in the living body but also those obtained by differentiation from pluripotent stem cells.
- hepatic stellate progenitor cells are cells derived from mesoderm cells that have self-replicating ability and have the ability to differentiate into hepatic stellate cells.
- hepatic stellate progenitor cells include not only those present in the living body but also those obtained by differentiation from pluripotent stem cells.
- proliferation ability refers to the ability of cells to proliferate.
- state of growth refers to the possibility of growth at steady state.
- steady state is a normal condition in a living body and a state in which the homeostasis of the living body is maintained. Such a state can be easily determined by those skilled in the art. For example, it can be confirmed by cell density analysis that the cell density is substantially constant and does not change, or that no expression of a cell proliferation marker is observed.
- “highly proliferating ability” means having a proliferating ability in a steady state.
- cell fraction refers to a group of cells containing a certain amount of cells to be sorted, isolated or concentrated.
- the cell fraction may contain other cells than cells to be sorted, isolated or enriched, and / or one or more chemical substances.
- the form of the cell fraction is not particularly limited, and may be, for example, a liquid containing cells, or a liquid in which the liquid is frozen.
- One embodiment of the present invention relates to a method for preparing hepatic progenitor cells, including a step of sorting hepatic progenitor cells from a cell fraction containing hepatic progenitor cells using a CPM positive phenotype as an index.
- the method for preparing hepatic progenitor cells of the present invention it is also referred to as “the method for preparing hepatic progenitor cells of the present invention”.
- the “cell fraction containing hepatic progenitor cells” used in the “method for preparing hepatic progenitor cells of the present invention” is not particularly limited, but any cell that differentiates into hepatic progenitor cells may be prepared as a raw material. . For example, it may be prepared by inducing differentiation of definitive endoderm cells or hepatic endoderm cells.
- the cell fraction may be prepared by extraction from a living body. For example, it may be prepared from the liver of an animal fetus.
- the preparation method further comprises a step of preparing a cell fraction containing hepatic progenitor cells.
- the definitive endoderm Cells or hepatic endoderm cells may be prepared by extraction from a living body, or may be prepared by inducing differentiation of pluripotent stem cells.
- the pluripotent stem cells used are not particularly limited, but are preferably mammalian pluripotent stem cells, more preferably primates (human, monkey).
- Rodents mouse, rat, guinea pig, etc.
- cats dogs, rabbits, sheep, pigs, cows, horses, donkeys, goats or ferrets, more preferably human pluripotent Stem cells, particularly preferably human iPS cells.
- the differentiation induction from pluripotent stem cells to hepatic progenitor cells via definitive endoderm cells and hepatic endoderm cells may be performed using a known method and is not particularly limited.
- the procedure is similar to the procedure for inducing differentiation of human hepatic progenitor cells from human iPS cells. It may be done.
- Carboxypeptidase M is a member of the carboxypeptidase family. CPM is expressed on the cell membrane surface and cleaves arginine and lysine residues from the C-terminus of peptides and proteins. Surprisingly, CPM was found to be a hepatic progenitor cell specific marker molecule. CPM is highly expressed in hepatic progenitor cells, but not expressed in pluripotent stem cells before differentiation, hepatocytes after differentiation or bile duct epithelial cells. Conventionally, ⁇ -fetoprotein (AFP) and HNF4a are known as markers specific to hepatic progenitor cells.
- AFP ⁇ -fetoprotein
- HNF4a are known as markers specific to hepatic progenitor cells.
- CPM is a membrane protein, it can be collected without destroying hepatic progenitor cells using this as an index.
- the process of sorting hepatic progenitor cells using the CPM positive phenotype as an index is not particularly limited.
- fluorescence activated cell sorting FACS
- MCS Magnetic Cell Sorting
- the preparation method may further include a step of growing the sorted hepatic progenitor cells.
- Cell culture conditions in the proliferation step are not particularly limited as long as they do not inhibit the proliferation of hepatic progenitor cells.
- One embodiment of the present invention relates to a method for isolating hepatic progenitor cells, comprising a step of sorting hepatic progenitor cells from a cell fraction containing hepatic progenitor cells using a CPM positive phenotype as an index.
- One embodiment of the present invention also relates to a method for concentrating hepatic progenitor cells, comprising a step of sorting hepatic progenitor cells from a cell fraction containing hepatic progenitor cells using a CPM positive phenotype as an index.
- One embodiment of the present invention can be prepared by the method for preparing hepatic progenitor cells of the present invention, has a CPM positive phenotype, has a proliferative ability, and has a differentiation ability into hepatocytes or bile duct epithelial cells. Relates to progenitor cells.
- the hepatic progenitor cell of the present invention it is also referred to as “the hepatic progenitor cell of the present invention”.
- the hepatic progenitor cells of the present invention can be stored frozen.
- the proliferation ability of the hepatic progenitor cells of the present invention after freezing and thawing does not decrease compared with the proliferation capacity of the hepatic progenitor cells of the present invention before cryopreservation.
- the “hepatic progenitor cells of the present invention” can be stored, for example, at ⁇ 80 ° C. for 3 months or more, 6 months or more, 9 months or more, or 12 months or more. Due to the high proliferative ability and cryopreservability of the hepatic progenitor cells of the present invention, hepatic progenitor cells can be thawed and cultured as necessary.
- the hepatic progenitor cells of the present invention can be subcultured.
- the hepatic progenitor cells of the present invention express ⁇ -fetoprotein (AFP), HNF4a, AFP, HNF4 ⁇ , HNF1 ⁇ , PROX1, TBX3, CD13, EpCAM and HHEX, which are known as markers for hepatic progenitor cells.
- One aspect of the present invention is a cell fraction containing 90% or more of hepatic progenitor cells with respect to total cells, wherein the hepatic progenitor cells have a CPM positive phenotype, have a proliferative ability
- the present invention relates to a cell fraction having the ability to differentiate into biliary epithelial cells.
- cell fraction containing hepatic progenitor cells of the present invention it is also referred to as “cell fraction containing hepatic progenitor cells of the present invention”.
- the “cell fraction containing the hepatic progenitor cells of the present invention” contains 90% or more of hepatic progenitor cells, preferably 91% or more, 92% or more, 93% with respect to all cells in the fraction. In the above, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more is included.
- the “cell fraction containing hepatic progenitor cells of the present invention” includes a concentrated fraction of hepatic progenitor cells of the present invention and a cell group obtained by culturing the concentrated fraction.
- the “cell fraction containing the hepatic progenitor cells of the present invention” is not particularly limited, and can be prepared, for example, according to the above-described method for preparing hepatic progenitor cells of the present invention.
- One embodiment of the present invention relates to a method for preparing hepatocytes, which includes a step of inducing differentiation of the hepatic progenitor cells of the present invention into hepatocytes.
- the method for preparing hepatocytes of the present invention it is also referred to as “the method for preparing hepatocytes of the present invention”.
- One embodiment of the “method for preparing hepatocytes of the present invention” comprises (1) a step of sorting hepatic progenitor cells from a cell fraction containing hepatic progenitor cells using a CPM positive phenotype as an index, and (2)
- the present invention relates to a method for preparing hepatocytes, comprising a step of inducing differentiation of the taken hepatic progenitor cells into hepatocytes.
- the induction of differentiation from hepatic progenitor cells to hepatocytes may be performed using a known method and is not particularly limited.
- a known method for example, as described in Non-Patent Document 2 (Si-Tayeb et al., Hepatology 2010, 51 (1), 297-305), it is known as an interleukin-6 (IL-6) family cytokine. May be performed by treating a medium containing human hepatic progenitor cells with Oncostatin M.
- IL-6 interleukin-6
- the preparation method may further include a step of proliferating hepatocytes obtained by inducing differentiation.
- the conditions for cell culture in the growth step are not particularly limited as long as the growth of hepatocytes is not inhibited.
- One embodiment of the present invention relates to a hepatocyte having proliferative ability that can be prepared by the method for preparing a hepatocyte of the present invention.
- the hepatocyte of the present invention it is also referred to as “the hepatocyte of the present invention”.
- the “hepatocytes of the present invention” express a drug metabolizing enzyme CYP (for example, CYP3A4, CYP2C19, CYP2C18, CYP2D6, CYP1A, CYP2C8) at a high level.
- CYP3A4 can be expressed at least twice as compared with hepatocytes prepared by subjecting a cell fraction having the same composition as the cell fraction to a differentiation induction step without sorting the progenitor cells.
- the “hepatocytes of the present invention” have higher albumin production ability and glycogen accumulation ability than the hepatic progenitor cells before differentiation induction.
- the hepatocytes of the present invention prepared by inducing differentiation of hepatic progenitor cells collected from the cell fraction containing hepatic progenitor cells using the CPM positive phenotype as an index are used as an index.
- a hepatocyte prepared by subjecting a cell fraction having the same composition as that of the cell fraction to a differentiation induction step without sorting hepatic progenitor cells it has higher albumin production ability and glycogen accumulation ability.
- One embodiment of the present invention relates to a cell fraction containing “the hepatocyte of the present invention”.
- the cell fraction is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more of the “hepatocytes of the present invention” are included.
- One embodiment of the present invention relates to a method for preparing bile duct epithelial cells, including a step of inducing differentiation of hepatic progenitor cells of the present invention into bile duct epithelial cells.
- the method for preparing bile duct epithelial cells of the present invention it is also referred to as “the method for preparing bile duct epithelial cells of the present invention”.
- One embodiment of the “method for preparing bile duct epithelial cells of the present invention” includes (1) a step of sorting hepatic progenitor cells from a cell fraction containing hepatic progenitor cells using a CPM positive phenotype as an index, and (2) The present invention relates to a method for preparing bile duct epithelial cells, comprising a step of inducing differentiation of the sorted hepatic progenitor cells into bile duct epithelial cells.
- differentiation induction from hepatic progenitor cells of the present invention into bile duct epithelial cells may be performed using a known method and is not particularly limited.
- the three-dimensional gel culture method described in Tanimizu ⁇ et al., Mol Biol Cell, 2007, 18 (4), 1472-1479 or Yanagida et al., PloS ONE 8, e67541 may be used.
- the preparation method may further include a step of proliferating bile duct epithelial cells obtained by inducing differentiation.
- Cell culture conditions in the proliferation step are not particularly limited as long as they do not inhibit the proliferation of bile duct epithelial cells.
- One embodiment of the present invention relates to a bile duct epithelial cell having proliferative ability, which can be prepared by the method for preparing a bile duct epithelial cell of the present invention.
- the biliary epithelial cell of the present invention it is also referred to as “the biliary epithelial cell of the present invention”.
- the “bile duct epithelial cell of the present invention” expresses CK7, CFTR, AQP1, TGR5, SOX9 and HNF6, which are known as markers for bile duct epithelial cells.
- One embodiment of the present invention relates to a cell fraction containing the “bile duct epithelial cell of the present invention”.
- the cell fraction is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more of the “bile duct epithelial cells of the present invention” are included.
- One aspect of the invention includes a step of sorting hepatic sinusoidal endothelial progenitor cells from a cell fraction containing hepatic sinusoidal progenitor progenitors using FLK1-positive, CD34-positive, and CD31-positive phenotypes as indices.
- the present invention relates to a method for preparing progenitor cells. Hereinafter, it is also referred to as “the method for preparing hepatic sinusoidal endothelial progenitor cells of the present invention”.
- the “cell fraction containing hepatic sinusoidal endothelial progenitor cells” used in “the method for preparing hepatic sinusoidal endothelial progenitor cells of the present invention” is not particularly limited, but any cell that differentiates into hepatic sinusoidal endothelial progenitor cells. It may be prepared from cells. For example, it may be prepared by inducing differentiation of mesoderm cells. For example, it may be prepared by inducing differentiation of hematopoietic stem cells. The cell fraction may be prepared by extraction from a living body. For example, it may be prepared from the liver of an animal fetus.
- the preparation method further comprises a step of preparing a cell fraction containing hepatic sinusoidal endothelial progenitor cells.
- the mesoderm cells When the “cell fraction containing hepatic sinusoidal endothelial progenitor cells” used in “the method for preparing hepatic sinusoidal endothelial progenitor cells of the present invention” is prepared by inducing differentiation of mesoderm cells, the mesoderm cells May be prepared by extraction from a living body or may be prepared by inducing differentiation of pluripotent stem cells. Differentiation from pluripotent stem cells to mesoderm cells can be performed according to a known method. For example, a method via an embryoid body (EB) may be used. Further, for example, it may be carried out according to the method described in Kattman S J, et al., Cell Stem Cell, 2011, 8, 228-40.
- EB embryoid body
- pluripotent stem cell is a mammalian pluripotent stem cell, More preferably, it is a primate (a human, a monkey, etc.), a rodent (a mouse, a rat, a guinea pig, etc.), a cat , Pluripotent stem cells of dogs, rabbits, sheep, pigs, cows, horses, donkeys, goats or ferrets, more preferably human pluripotent stem cells, particularly preferably human iPS cells.
- a primate a human, a monkey, etc.
- rodent a mouse, a rat, a guinea pig, etc.
- a cat Pluripotent stem cells of dogs, rabbits, sheep, pigs, cows, horses, donkeys, goats or ferrets, more preferably human pluripotent stem cells, particularly preferably human iPS cells.
- the differentiation induction of mesoderm cells into hepatic sinusoidal endothelial progenitor cells may be performed using a known method and is not particularly limited. For example, it may be carried out according to the method described in Kattman S J, et al., Cell Stem Cell, 2011, 8, 228-40.
- One embodiment of the “method for preparing hepatic sinusoidal endothelial progenitor cells of the present invention” includes (1) a step of inducing differentiation of mesoderm cells to prepare a cell fraction containing hepatic sinusoidal endothelial progenitor cells, (2)
- the present invention relates to a method for preparing hepatic sinusoidal progenitor progenitor cells, comprising a step of sorting hepatic sinusoidal progenitor progenitor cells using FLK1-positive, CD34-positive and CD31-positive phenotypes as indices.
- FLK1, CD34, and CD31 are known as vascular endothelial cell markers. Surprisingly, it was found that the combination of FLK1, CD34 and CD31 is a specific marker for hepatic sinusoidal progenitor cells.
- the process of sorting hepatic sinusoidal progenitor progenitors using FLK1-positive, CD34-positive and CD31-positive phenotypes as an index is not particularly limited.
- fluorescence activated cell sorting FACS
- MCS Magnetic Cell Sorting
- the preparation method may further include a step of growing the sorted hepatic sinusoidal progenitor progenitor cells.
- the conditions for cell culture in the proliferation step are not particularly limited as long as the proliferation of hepatic sinusoidal endothelial precursor cells is not inhibited.
- One aspect of the present invention includes a step of sorting hepatic sinusoidal progenitor cells from a cell fraction containing hepatic sinusoidal progenitor progenitors using FLK1-positive, CD34-positive, and CD31-positive phenotypes as indices.
- the present invention relates to a method for isolating endothelial progenitor cells.
- One embodiment of the present invention includes a step of sorting hepatic sinusoidal progenitor cells from a cell fraction containing hepatic sinusoidal progenitor cells using FLK1-positive, CD34-positive, and CD31-positive phenotypes as indices.
- the present invention relates to a method for concentrating sinusoidal endothelial progenitor cells.
- One aspect of the invention can be prepared by the method for preparing hepatic sinusoidal endothelial progenitor cells of the present invention, has FLK1-positive, CD34-positive and CD31-positive phenotypes, has a proliferative ability, and is into hepatic sinusoidal endothelial cells.
- the present invention relates to hepatic sinusoidal endothelial progenitor cells having differentiating ability. Hereinafter, it is also referred to as “hepatic sinusoidal progenitor cell of the present invention”.
- the hepatic sinusoidal endothelial progenitor cells of the present invention can be cryopreserved. After freezing and thawing, the ability of the hepatic sinusoidal endothelial progenitor cells of the present invention to proliferate does not decrease compared to the ability of the hepatic sinusoidal endothelial progenitor cells of the present invention to proliferate before cryopreservation.
- the “hepatic sinusoidal progenitor cells of the present invention” can be stored, for example, at ⁇ 80 ° C. for 3 months or more, 6 months or more, 9 months or more, or 12 months or more.
- hepatic sinusoidal endothelial progenitor cells of the present invention Due to the excellent proliferative ability and cryopreservability of the hepatic sinusoidal endothelial progenitor cells of the present invention, it is possible to appropriately cultivate hepatic sinusoidal endothelial progenitor cells as needed.
- the hepatic sinusoidal progenitor cells of the present invention can be subcultured.
- One aspect of the present invention is a cell fraction containing 90% or more of hepatic sinusoidal progenitor progenitors with respect to all cells, wherein the hepatic sinusoidal progenitor progenitors are FLK1-positive, CD34-positive and CD31-positive phenotypes. And a cell fraction having proliferation ability and differentiation ability into hepatic sinusoidal endothelial cells.
- a cell fraction containing the hepatic sinusoidal progenitor cells of the present invention it is also referred to as “a cell fraction containing the hepatic sinusoidal progenitor cells of the present invention”.
- the “cell fraction containing hepatic sinusoidal progenitor progenitor cells of the present invention” contains 90% or more of hepatic sinusoidal progenitor progenitor cells, preferably 91% or more, with respect to the total cells in the fraction. 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
- the “cell fraction containing hepatic sinusoidal progenitor progenitor cells of the present invention” includes a concentrated fraction of hepatic sinusoidal endothelial progenitor cells of the present invention and a cell group obtained by culturing the concentrated fraction. .
- the “cell fraction containing the hepatic sinusoidal progenitor progenitor cells of the present invention” is not particularly limited, and can be prepared, for example, according to the “method for preparing hepatic sinusoidal endothelial progenitor cells of the present invention” described above.
- One aspect of the invention is (1) preparing a cell fraction containing hepatic sinusoidal progenitor cells by inducing differentiation of the hepatic sinusoidal progenitor cells of the present invention using a TGF- ⁇ inhibitor, (2 And a method of preparing hepatic sinusoidal endothelial cells from the cell fraction containing hepatic sinusoidal endothelial cells using CD31-positive and Fc ⁇ R II-positive phenotypes as an index.
- the method for preparing hepatic sinusoidal endothelial cells of the present invention is also referred to as “the method for preparing hepatic sinusoidal endothelial cells of the present invention”.
- One embodiment of the “method for preparing hepatic sinusoidal endothelial cells of the present invention” is as follows: (1) From a cell fraction containing hepatic sinusoidal endothelial progenitor cells, FLK1 positive, CD34 positive and CD31 positive phenotypes are used as indicators.
- a step of sorting out sinusoidal progenitor cells (2) a step of preparing hepatic sinusoidal endothelial progenitor cells by inducing differentiation of the hepatic sinusoidal progenitor cells of the present invention using a TGF- ⁇ inhibitor, (3)
- the present invention relates to a method for preparing hepatic sinusoidal endothelial cells, comprising a step of sorting hepatic sinusoidal endothelial cells from the cell fraction containing hepatic sinusoidal endothelial cells using CD31 positive and Fc ⁇ R II positive phenotypes as indices.
- TGF- ⁇ inhibitor refers to a drug for inhibiting the function or signal transduction of TGF- ⁇ , which is a transforming growth factor, such as a low molecular weight compound, an antibody, or an antisense compound. It may be in the form of
- the TGF- ⁇ inhibitor used in the method for preparing hepatic sinusoidal endothelial cells of the present invention is not particularly limited.
- A83-01 (3- (6-methyl-2-pyridinyl) -N-phenyl- 4- (4-quinolinyl) -1H-pyrazolo-1-carbothioamide), I616451 (3- (pyridin-2-yl) -4- (4-quinonyl))-1H-pyrazole), LDN193189 (4- (6 -(4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl) quinoline), SB431542 (4- [4- (1,3-benzodioxol-5-yl) ) -5-pyridin-2-yl-1H-imidazol-2-yl] benzamide), SB-505124 (2- (5-benzo [1,3] dioxol-5-yl-2-ter) -Butyl-3H-imidazol-4-yl) -6-methylpyridine hydrochloride hydrate), SD-208 (2- (5-chloride
- the process of sorting hepatic sinusoidal endothelial cells using CD31-positive and Fc ⁇ R II-positive phenotypes as an index is not particularly limited.
- fluorescence activated cell sorting FACS
- MACS Magnetic Cell Sorting
- the preparation method may further include a step of proliferating hepatic sinusoidal endothelial cells obtained by inducing differentiation.
- the conditions for cell culture in the growth step are not particularly limited as long as the growth of hepatic sinusoidal endothelial cells is not inhibited.
- One aspect of the present invention relates to hepatic sinusoidal endothelial cells that can be prepared by the “method for preparing hepatic sinusoidal endothelial cells of the present invention”, have CD31 positive and Fc ⁇ RII positive phenotypes, and have proliferative ability. Hereinafter, it is also referred to as “hepatic sinusoidal endothelial cell of the present invention”.
- the hepatic sinusoidal endothelial cells of the present invention also express Stab2, Lyve1 and factor VIII, which are known as markers for hepatic sinusoidal endothelial cells.
- the “hepatic sinusoidal endothelial cell of the present invention” has the ability to take up acetylated LDL, which is a characteristic function of vascular endothelial cells. Moreover, the hepatic sinusoidal endothelial cells of the present invention have the ability to take in hyaluronic acid, which is a function characteristic of hepatic sinusoidal endothelial cells. The “hepatic sinusoidal endothelial cell of the present invention” has the ability to form a vascular network.
- hemophilia can be treated by transplanting the hepatic sinusoidal endothelial cells of the present invention into a hemophilia patient.
- One aspect of the present invention relates to a cell fraction containing “the hepatic sinusoidal endothelial cell of the present invention”.
- the cell fraction is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or It contains 99% or more of “the liver sinusoidal endothelial cells of the present invention”.
- One aspect of the present invention is a cell fraction containing 90% or more hepatic sinusoidal endothelial cells with respect to the total cells, wherein the hepatic sinusoidal endothelial cells have a CD31 positive and Fc ⁇ R II positive phenotype, It relates to a cell fraction capable of proliferating.
- the cell fraction is preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the hepatic sinusoidal endothelium. Contains cells.
- One embodiment of the present invention relates to a method for preparing hepatic stellate progenitor cells, comprising a step of sorting hepatic stellate progenitor cells from a cell fraction containing hepatic stellate progenitor cells using an ALCAM positive phenotype as an index.
- the method for preparing hepatic stellate progenitor cells of the present invention it is also referred to as “the method for preparing hepatic stellate progenitor cells of the present invention”.
- the “cell fraction containing hepatic stellate progenitor cells” used in the “method for preparing hepatic stellate progenitor cells of the present invention” is not particularly limited, but is prepared using any cell that differentiates into hepatic stellate progenitor cells as a raw material. May be. For example, it may be prepared from a mesoderm cell population containing hepatic stellate progenitor cells. Further, for example, it may be prepared by inducing differentiation of mesenchymal stem cells (Mesenchymal Stem Cells: MSC). The cell fraction may be prepared by extraction from a living body. For example, it may be prepared from the liver of an animal fetus. In one embodiment of the method for preparing hepatic stellate progenitor cells of the present invention, the preparation method further comprises a step of preparing a cell fraction containing hepatic progenitor cells.
- the mesoderm cells are prepared by extraction from a living body. It may be prepared by differentiation induction from pluripotent stem cells. Differentiation from pluripotent stem cells to mesoderm cells can be performed according to a known method. For example, a method via an embryoid body (EB) may be used. Further, for example, it may be carried out according to the method described in Kattman S J, et al., Cell Stem Cell, 2011, 8, 228-40.
- EB embryoid body
- the pluripotent stem cell is not particularly limited, but is preferably a mammalian pluripotent stem cell, more preferably a primate (human, monkey, etc.), a rodent (mouse, rat, guinea pig, etc.), a cat, A pluripotent stem cell of a dog, rabbit, sheep, pig, cow, horse, donkey, goat or ferret, more preferably a human pluripotent stem cell, particularly preferably a human iPS cell.
- ALCAM Activated leukocyte cell adhesion molecule
- the step of sorting hepatic stellate progenitor cells using ALCAM-positive phenotype as an index is not particularly limited.
- FACS fluorescence activated cell sorting
- MCS magnetic cell sorting
- the preparation method may further include a step of growing the sorted hepatic stellate progenitor cells.
- Cell culture conditions in the proliferation step are not particularly limited as long as the proliferation of hepatic stellate progenitor cells is not inhibited.
- One embodiment of the present invention relates to a method for isolating hepatic stellate progenitor cells, including a step of sorting hepatic stellate progenitor cells from a cell fraction containing hepatic stellate progenitor cells using an ALCAM positive phenotype as an index.
- One embodiment of the present invention also relates to a method for concentrating hepatic stellate progenitor cells, comprising a step of sorting hepatic stellate progenitor cells from a cell fraction containing hepatic stellate progenitor cells using an ALCAM positive phenotype as an index.
- One aspect of the invention is a hepatic stellate progenitor cell that can be prepared by the method for preparing hepatic stellate progenitor cells of the present invention, has an ALCAM-positive phenotype, has proliferative ability, and has the ability to differentiate into hepatic stellate cells. About. Hereinafter, it is also referred to as “the hepatic stellate progenitor cell of the present invention”.
- the hepatic stellate progenitor cells of the present invention can be stored frozen. After freezing and thawing, the proliferative ability of the hepatic stellate progenitor cells of the present invention does not decrease compared to the proliferative ability of the hepatic stellate progenitor cells of the present invention before cryopreservation.
- the “hepatic stellate progenitor cells of the present invention” can be stored, for example, at ⁇ 80 ° C. for 3 months or more, 6 months or more, 9 months or more, or 12 months or more. Due to the excellent proliferative ability and cryopreservability of the hepatic stellate progenitor cells of the present invention, hepatic stellate progenitor cells can be appropriately cultured in large quantities as needed.
- One aspect of the invention is a cell fraction containing 90% or more hepatic stellate progenitor cells with respect to all cells, wherein the hepatic stellate progenitor cells have an ALCAM positive phenotype, have a proliferative ability
- the present invention relates to a cell fraction having the ability to differentiate into stellate cells.
- a cell fraction containing hepatic stellate progenitor cells of the present invention it is also referred to as “a cell fraction containing hepatic stellate progenitor cells of the present invention”.
- the “cell fraction containing hepatic stellate progenitor cells of the present invention” contains 90% or more of hepatic stellate progenitor cells, preferably 91% or more, preferably 92% or more, based on the total cells in the fraction. 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
- the “cell fraction containing the hepatic stellate progenitor cells of the present invention” includes a concentrated fraction of the hepatic stellate progenitor cells of the present invention and a cell group obtained by culturing the concentrated fraction.
- the cell fraction containing the hepatic stellate progenitor cells of the present invention is not particularly limited, and can be prepared, for example, according to the “method for preparing hepatic stellate progenitor cells of the present invention” described above.
- One aspect of the present invention relates to a method for preparing hepatic stellate cells, including a step of inducing differentiation of hepatic stellate progenitor cells into hepatic stellate cells using a Rock inhibitor.
- the method for preparing hepatic stellate cells of the present invention it is also referred to as “the method for preparing hepatic stellate cells of the present invention”.
- Rho-associated protein-kinase is a serine-threonine protein kinase that has been identified as a target protein of the low-molecular-weight GTP-binding protein Rho and has various physiological functions such as smooth muscle contraction and cell shape change. It is known to be involved.
- Rock inhibitor refers to a drug for inhibiting the function of Rock, and may be in the form of a low molecular weight compound, an antibody, an antisense compound, or the like.
- the Rock inhibitor used in the method for preparing hepatic stellate cells of the present invention is not particularly limited.
- Y27632 ((R)-(+)-trans-N- (4-pyridyl) -4- (1 -Aminoethyl) -cyclohexanecarboxamide), Fasudil (hexahydro-1- (5-isoquinolinesulfonyl) -1H-1,4-diazepine), H-1152 ((S)-(+)-2-methyl- 1-[(4-methyl-5-isoquinolinyl) sulfonyl] -hexahydro-1H-1,4-diazepine) and the like.
- Y27632 is preferable.
- the hepatic stellate progenitor cells used in the “method for preparing hepatic stellate cells of the present invention” are not particularly limited, but may be prepared using any cells that differentiate into hepatic stellate progenitor cells as a raw material. For example, it may be prepared from a mesoderm cell population containing hepatic stellate progenitor cells. For example, it may be prepared according to the above-described “method for preparing hepatic stellate progenitor cells of the present invention”.
- the hepatic stellate progenitor cells may be prepared by extraction from a living body. In one embodiment of the method for preparing hepatic stellate cells of the present invention, the preparation method further comprises a step of preparing hepatic stellate progenitor cells.
- One embodiment of the method for preparing hepatic stellate cells of the present invention includes (1) a step of sorting hepatic stellate progenitor cells from a cell fraction containing hepatic stellate progenitor cells using an ALCAM positive phenotype as an index, and (2) Rock.
- the present invention relates to a method for preparing hepatic stellate cells, comprising a step of inducing differentiation of hepatic stellate progenitor cells into hepatic stellate cells using an inhibitor.
- the hepatic stellate progenitor cells used are “the hepatic stellate progenitor cells of the present invention”.
- the preparation method may further include a step of growing hepatic stellate cells obtained by inducing differentiation.
- Cell culture conditions in the proliferation step are not particularly limited as long as they do not inhibit the proliferation of hepatic stellate cells.
- One embodiment of the present invention relates to a hepatic stellate cell that can be prepared by the method for preparing a hepatic stellate cell of the present invention and has a proliferation ability.
- the hepatic stellate cell of the present invention it is also referred to as “the hepatic stellate cell of the present invention”.
- One embodiment of the present invention relates to a cell fraction containing “the hepatic stellate cells of the present invention”.
- the cell fraction is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or It contains 99% or more of the “hepatic stellate cells of the present invention”.
- One aspect of the present invention includes the step of co-culturing the hepatic progenitor cells of the present invention with at least one hepatic non-parenchymal cell selected from the group consisting of hepatic sinusoidal endothelial cells, hepatic stellate cells, and hepatic mesothelial cells.
- the present invention relates to a method for preparing a hepatocyte tissue model. Hereinafter, it is also referred to as “a method for preparing a hepatocyte tissue model of the present invention”.
- the hepatic sinusoidal endothelial cells used in the “method for preparing a hepatocyte tissue model of the present invention” may be any of liver, sinusoidal endothelial cells of mammals, birds, fish, reptiles and amphibians, and are not particularly limited.
- it is a mammalian hepatic sinusoidal endothelial cell, more preferably a primate (human, monkey, etc.), rodent (mouse, rat, guinea pig, etc.), cat, dog, rabbit, sheep, pig, cow, Hepatic sinusoidal endothelial cells of horses, donkeys, goats or ferrets, particularly preferably human hepatic sinusoidal endothelial cells.
- the hepatic sinusoidal endothelial cell may be the hepatic sinusoidal endothelial cell of the present invention.
- the hepatic sinusoidal endothelial cells may be prepared from any cells that differentiate into hepatic sinusoidal endothelial cells. For example, it may be prepared by inducing differentiation of pluripotent stem cells, mesoderm cells, or hepatic sinusoidal endothelial cells.
- the hepatic sinusoidal endothelial cells may be prepared by extraction from a living body. For example, it may be prepared from the liver of an animal fetus.
- the hepatic sinusoidal endothelial cells may be prepared according to the “method for preparing hepatic sinusoidal endothelial cells of the present invention”.
- the hepatic stellate cells used in the “method for preparing a hepatocyte tissue model of the present invention” may be any of mammalian, avian, fish, reptile, and amphibian hepatic stellate cells, and is not particularly limited.
- it is a mammalian hepatic stellate cell, more preferably a primate (human, monkey, etc.), rodent (mouse, rat, guinea pig, etc.), cat, dog, rabbit, sheep, pig, cow, horse, Donkey, goat or ferret hepatic stellate cells, particularly preferably human hepatic stellate cells.
- the hepatic stellate cell may be the hepatic stellate cell of the present invention.
- the hepatic stellate cells may be prepared using any cells that differentiate into hepatic stellate cells as a raw material. For example, it may be prepared by inducing differentiation of pluripotent stem cells, mesoderm cells, or hepatic stellate progenitor cells.
- the cell fraction may be prepared by extraction from a living body. For example, it may be prepared from the liver of an animal fetus.
- the hepatic stellate cells may be prepared according to the “method for preparing hepatic stellate cells of the present invention”.
- the hepatic mesothelial cells used in the “method for preparing a hepatocyte tissue model of the present invention” may be any of mammalian, avian, fish, reptile, and amphibian hepatic mesothelial cells, and are not particularly limited.
- it is a mammalian mesothelial cell, preferably a primate (human, monkey, etc.), rodent (mouse, rat, guinea pig, etc.), cat, dog, rabbit, sheep, pig, cow, horse, Donkey, goat or ferret liver mesothelial cells.
- the hepatic mesothelial cells may be prepared using any cells that differentiate into hepatic mesothelial cells as a raw material. For example, it may be prepared by inducing differentiation of pluripotent stem cells.
- the hepatic mesothelial cells may be prepared by extraction from a living body. For example, it may be prepared from the liver of an animal fetus.
- the conditions in the step of co-culturing are not particularly limited as long as they promote differentiation induction of hepatic progenitor cells.
- two-dimensional culture planar culture
- it is three-dimensionally cultured.
- three-dimensional culture is performed.
- One aspect of the present invention includes hepatocytes and at least one hepatic non-parenchymal cell selected from the group consisting of hepatic sinusoidal endothelial cells, hepatic stellate cells, and hepatic mesothelial cells.
- the present invention relates to a hepatocyte tissue model that can be prepared in “Model Preparation Method”. Hereinafter, it is also referred to as “the hepatocyte tissue model of the present invention”.
- One embodiment of the present invention includes a hepatic progenitor cell of the present invention, a cell fraction containing the hepatic progenitor cell of the present invention, a hepatocyte of the present invention, a bile duct epithelial cell of the present invention, a hepatic sinusoid endothelial progenitor cell of the present invention, Cell fraction containing hepatic sinusoidal progenitor cells of the invention, hepatic sinusoidal endothelial cells of the invention, hepatic stellate progenitor cells of the invention, cell fraction containing hepatic stellate progenitor cells of the invention, hepatic stellate cells of the invention Or a pharmaceutical composition comprising the hepatocyte tissue model of the present invention.
- the pharmaceutical composition of the present invention it is also referred to as “the pharmaceutical composition of the present invention”.
- composition of the present invention may further contain a pharmaceutically acceptable carrier, diluent, buffer, excipient, or a combination thereof.
- the pharmaceutical composition is a material for cell transplantation treatment or a composition for cell transplantation treatment, or a material for regenerative medicine or a composition for regenerative medicine.
- the administration site of the pharmaceutical composition of the present invention is not particularly limited, and may be, for example, in the liver, in the spleen, in the portal vein, in the intestinal tract, in the abdominal cavity, under the kidney capsule, or in the lymph node.
- One embodiment of the present invention relates to a method for screening a liver disease therapeutic agent, comprising administering a candidate for a liver disease therapeutic agent to the hepatocytes of the present invention or the hepatocyte tissue model of the present invention.
- the screening method for the therapeutic agent for liver disease of the present invention it is also referred to as “the screening method for the therapeutic agent for liver disease of the present invention”.
- liver disease in the “screening method for the therapeutic agent for liver disease of the present invention” is not particularly limited.
- One embodiment of the present invention relates to a method for evaluating the hepatotoxicity of a drug, comprising administering the drug to “the hepatocyte of the present invention” or “the hepatocyte tissue model of the present invention”.
- the method for evaluating the hepatotoxicity of the drug of the present invention it is also referred to as “the method for evaluating the hepatotoxicity of the drug of the present invention”.
- the method for evaluating the hepatotoxicity of the drug of the present invention is not particularly limited, and includes, for example, examining the inhibitory activity of CYP (eg, CYP3A4, CYP2C19, CYP2C18, CYP2D6, CYP1A, CYP2C8).
- CYP eg, CYP3A4, CYP2C19, CYP2C18, CYP2D6, CYP1A, CYP2C8.
- One aspect of the present invention includes (1) a step of infecting a hepatic progenitor cell in “the hepatic progenitor cell of the present invention” or “a cell fraction containing the hepatic progenitor cell of the present invention” with (2) a pathogen.
- the present invention relates to a method for preparing hepatocytes of an infectious liver disease model, comprising preparing differentiation of the infected hepatic progenitor cells to prepare hepatocytes of the infectious disease model.
- One embodiment of the present invention also relates to a method for preparing hepatocytes of an infectious disease model, which includes the step of infecting hepatocytes of the present invention with a pathogen.
- these preparation methods are also referred to as “the preparation method of hepatocytes of the infectious disease model of the present invention”.
- the pathogen used in the “method for preparing hepatocytes of an infectious disease model of the present invention” is not particularly limited, and examples thereof include hepatitis viruses (eg, hepatitis A virus (HAV), hepatitis B virus (HBV)). , Hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), hepatitis F virus (HFV), hepatitis G virus (HGV), hepatitis TT virus (HTTV)), Examples include malaria parasites.
- HAV hepatitis A virus
- HBV Hepatitis B virus
- HCV Hepatitis C virus
- HDV hepatitis D virus
- HEV hepatitis E virus
- HV hepatitis F virus
- HGV hepatitis G virus
- HTTV hepatitis TT virus
- infectious disease depends on the infectious agent, but for example hepatitis (eg, hepatitis A, hepatitis B, hepatitis C, etc.) , Hepatitis D, hepatitis E, hepatitis F, hepatitis G, hepatitis TT) and malaria.
- hepatitis eg, hepatitis A, hepatitis B, hepatitis C, etc.
- Hepatitis D hepatitis E, hepatitis F, hepatitis G, hepatitis TT
- One embodiment of the present invention relates to an infectious disease model hepatocyte that can be prepared by the method for preparing an infectious disease model hepatocyte of the present invention.
- hepatocytes of the infectious disease model of the present invention it is also referred to as “hepatocytes of the infectious disease model of the present invention”.
- One aspect of the present invention includes (1) a step of infecting a hepatic progenitor cell of the present invention with a pathogen, (2) a hepatic sinusoidal endothelial cell, hepatic stellate cell, and hepatic mesothelial cell infected with the hepatic progenitor cell infected with the pathogen.
- the present invention relates to a method for preparing an infectious disease model tissue comprising a step of co-culturing with at least one hepatic non-parenchymal cell selected from the group consisting of: Hereinafter, it is also referred to as “a method for preparing an infectious disease model tissue of the present invention”.
- hepatic sinusoidal endothelial cells, hepatic stellate cells and hepatic mesothelial cells used in the “method for preparing an infectious liver disease model tissue of the present invention” are not particularly limited, and “the method for preparing the hepatocyte tissue model of the present invention” May be the same as those mentioned in.
- the pathogen used in the “preparation method of the infectious disease model tissue of the present invention” and the target disease are not particularly limited, and are the same as those mentioned in the “hepatocytes of the infectious disease model of the present invention”. Good.
- infectious disease model tissue that can be prepared by the method for preparing an infectious disease model tissue of the present invention.
- infectious disease model tissue of the present invention it is also referred to as “infectious disease model tissue of the present invention”.
- One aspect of the present invention includes administering a candidate for a therapeutic agent for infectious liver disease to “hepatocytes of the infectious disease model of the present invention” or “infectious disease model tissue of the present invention”.
- the present invention relates to a screening method for therapeutic agents for liver diseases.
- One embodiment of the present invention includes a hepatic progenitor cell of the present invention, a cell fraction containing the hepatic progenitor cell of the present invention, a hepatocyte of the present invention, a bile duct epithelial cell of the present invention, a hepatic sinusoid endothelial progenitor cell of the present invention, Cell fraction containing hepatic sinusoidal progenitor cells of the invention, hepatic sinusoidal endothelial cells of the invention, hepatic stellate progenitor cells of the invention, cell fraction containing hepatic stellate progenitor cells of the invention, hepatic stellate cells of the invention Or a kit containing the hepatocyte tissue model of the present invention.
- kit of the present invention it is also referred to as “kit of the present invention”.
- the kit of the present invention is not particularly limited.
- the hepatocytes of the present invention, the bile duct epithelial cells of the present invention, the hepatic sinusoidal endothelial cells of the present invention, the hepatic stellate cells of the present invention, or the hepatocyte tissue model of the present invention are prepared. Therefore, for the preparation of the pharmaceutical composition of the present invention, for the screening method of the therapeutic agent for liver disease of the present invention, for the evaluation method of hepatotoxicity of the drug of the present invention, for the infectious disease model of the present invention. May be used for the preparation of hepatocytes, for the preparation of the infectious disease model tissue of the present invention, or for the screening method for the therapeutic agent for infectious liver disease.
- the kit of the present invention may include instructions for using the kit.
- the cultured cells were fixed with 10% formalin solution (Wako Pure Chemical Industries, Ltd.) for 10 minutes and then permeabilized with 0.2% Triton-X100 (Wako Pure Chemical Industries, Ltd.) for 15 minutes. After blocking with 5% skim milk solution (BD Biosciences), the primary antibody was reacted overnight at 4 ° C. After washing with PBS, the fluorescently labeled secondary antibody was reacted at room temperature for 2 hours and counterstained with Hoechst33342 (Sigma-Aldrich Corporation, St. Louis, US).
- FcR block reagent Miltenyi Biotech, Bergisch-Gladbach, Germany
- the cells were labeled with anti-FITC microbeads (Miltenyi Biotech) and anti-PE microbeads (Miltenyi Biotech) as necessary, and the target cells were concentrated with an autoMACS Pro separator (Mi1tenyi Biotech). Then, it isolate
- the primary antibody, secondary antibody and microbeads used in this example are shown below.
- Example 1 Preparation of human hepatic progenitor cells
- Human iPS cells (454E2 strain, RIKEN Cell Bank) were described in Non-Patent Document 2 (Si-Tayeb et al., Hepatology 2010, 51 (1), 297-305). Differentiation was induced into human hepatic progenitor cells by the following procedure. Human iPS cells were cultured in an RPMI medium containing B27 and 100 ng / ml of activin A for 5 days under an environment of 5% CO 2 and ambient oxygen.
- the cells were cultured for 5 days in an RPMI / B27 medium supplemented with 20 ng / ml BMP4 and 10 ng / ml FGF2 in a 4% O 2 /5% CO 2 environment.
- a cell group containing human hepatic progenitor cells was cultured in an RPMI / B27 medium supplemented with 20 ng / ml hepatocyte growth factor (HGF) for 10 days or more in a 4% O 2 /5% CO 2 environment. Obtained.
- human hepatic progenitor cells which are CPM positive cells, and CPM negative cells were separated using a MoFlo XDP cell sorter (Beckman Coulter). The separation could be performed in the same manner using an autoMACS Pro separator (Miltenyi Biotech).
- FIG. 1 shows the results of flow cytometry analysis showing the transition of the CPM positive cell fraction over time when differentiation was induced from human iPS cells to hepatocytes.
- FIG. 2 shows the results of flow cytometry analysis when cells were separated from a cell group (IH) containing hepatic progenitor cells using CPM positive as an index.
- IH cell group
- a cell fraction having a maximum CPM positive cell content of about 40% was obtained.
- a concentrated cell fraction having a CPM positive cell content of 97.7% was obtained.
- the content of CPM positive cells decreased.
- FIG. 3 shows the results of immunocytochemical staining of isolated CPM positive cells. Expression of AFP and HNF4 ⁇ , known as hepatic progenitor cell markers, was observed.
- FIG. 4 shows a comparison result of expression levels of cell markers in CPM positive cells and CPM negative cells obtained by quantitative RT-PCR.
- CPM positive cells significantly expressed AFP, HNF4 ⁇ , HNF1 ⁇ , PROX1, TBX3, CD13, EpCAM and HHEX, which are known as hepatic progenitor cell markers, as compared with CMP negative cells.
- CD133 known as a bile duct cell marker, was significantly lower in CPM positive cells than in CPM negative cells.
- CPM positive cells formed colonies by culturing on MEF feeder cells.
- CPM negative cells were cultured under the same conditions, no such colonies were formed.
- CPM positive cells became confluent on day 7 after cell seeding (FIG. 5).
- the cells could be subcultured in vitro and proliferated 1000 times or more after 5 passages (FIG. 5).
- the properties of the hepatic progenitor cells before cryopreservation were maintained.
- Example 2 Differentiation induction from human hepatic progenitor cells to human hepatocytes As described in Non-Patent Document 2, CPM-positive cells grown in Example 1 (2) and reaching confluence were treated with HCM Single Quots (EGF And oncostatin M (20 ng / ml) (PeproTech) supplemented with Hepatocyte Basal Medium (Lonza) for 5-10 days to induce differentiation into human hepatocytes Went.
- HCM Single Quots EGF And oncostatin M (20 ng / ml) (PeproTech) supplemented with Hepatocyte Basal Medium (Lonza) for 5-10 days to induce differentiation into human hepatocytes Went.
- the human hepatocytes obtained in (1) showed higher expression of CYP3A4, CYP2C19, CYP2C18, CYP2D6, CYP1A2 and CYP2C8 compared to human hepatocytes prepared by the conventional method. It was also found that the expression of CYP3A4 was further increased by adding rifampicin.
- the human hepatocytes prepared by the method of the present invention are distinguished from human iPS cell-derived hepatocytes obtained by inducing differentiation of hepatic progenitor cells without sorting using CPM positive as an index. .
- Example 3 Differentiation induction from human hepatic progenitor cells to human bile duct epithelial cells Tanimizu et al., Mol Biol Cell, 2007, 18 (4), 1472-1479, and Yanagida et al., PloS ONE 8, e67541
- the three-dimensional gel culture method was slightly modified to induce differentiation into human biliary epithelial cells. After CPM positive cells were grown according to Example 1 (2), the cells were harvested from a 2: 3 mixture of growth factor reduced Matrigel (Corning) and collagen type I (Nitta Gelatin). The resulting gel was resuspended at a density of 1 ⁇ 10 5 cells / 50 ⁇ l.
- the cell suspension was then added to a 24-well plate (Corning) and incubated for 2 hours at 37 ° C. until solidified. Thereafter, the cells were cultured for 7 days in the presence of R-spondin-1 (40 ng / ml) and WNT-3a (40 ng / ml) (PeproTech) to induce differentiation into human biliary epithelial cells.
- R-spondin-1 40 ng / ml
- WNT-3a 40 ng / ml
- Human biliary epithelial cells formed cysts.
- the human bile duct epithelial cells obtained in the above (1) are compared with human bile duct epithelial cells obtained by inducing differentiation of hepatic progenitor cells without sorting using CPM positive as an index.
- CK7, CFTR, AQP1, TGR5, SOX9 and HNF6 were highly expressed.
- Example 4 Preparation of human hepatic sinusoidal endothelial progenitor cells (1) Differentiation induction from human iPS cells to human mesoderm cells Mouse fetal fibroblasts (MEF: Mouse) treated with mitomycin C of human iPS cells (454E2 strain, RIKEN Cell Bank) Embryo Fibroblast,) was cultured as a feeder. MEF was prepared from a fetal mouse (ICR mouse, Nippon SLC Co., Ltd.). Prior to the start of differentiation induction, human iPS cells were replated on a gelatin-coated dish and incubated for 30 minutes to remove MEF.
- embryoid body formation culture was performed on an Ultra-Low Attachment plate (Corning). Embryon body formation culture was performed using Stempro-34 SFM (Life Technologies) as a basic medium with 10 ⁇ M Y27632 (Wako Pure Chemical Industries, Ltd.), 2 ng / ml BMP4 (Life Technologies) from day 0 to day 1 of induction.
- hepatic sinusoidal endothelial progenitor cell marker molecules such as STAB2 and LYVE1 were also highly expressed at the stage of hepatic sinusoidal endothelial progenitor cells (FIG. 11). From the above results, it was suggested that the induced cells include cells having the characteristics of hepatic sinusoidal endothelial progenitor cells.
- the cells maintained the expression of hepatic sinusoidal endothelial progenitor cell marker molecule after 5 passages (FIG. 16). Furthermore, long-term cryopreservation was possible, and CD31 was uniformly expressed even after 30 days of cryopreservation (FIG. 17).
- Example 5 (1) by preparative human liver sinusoidal endothelial progenitor cells preparation of human liver sinusoidal endothelial cells above Example 4 (2), and re-seeded in plates fibronectin coated at a density of 20,000 cells / cm 2 Cultured in human hepatic sinusoidal endothelial cell induction medium supplemented with 50 ⁇ g / ml VEGF and 1.5 ⁇ M A83-01 (Tocris), a TGF- ⁇ inhibitor, in EGM-2 (Lonza, Basel, Switzerland) Thus, a cell fraction containing human hepatic sinusoidal endothelial cells was obtained. All differentiation induction was performed in a 5% CO 2 , 4% O 2 environment. In order to induce mature hepatic sinusoidal endothelial cells with high efficiency, CD31-positive and FcR ⁇ II-positive cells were then separated from the cell fraction using a MoFlo XDP cell sorter (Beckman Coulter, Inc).
- Example 4 Human hepatic sinusoidal progenitor cells sorted in Example 4 (2) and human hepatic sinusoidal endothelial cells prepared in Example 5 (1) (before sorting)
- the expression level of hepatic sinusoidal endothelial cell marker was analyzed by quantitative RT-PCR. The results are shown in FIG. Addition of a TGF- ⁇ inhibitor promoted the expression of hepatic sinusoidal endothelial cell markers STAB2, FcR ⁇ IIB and F8 in the cells.
- CD31-positive FcR ⁇ II-positive hepatic sinusoidal endothelial cells sorted in Example 5 (1) exhibited an endothelial cell-like morphology (FIG. 20).
- CD31-positive FcR ⁇ II-positive cells expressed STAB2, FcR ⁇ IIB, and blood coagulation factor VIII (factor VIII) higher than CD31-positive FcR ⁇ II-negative cells and HUVEC (FIG. 21).
- Example 6 Preparation of human hepatic stellate progenitor cells
- Human mesoderm cells were differentiated from human iPS cells in the same manner as described in Example 4 (1).
- Human hepatic stellate progenitor cells contained in the mesoderm cell population were separated into ALCAM positive cells (human hepatic stellate progenitor cells) and ALCAM negative cells with a MoFlo XDP cell sorter (Beckman Coulter, Inc) using ALCAM positive as an index.
- ALCAM positive cells expressed hepatic stellate progenitor cell-specific marker molecules such as HGF, CYGB, NGFR, and desmin (DES) at a higher level than ALCAM negative cells.
- Example 7 (1) Preparation of human hepatic stellate cells Cell matrix Type I-C (Nitta Gelatin, Osaka, Japan) coated on a plate coated with human hepatic stellate progenitor cells prepared in Example 6 at a density of 15,000 cells / cm 2 . Then, differentiation was induced by culturing in MSCGM (Lonza) for 5 days in a hepatic stellate cell induction medium supplemented with 10 ⁇ M Y27632, a Rock inhibitor, to obtain human hepatic stellate cells. Differentiation induction was performed in an environment of 5% CO 2 and 20% O 2 .
- Vitamin A uptake ability It is known that hepatic stellate cells take up and store pitamine A in the cell. Therefore, vitamin A was added to the medium and the uptake ability was analyzed. Vitamin A (Retinoid (Sigma-Aldrich Corporation, St. Louis, US)) was added at 10 ⁇ M to the culture system of human iPS cell-derived hepatic stellate cells. Cells were dissociated using 0.05% trypsin / 0.5 mM EDTA, and autofluorescence of vitamin A in the cells was detected by flow cytometry using CantoII (BD Biosciences). hMSC was used as a control. Autofluorescence was detected only in human iPS cell-derived hepatic stellate cells, and it was confirmed that pitamine A was incorporated into the cells (FIG. 29).
- lipid droplets were confirmed in the human iPS cell-derived hepatic stellate cells prepared in Example 7 (1) as compared with hMSC (FIG. 30).
- Example 8 Preparation of human hepatocyte tissue model (co-culture of human liver non-parenchymal cells and human liver progenitor cells) Human hepatic sinusoidal endothelial cells prepared in Example 5 and human hepatic stellate cells prepared in Example 7 were seeded so as to be confluent on a plate coated with cell matrix Type I-C (Nitta Ge1atin, Osaka, Japan). Then, a feeder was produced. The next day, feeder cells were treated with mitomycin C. As feeder cell controls, human umbilical vein endothelial cells (HUVEC, obtained from Lonza) and human bone marrow-derived mesenchymal stem cells (hMSC, obtained from Lonza) were used. These feeder cells were seeded with the hepatic progenitor cells prepared in Example 1 and co-cultured for 10 days.
- HUVEC human umbilical vein endothelial cells
- hMSC human bone marrow-derived mesenchymal stem cells
- Example 9 Preparation of human hepatocyte tissue model (co-culture of mouse liver non-parenchymal cells and human hepatic progenitor cells)
- Digestion and hemolysis treatment of mouse fetal liver A liver was surgically extracted from a fetal mouse mouse (C57BL / 6 mouse, Nippon SLC Co., Ltd.) at 14.5 days of age using small scissors and tweezers. The excised liver tissue was minced with a small scissors. The liver tissue was transferred to 10-20 ml of LPM (Thermo Fisher Scientific) and allowed to stand in a 37 ° C. bath for 5 minutes. Thereafter, the solution was replaced with Liver Digest Medium (Thermo Fisher Scientific), and the mixture was stirred in a 37 ° C.
- LPM Thermo Fisher Scientific
- the liver tissue was mechanically digested by pipetting using a disposable pipette. After centrifuging at 1800 rpm for 3 minutes and removing the supernatant, the suspension was suspended in DMEM supplemented with 10% FBS. Pass the cell suspension through a 70 ⁇ m cell strainer, centrifuge at 1800 rpm for 3 minutes, remove the supernatant, suspend the resulting cells in 5 ml ammonium chloride solution, react at 4 ° C for 6 minutes, Hemolysis treatment was performed. DMEM supplemented with 10% FBS was added, passed through a 70 ⁇ m cell strainer, centrifuged at 1800 rpm for 3 minutes, and the supernatant was removed to recover the cells.
- the gel-derived non-parenchymal cells of Example 9 (3) are seeded on the gel, and after culture, this can be used as feeder cells together with the hepatic progenitor cells prepared in Example 1 to maintain and proliferate hepatic progenitor cells.
- Maintenance medium and hepatic progenitor cells were co-cultured in differentiation medium that induced mature hepatocytes.
- the DMEM-F12 medium used for culturing hepatic progenitor cells in Example 1 (2) was used.
- HCM SingleQuots Kit without LONZA and rhEGF only
- 20 ng / ml rhOSM Proprotech
- 1% ITS premix Life Technology
- 0.5 ⁇ M dexamethasone 0.5 ⁇ M A83-01 (Tocris) It was.
- hepatic progenitor cells were cultured alone in the maintenance medium and differentiation medium.
- the expression of genes involved in hepatocyte differentiation was analyzed by quantitative RT-PCR (FIG. 32). Specifically, the expression levels of ALB, a secreted protein of hepatocytes, CYP3A4, an enzyme involved in drug metabolism, and ABCB11 (BileBSalt Export Pump), an ABC transporter involved in bile acid excretion, were compared. The expression level of various hepatocyte markers did not change when cultured in a maintenance medium, but when cultured in a differentiation medium, increased expression was confirmed and the highest expression was observed in co-culture with non-parenchymal cells. Amount indicated.
- HNF4A and CEBPA are transcription factors that control the differentiation from hepatic progenitor cells to hepatocytes, increased in coculture with hepatic stellate cells and hepatic mesothelial cells, compared to iPS hepatocyte single culture. Therefore, it was suggested that hepatic stellate cells and hepatic mesothelial cells have a role in promoting hepatocyte differentiation.
- Albumin (ALB) which is one of hepatocyte differentiation markers, showed 1.8 times the expression level in co-culture with hepatic stellate cells compared to single culture.
- the expression of the enzyme CPS1, which is involved in ammonia metabolism, which is one of the liver functions, is increased under co-culture conditions with hepatic stellate cells and hepatic mesothelial cells, and is cultured alone under co-culture conditions with hepatic stellate cells. 3.8 times the expression level.
- Co-culture three-dimensional culture (spheroid culture)
- the cells were cultured in 96-well-EZsphere (IWAKI) at a density of 5 ⁇ 10 4 cells / well.
- Liver sinusoidal endothelial cells (3 ⁇ 10 4 cells / well), hepatic stellate cells (3 ⁇ 10 4 cells / well) or hepatic mesothelial cells (3 ⁇ 10 3 cells) of Example 9 (4) to (6) above / well
- half the medium was changed on the second day of culture.
- FIG. 37 shows the results of expression analysis of genes involved in hepatocyte differentiation when the mouse non-parenchymal cells of Example 9 (3) and the human iPS cell-derived hepatic progenitor cells of Example 1 were subjected to planar culture and three-dimensional culture. Show. Liver function markers were highly expressed in the three-dimensional culture system.
- Example 10 Preparation of human hepatitis B disease model hepatocytes Human hepatic progenitor cells prepared in Example 1 (1) and human hepatocytes prepared in Example 2 (2) were infected with HBV. On the 16th day after HBV infection, the culture supernatant and cells were collected.
- HBs antigen was observed in the culture supernatants of hepatic progenitor cells and hepatocytes infected with HBV. In particular, HBs antigen was high in hepatocytes.
- HBV DNA was observed in hepatic progenitor cells and hepatocytes. The value was particularly high in hepatocytes.
- Example 11 Analysis of production of substances necessary for pathogen infection Human hepatocytes prepared in Example 2 (1) (hereinafter also referred to as “CPM + -Hep”), and hepatic progenitors from iPS cells in Example 1 (1)
- CPM + -Hep Human hepatocytes prepared in Example 2 (1)
- CPM + -Hep Human hepatocytes prepared in Example 2 (1)
- hepatic progenitors from iPS cells in Example 1
- iPSCs-Hep CPM positive as an index
- any of the hepatocytes tested was tested for molecules required for HBV infection (CD81), molecules required for HCV infection (CD81, CLDN1, OCLN), molecules required for HBV infection (CD81, SCARB). It was expressed. The expression level of CPM + -Hep was higher than that of iPSCs-Hep.
- This result shows that human iPS cell-derived hepatocytes obtained by inducing differentiation of hepatic progenitor cells sorted using CPM positivity as an indicator are sorted using CPM positivity as an indicator.
- human iPS cell-derived hepatocytes obtained by inducing differentiation of hepatic progenitor cells without performing the above it can be suitably used for preparing an infectious disease model.
- Example 12 Analysis of mouse fetal liver cells (mouse liver progenitor cells)
- the livers of 12.5 day-old mouse embryos (C57BL / 6 mice) were collected. The liver was chopped and digested with Liver Digestion Medium (Life Technologies, California, US) for 15 minutes. Digested fetal mouse liver cells were passed through a 40 ⁇ m cell strainer (BD bioscience, New Jersey, US) to obtain a single cell suspension. The cells were blocked with an Fc blocking reagent and incubated with PE-labeled anti-CPM antibody and FITC-labeled anti-DLK1 antibody. PE and FITC labeled anti-isotype controls were used as negative controls. CPM positive (CPM +) and CPM negative (CPM-) cells were isolated with a MoFlo XDP cell sorter (Beckman Coulter, Inc, California, US).
- CPM CPM positive cells
- mouse mature hepatocytes mouse bile duct epithelial cells
- mouse bile duct epithelial cells The expression analysis of CPM in the collected CPM positive cells (liver progenitor cells), mouse mature hepatocytes, and mouse bile duct epithelial cells was performed by quantitative RT-PCR. The results are shown in FIG. In hepatic progenitor cells, CPM was specific for hepatic progenitor cells and was hardly expressed in hepatocytes and bile duct epithelial cells.
- Example 13 Analysis of mouse fetal liver cells (mouse liver sinusoidal endothelial progenitor cells) The livers of 12.5 day-old mouse embryos (C57BL / 6 mice) were collected. The liver was chopped and digested with Liver Digestion Medium (Life Technologies, California, US) for 10 minutes. The digested fetal mouse liver cells were hemolyzed and passed through a 70 ⁇ m cell strainer (BD bioscience, New Jersey, US).
- Liver Digestion Medium Life Technologies, California, US
- FITC-labeled anti-CD31 antibody (BD Biosciences), PE-labeled anti-F1k1 antibody (eBioscience, San Diego, USA), biotin-labeled anti-CD34 antibody (eBioscience), BV395-labeled anti-CD45 antibody Labeled with (BD bioscience) for 30 minutes.
- the cells were washed and labeled with streptavidin APC (BD Biosciences) and anti-FITC microbeads (Miltenyi Biotech) for 20 minutes.
- CD45 ⁇ CD31 + F1k1 + CD34 +/ ⁇ cells were isolated using a MoFlo XDP cell sorter (Beckman Coulter, Inc, California, US).
- hepatic sinusoidal endothelial progenitor cells in mouse fetal liver express vascular endothelial cell marker molecules such as Flk1, CD31, and CD34.
- vascular endothelial cell marker molecules such as Flk1, CD31, and CD34.
- CD45 ⁇ Flk1 + CD31 + vascular endothelial cells are present in the mouse liver at 12.5 days of gestation, and subpopulation of CD45 ⁇ F1k1 + CD31 + CD34 +/ ⁇ is present in the fraction. was found to exist (FIG. 42).
- CD45 ⁇ FLK1 + CD31 + CD34 + cells were compared with CD45 ⁇ FLK1 + CD31 + CD34 ⁇ cells, and hepatic sinusoidal endothelium such as Stab2, Flt4 and Lyve1
- the progenitor cell specific marker gene was highly expressed (FIG. 43).
- the FLK1 + CD31 + CD34 + cells sorted above were seeded at 15,000 cells / cm 2 on a fibronectin-coated plate and cultured in an endothelial cell medium. On the 7th day of culture, the cells grew about 3 times (FIG. 44). The number of cells was calculated using a hemocytometer after dissociating the cells with 0.05% trypsin / 0.5 mM EDTA.
- the method of the present invention it is possible to efficiently prepare uniform and highly functional hepatocytes, hepatic non-parenchymal cells and their progenitor cells.
- the obtained hepatocytes can be used, for example, for drug discovery screening.
- the obtained hepatocytes, hepatic non-parenchymal cells and their progenitor cells can be used for cell therapy, for example.
- the obtained hepatocytes, hepatic non-parenchymal cells and their progenitor cells can be used for the preparation of a disease model.
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Cell Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Toxicology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Developmental Biology & Embryology (AREA)
- Rheumatology (AREA)
- Animal Behavior & Ethology (AREA)
- Physiology (AREA)
- Virology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Nutrition Science (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
[1]
肝前駆細胞を含む細胞画分から、CPM陽性の表現型を指標として肝前駆細胞を分取する工程を含む、肝前駆細胞の調製方法。
[2]
胚体内胚葉細胞又は肝内胚葉細胞を分化誘導して肝前駆細胞を含む前記細胞画分を調製する工程をさらに含む、[1]に記載の方法。
[3]
多能性幹細胞を分化誘導して前記胚体内胚葉細胞又は前記肝内胚葉細胞を調製する工程をさらに含む、[1]に記載の方法。
[4]
前記多能性幹細胞がヒトiPS細胞である、[2]又は[3]に記載の方法。
[5]
[1]~[4]のいずれか1つに記載の方法で調製され得、CPM陽性の表現型を有し、増殖能を有し、肝細胞又は胆管上皮細胞への分化能を有する、肝前駆細胞。
[6]
凍結保存し、解凍した後における前記肝前駆細胞の増殖能が、凍結保存前における肝前駆細胞の増殖能と比較して低下しない、[5]に記載の肝前駆細胞。
[7]
全細胞に対して90%以上の肝前駆細胞を含む細胞画分であって、前記肝前駆細胞はCPM陽性の表現型を有し、増殖能を有し、肝細胞又は胆管上皮細胞への分化能を有する、細胞画分。
[8]
[5]又は[6]に記載の肝前駆細胞を肝細胞へと分化誘導する工程を含む、肝細胞の調製方法。
[9]
[8]に記載の方法で調製され得る、増殖能を有する肝細胞。
[10]
[5]又は[6]に記載の肝前駆細胞を胆管上皮細胞へと分化誘導する工程を含む、胆管上皮細胞の調製方法。
[11]
[10]に記載の方法で調製され得る、増殖能を有する胆管上皮細胞。
[12]
肝類洞内皮前駆細胞を含む細胞画分から、FLK1陽性、CD34陽性及びCD31陽性の表現型を指標として肝類洞内皮前駆細胞を分取する工程を含む、肝類洞内皮前駆細胞の調製方法。
[13]
中胚葉細胞を分化誘導して肝類洞内皮前駆細胞を含む前記細胞画分を調製する工程をさらに含む、[12]に記載の方法。
[14]
多能性幹細胞を分化誘導して前記中胚葉細胞を調製する工程をさらに含む、[13]に記載の方法。
[15]
前記多能性幹細胞がヒトiPS細胞である、[14]に記載の方法。
[16]
[12]~[15]のいずれか1つに記載の方法で調製され得、FLK1陽性、CD34陽性及びCD31陽性の表現型を有し、増殖能を有し、肝類洞内皮細胞への分化能を有する、肝類洞内皮前駆細胞。
[17]
全細胞に対して90%以上の肝類洞内皮前駆細胞を含む細胞画分であって、前記肝類洞内皮前駆細胞はFLK1陽性、CD34陽性及びCD31陽性の表現型を有し、増殖能を有し、肝類洞内皮細胞への分化能を有する、細胞画分。
[18]
(1)TGF-β阻害剤を用いて[16]に記載の肝類洞内皮前駆細胞を分化誘導して、肝類洞内皮細胞を含む細胞画分を調製する工程、及び
(2)肝類洞内皮細胞を含む前記細胞画分からCD31陽性およびFcγR II陽性の表現型を指標として肝類洞内皮細胞を分取する工程
を含む、肝類洞内皮細胞の調製方法。
[19]
[18]に記載の方法で調製され得、CD31陽性およびFcγR II陽性の表現型を有し、増殖能を有する、肝類洞内皮細胞。
[20]
肝星前駆細胞を含む細胞画分からALCAM陽性の表現型を指標として肝星前駆細胞を分取する工程を含む、肝星前駆細胞の調製方法。
[21]
多能性幹細胞を分化誘導して肝星前駆細胞を含む前記細胞画分を調製する工程をさらに含む、肝星前駆細胞の調製方法。
[22]
前記多能性幹細胞がヒトiPS細胞である、[21]に記載の方法。
[23]
[20]~[22]のいずれか1つに記載の方法で調製され得、ALCAM陽性の表現型を有し、増殖能を有し、肝星細胞への分化能を有する、肝星前駆細胞。
[24]
全細胞に対して90%以上の肝星前駆細胞を含む細胞画分であって、前記肝星前駆細胞はALCAM陽性の表現型を有し、増殖能を有し、肝星細胞への分化能を有する、細胞画分。
[25]
Rock阻害剤を用いて、肝星前駆細胞を肝星細胞へと分化誘導する工程を含む、肝星細胞の調製方法。
[26]
前記肝星前駆細胞が、[23]に記載の肝星前駆細胞である、[25]に記載の方法。
[27]
[25]又は[26]に記載の方法で調製され得、増殖能を有する、肝星細胞。
[28]
[5]又は[6]に記載の肝前駆細胞を、肝類洞内皮細胞、肝星細胞及び肝中皮細胞から成る群から選択される少なくとも1つの肝非実質細胞と共培養する工程を含む、肝細胞組織モデルの調製方法。
[29]
前記肝類洞内皮細胞が、[19]に記載の肝類洞内皮細胞である、[28]に記載の方法。
[30]
前記肝星細胞が、[27]に記載の肝星細胞である、[28]又は[29]に記載の方法。
[31]
肝細胞と、肝類洞内皮細胞、肝星細胞及び肝中皮細胞から成る群から選択される少なくとも1つの肝非実質細胞とを含み、[28]~[30]のいずれか1つに記載の方法で調製され得る、肝細胞組織モデル。
[32]
[5]又は[6]に記載の肝前駆細胞、[7]に記載の細胞画分、[9]に記載の肝細胞、[11]に記載の胆管上皮細胞、[16]に記載の肝類洞内皮前駆細胞、[17]に記載の細胞画分、[19]に記載の肝類洞内皮細胞、[23]に記載の肝星前駆細胞、[24]に記載の細胞画分、[27]に記載の肝星細胞、或いは[31]に記載の肝細胞組織モデルを含む、医薬組成物。
[33]
[9]に記載の肝細胞又は[31]に記載の肝細胞組織モデルに肝疾患治療剤の候補品を投与することを含む、肝疾患治療剤のスクリーニング方法。
[34]
[9]に記載の肝細胞又は[31]に記載の肝細胞組織モデルに薬剤を投与することを含む、薬剤の肝毒性の評価方法。
[35]
(1)[5]若しくは[6]に記載の肝前駆細胞、又は[7]に記載の細胞画分中の肝前駆細胞に病原体を感染させる工程、
(2)病原体を感染した前記肝前駆細胞を分化誘導して病原体感染疾患モデルの肝細胞を調製すること
を含む、感染性疾患モデルの肝細胞の調製方法。
[36]
[9]に記載の肝細胞に病原体を感染させる工程を含む、感染性疾患モデルの肝細胞の調製方法。
[37]
前記病原体が、肝炎ウィルス又はマラリア原虫である、[35]又は[36]に記載の調製方法。
[38]
[35]~[37]に記載の方法によって調製され得る、感染性疾患モデルの肝細胞。
[39]
(1)[5]又は[6]に記載の肝前駆細胞、又は[7]に記載の細胞画分中の肝前駆細胞に病原体を感染させる工程、
(2)病原体を感染した前記肝前駆細胞を、肝類洞内皮細胞、肝星細胞及び肝中皮細胞から成る群から選択される少なくとも1つの肝非実質細胞と共培養する工程
を含む、感染性疾患モデル組織の調製方法。
[40]
前記病原体が、肝炎ウィルス又はマラリア原虫である、[39]に記載の調製方法。
[41]
[39]又は[40]に記載の方法によって調製され得る、感染性疾患モデル組織。
[42]
[38]に記載の感染性疾患モデルの肝細胞又は[41]に記載の感染性疾患モデル組織に感染性肝疾患治療剤の候補品を投与することを含む、感染性肝疾患治療剤のスクリーニング方法。
[43]
[5]又は[6]に記載の肝前駆細胞、[7]に記載の細胞画分、[9]に記載の肝細胞、[11]に記載の胆管上皮細胞、[16]に記載の肝類洞内皮前駆細胞、[17]に記載の細胞画分、[19]に記載の肝類洞内皮細胞、[23]に記載の肝星前駆細胞、[24]に記載の細胞画分、[27]に記載の肝星細胞、或いは[31]に記載の肝細胞組織モデルを含む、キット。
Cytofix/Cytoperm Fixtation/permeabi1ization kit(BD Biosciences)を用いて細胞を固定および透過処理し、細胞質タンパク質を抗原抗体法により標識した。その後、MoF1o XDP セルソーター ((Beckman Coulter, Inc) を用いてフローサイトメトリー解析を行った。
TRIzol reagent(Life Technologies)もしくはNucleoSpin RNA XS (MACHEREY-NAGAL, Duren, Germany)を用いてRNAを抽出した。残存しているゲノムDNAを、DNaseI (Life Techno1ogies)を用いて消化後、PrimeScript II 1st strand cDNA Synthesis Kit (Takara bio, Shiga, Japan)を用いて一本鎖cDNAを合成した。定量RT-PCRは、SYBR Premix EX TaqII(Takara bio, Shiga, Japan)を用いて行い、データはβ-アクチンを標準化コントロールとしてddCt法に従って算出した。
培養細胞を10%ホルマリン溶液(Wako Pure Chemical Industries, Ltd.)で10分間固定後、0.2% Triton-X100(Wako Pure Chemical Industries, Ltd.)で15分間浸透化処理した。5%スキムミルク溶液(BD Biosciences)でブロッキング後、一次抗体を4℃で一晩反応させた。PBSで洗浄後、蛍光標識された二次抗体を室温で2時間反応させ、Hoechst33342(Sigma-Aldrich Corporation, St. Louis, US)で対比染色した。
実施例4(2)の肝類洞内皮前駆細胞の分離、実施例5(1)の肝類洞内皮細胞の分離、及び実施例6の肝星前駆細胞の分離を以下の通り行った。
0.05%のトリプシン/0.5mMのEDTA、若しくはAccumax(Innovative Cell Technologies, Inc.)を用いて解離し、0.03%BSA-PBSで懸濁した。調製した細胞を、FcR block reagent(Miltenyi Biotech, Bergisch-Gladbach, Germany)で20分間ブロッキングした後、各細胞に特異的な抗原を認識する一次抗体で、30分間標識した。細胞を洗浄後、必要に応じて抗FITCマイクロビーズ(Miltenyi Biotech)、抗PEマイクロビーズ(Miltenyi Biotech)で20分間標識し、autoMACS Pro セパレーター(Mi1tenyi Biotech)で目的の細胞を濃縮した。その後、MoF1o XDP セルソーター(Beckman Coulter, Inc)を用いて分離した。
(1)ヒト肝前駆細胞の調製
ヒトiPS細胞(454E2株、RIKEN Cell Bank)を、非特許文献2(Si-Tayeb et al., Hepatology 2010, 51(1), 297-305)に記載された以下の手順により、ヒト肝前駆細胞へと分化誘導した。B27及び100ng/mlのアクチビンAを含むRPMI培地中で、ヒトiPS細胞を5%CO2、周囲酸素環境下で5日間培養した。その後、20ng/mlのBMP4及び10ng/mlのFGF2を添加したRPMI/B27培地中で4%O2/5%CO2環境下で5日間培養した。その後、20ng/mlの肝細胞増殖因子(HGF)を添加したRPMI/B27培地中で10日間以上4%O2/5%CO2環境下で培養することでヒト肝前駆細胞を含む細胞群を得た。当該細胞群から、MoFlo XDP セルソーター (Beckman Coulter)を用いて、CPM陽性細胞であるヒト肝前駆細胞と、CPM陰性の細胞とを分離した。当該分離は、autoMACS Pro セパレーター (Miltenyi Biotech)を用いても同様に行うことができた。
10% FBS(JRH Biosciences)、ペニシリン-ストレプトマイシン-グルタミン、ITS、N-2サプリメント、MEM非必須アミノ酸溶液、L-グルタミン(Life Technologies)、アスコルビン酸 (1 mM)、ニコチンアミド (10 mM)、N-アセチル-システイン (0.2 mM) (Sigma-Aldrich)、デキサメタゾン(1x10-7M)、HGF (20 ng/ml)、EGF (10 ng/ml) (PeproTech)、 Y-27632(5 μM) (Wako) 及びA83-01(2.5 μM) (Tocris) を添加したDMEM-F12(Sigma-Aldrich)中で、マイトマイシンCで処理したMEFフィーダー細胞(2.0x104細胞/cm2)上にて、上記で分取されたCPM陽性細胞を培養した。
(1)ヒト肝前駆細胞からヒト肝細胞への分化誘導
非特許文献2に記載された通りに、実施例1(2)で増殖させてコンフルエントに達したCPM陽性細胞を、HCM Single Quots (EGFを除く)及びオンコスタチンM (20 ng/ml) (PeproTech)を添加した肝細胞基本培地(Hepatocyte Basal Medium) (Lonza) 中で5~10日間、インキュベートすることで、ヒト肝細胞への分化誘導を行った。
分化誘導前の肝前駆細胞と分化誘導して得られた肝細胞について、明視野顕微鏡による観察、免疫細胞化学染色によるアルブミンの染色、及びPAS染色によるグリコーゲンの蓄積を観察した結果を示す(図6)。PAS染色は、コールドシッフ試薬(Cold Schiff's Reagent)(Wako Pure Chemical Industires, Ltd.)を用いて、標準的なプロトコルにしたがって行われた。分化誘導前の肝前駆細胞と比較して分化誘導して得られた肝細胞において、アルブミンの高い産生及びグリコーゲンの高い蓄積が確認された。
上記(1)で得られたヒト肝細胞と、従来法である非特許文献2(Si-Tayeb et al., Hepatology 2010, 51 (1), 297-305)に記載された手順でヒトiPS細胞から誘導されたヒト肝細胞(すなわち、CPM陽性を指標とした分取を行わずに肝前駆細胞を分化誘導して得られたヒトiPS細胞由来肝細胞)とにおけるCYP450のmRNA発現を定量RT-PCRを用いて分析した。結果を図7及び8に示す。上記(1)で得られたヒト肝細胞は、従来法で調製されたヒト肝細胞と比較して、CYP3A4、CYP2C19、CYP2C18、CYP2D6、CYP1A2及びCYP2C8の高い発現を示した。また、リファンピシンを添加することで、CYP3A4の発現がさらに増大することが分かった。本発明の方法で調製されたヒト肝細胞は、その特性において、CPM陽性を指標とした分取を行わずに肝前駆細胞を分化誘導して得られたヒトiPS細胞由来肝細胞と区別される。
(1)ヒト肝前駆細胞からヒト胆管上皮細胞への分化誘導
Tanimizu et al., Mol Biol Cell, 2007, 18(4), 1472-1479、及びYanagida et al., PloS ONE 8, e67541に記載された三次元ゲル培養法を少し修正して、ヒト胆管上皮細胞への分化誘導を行った。実施例1(2)に従って、CPM陽性細胞を増殖させた後、細胞を回収し、増殖因子削減マトリゲル(growth factor reduced Matrigel)(Corning)とコラーゲンタイプI(Nitta Gelatin)との2:3混合物からなるゲル中で1x105細胞/50μlの密度で再懸濁した。細胞懸濁液をその後24ウェルプレート(Corning)へ添加し、固体化するまで37℃で2時間インキュベートした。その後、細胞をR-スポンジン-1(40 ng/ml)及びWNT-3a(40 ng/ml) (PeproTech)の存在下、7日間培養することで、ヒト胆管上皮細胞へと分化誘導した。ヒト胆管上皮細胞は、シストを形成した。
得られたヒト胆管上皮細胞において、肝前駆細胞マーカーであるAFPの発現量は低下した(図9)。一方、胆管上皮細胞特異的マーカーの発現が確認された(図10)。CPM陽性を指標とした分取を行わずに肝前駆細胞を分化誘導して得られたヒト胆管上皮細胞と、上記(1)で調製されたヒト胆管上皮細胞とにおける胆管上皮細胞マーカーのmRNA発現を定量RT-PCRを用いて分析した(図10)。上記(1)で得られたヒト胆管上皮細胞は、CPM陽性を指標とした分取を行わずに肝前駆細胞を分化誘導して得られたヒト胆管上皮細胞と比較して、胆管上皮細胞マーカーであるCK7、CFTR、AQP1、TGR5、SOX9及びHNF6の高い発現を示した。
ヒト肝類洞内皮前駆細胞の調製
(1)ヒトiPS細胞からヒト中胚葉細胞への分化誘導
ヒトiPS細胞(454E2株、RIKEN Cell Bank)を、マイトマイシンC処理したマウス胎仔線維芽細胞 (MEF:Mouse Embryo Fibroblast、)をフィーダーとして培養した。なお、MEFは、胎仔マウス(ICRマウス、日本エスエルシー株式会社)から調製した。分化誘導開始前に、ヒトiPS細胞をゼラチンコートディッシュに再播種し、30分間インキュベートすることでMEFを取り除いた。ヒトiPS細胞から中胚葉へ分化誘導するため、Ultra-Low Attachment plate (Corning) 上で胚葉体形成培養を行った。胚葉体形成培養を、Stempro-34 SFM (Life Technologies) を基本培地として、誘導開始0日から1日に10μMのY27632(Wako Pure Chemical Industries, Ltd.)、2ng/mlのBMP4(Life Technologies)を加え、誘導開始1日~4日に5ng/mlのアクチビンA(Pepro Tech, New Jersey, US)、5ng/mlのbFGF(Life Technologies社)、30ng/mlのBMP4(Life Technologies社)を加え、誘導開始4日から6日に10ng/mlのVEGF(Pepro Tech)、5.4μMのSB431542(Tocris, Bristol, UK)、0.5μMのドルソモルフィン(Tocris)を加えることで行った。6日間培養して中胚葉細胞へと分化誘導した。
(1)で得られた胚葉体をあらかじめゼラチンコートしたプレートに移し、EGM-2(Lonza, Basel, Switzerland)に50ng/m1のVEGFを添加した内皮細胞培地で7日間培養した。CD31陽性、FLK1陽性及びCD34陽性のヒト肝類洞内皮前駆細胞を、MoFlo XDP cell sorter (Beckman Coulter, Inc)で分離した。すべての分化誘導を5%CO2、4%O2環境下で行った。
ヒトiPS細胞、上記(1)で誘導されたヒト中胚葉細胞、上記(2)で誘導されたヒト肝類洞内皮前駆細胞(細胞分離前)における、マーカー分子の遺伝子発現量を定量RT-PCRにより分析した。結果を図11に示す。未分化マーカーであるOCT4は、iPS細胞において高く発現し、分化の進行とともに減少した。中胚葉マーカーであるMESP1は、中胚葉細胞の分化ステージで最も高い発現を示した。CD31やVE-カドヘリン(VE-Cad)といった血管内皮細胞マーカー分子は、肝類洞内皮前駆細胞のステージで高発現した。さらに、STAB2やLYVE1といった肝類洞内皮前駆細胞マーカー分子も肝類洞内皮前駆細胞のステージで高発現した(図11)。以上の結果から、誘導された細胞中には肝類洞内皮前駆細胞の特徴を有する細胞が含まれることが示唆された。
ヒト中胚葉から分化誘導されたヒト肝類洞内皮前駆細胞を含む細胞群(pre-sorted)、pre-sorted群から分離されたFLK1+CD31+CD34+細胞(CD34+)、FLK1+CD31+CD34-細胞(CD34-)における、ヒト肝類洞内皮前駆細胞のマーカー分子(STAB2及びLYVE1)の発現量を定量RT-PCRにより分析した。結果を図13に示す。CD34+細胞群は、CD34-細胞群と比較して、STAB2やLYVE1といった肝類洞内皮前駆細胞マーカー分子を高発現していた。当該結果から、FLK1+CD31+CD34+の表現型の組み合わせが、ヒト肝類洞内皮前駆細胞を分取するためのマーカーとして利用できることが明らかとなった。
上記実施例4(2)で分取したFLK1+CD31+CD34+細胞を、フィブロネクチンコートしたプレートに15,000 細胞/cm2で播種し、内皮細胞培地で培養した。FLK1+CD31+CD34+細胞は、内皮細胞様の形態を維持し(図14)、高い増殖能を有しており(図15上)、数回の継代培養も可能であった(図15下)。なお細胞数は、0.05%トリプシン/0.5mM EDTAを用いて細胞を解離し、血球計算盤を用いて算出した。また、定量RT-PCR解析により、この細胞は5継代後も肝類洞内皮前駆細胞マーカー分子の発現を維持した(図16)。さらに、長期凍結保存が可能であり、30日間の凍結保存後もCD31を均一に発現した(図17)。
(1)ヒト肝類洞内皮細胞の調製
上記実施例4(2)で分取されたヒト肝類洞内皮前駆細胞を、20,000細胞/cm2の密度でフィブロネクチンコートしたプレートに再播種し、EGM-2(Lonza, Basel, Switzerland)に50ng/m1のVEGF、TGF-β阻害剤である1.5μMのA83-01(Tocris)を添加したヒト肝類洞内皮細胞誘導培地で14日間培養することで、ヒト肝類洞内皮細胞を含む細胞画分を得た。すべての分化誘導を5%CO2、4%O2環境下で行った。高効率に成熟した肝類洞内皮細胞を誘導するため、当該細胞画分からその後、MoFlo XDP セルソーター(Beckman Coulter, Inc)を用いて、CD31陽性、FcRγII陽性細胞を分離した。
上記実施例4(2)で分取されたヒト肝類洞内皮前駆細胞と上記実施例5(1)で調製されたヒト肝類洞内皮細胞(分取前)における、肝類洞内皮細胞マーカーの発現量を定量RT-PCRにより分析した。結果を図18に示す。TGF-β阻害剤の添加により、肝類洞内皮細胞マーカーであるSTAB2、FcRγIIB及びF8の発現が細胞中で促進された。
実施例5(1)で分取されたCD31陽性FcRγII陽性の肝類洞内皮細胞は、内皮細胞様形態を呈した(図20)。また、CD31陽性FcRγII陽性細胞は、CD31陽性FcRγII陰性細胞やHUVECと比較して、STAB2、FcRγIIB、血液凝固第VIII因子(ファクターVIII)を高発現していた(図21)。
CD31陽性FcRγII陽性の肝類洞内皮細胞を含む培養培地に 5μg/mlのDiI-Ac-LDL(AlfaAesar, Massachusetts, US)もしくは 25μg/mlのフルオレセインアミン標識されたヒアルロン酸ナトリウム(FAHA-L1)(PG Research, Tokyo, Japan)を添加し、30℃で4時間インキュベートした。4時間後、PBSで洗浄し、Hoechst33342で対比染色した。結果を図22及び23に示す。肝類洞内皮細胞におけるアセチル化LDLの取り込み能はHUVECと比べて同等程度であったのに対し、STAB2を介して行われるヒアルロン酸の取り込みは、肝類洞内皮細胞のみで確認された。
免疫細胞化学染色およびフローサイトメトリー解析(図24及び25)により、ファクターVIIIのタンパク質レベルでの発現が認められた。
ヒト肝星前駆細胞の調製
実施例4(1)に記載された手順と同様にして、ヒトiPS細胞からヒト中胚葉細胞を分化誘導した。当該中胚葉細胞集団に含まれるヒト肝星前駆細胞を、ALCAM陽性を指標として、MoFlo XDP セルソーター(Beckman Coulter, Inc)でALCAM陽性細胞(ヒト肝星前駆細胞)とALCAM陰性細胞に分離した。ヒトiPS細胞由来中胚葉細胞の分離前、ALCAM陽性細胞及びALCAM陰性細胞における、肝星前駆細胞マーカー分子の発現量を定量RT-PCRにより分析した。結果を図27に示す。ALCAM陽性細胞はALCAM陰性細胞と比較して、HGF、CYGB、NGFR、デスミン(DES)といった肝星前駆細胞特異的マーカー分子を高発現していた。
(1)ヒト肝星細胞の調製
セルマトリックス TypeI-C(Nitta Gelatin, Osaka, Japan)をコートしたプレートに、実施例6で調製されたヒト肝星前駆細胞を15,000細胞/cm2の密度で播種し、MSCGM(Lonza)に、Rock阻害剤である10μMのY27632を添加した肝星細胞誘導培地で5日間培養することで分化誘導し、ヒト肝星細胞を得た。分化誘導は5%CO2、20%O2の環境下で行った。
上記実施例7(1)で調製されたヒトiPS細胞由来肝星細胞における肝星細胞マーカー分子の発現量を定量RT-PCRにより分析した。結果を図28に示す。ヒトiPS細胞由来肝星細胞は、ヒト骨髄由来間葉系幹細胞(hMSC)と比較して肝星細胞に特異的に発現するHGF、NGFR、CYGB、LRATを高発現した。
肝星細胞は、細胞内にピタミンAを取り込み、貯蔵することが知られている。そこで、ビタミンAを培地中に添加し、取り込み能を解析した。ヒトiPS細胞由来肝星細胞の培養系にビタミン A (レチノイド(Sigma-Aldrich Corporation,St. Louis,US))を10μMで添加した。0.05%トリプシン/0.5mM EDTAを用いて細胞を解離し、CantoII (BD Biosciences)用い、フローサイトメトリーによって細胞内のビタミンAの自家蛍光を検出した。hMSCをコントロールとして用いた。ヒトiPS細胞由来肝星細胞のみにおいて自家蛍光が検出され、細胞内にピタミンAを取り込んでいることが確認された(図29)。
ヒト肝細胞組織モデルの調製(ヒト肝非実質細胞とヒト肝前駆細胞との共培養)
実施例5で調製したヒト肝類洞内皮細胞、及び実施例7で調製したヒト肝星細胞を、セルマトリックスTypeI-C(Nitta Ge1atin, Osaka, Japan)でコートしたプレートにコンフルエントになるように播種し、フィーダーを作製した。翌日フィーダー細胞をマイトマイシンC処理した。フィーダー細胞のコントロールとして、ヒト臍帯静脈内皮細胞(HUVEC,Lonza社から入手)とヒト骨髄由来間葉系幹細胞(hMSC,Lonza社から入手)を用いた。これらのフィーダー細胞に実施例1で調製した肝前駆細胞を播種し、10日間共培養した。
ヒト肝細胞組織モデルの調製(マウス肝非実質細胞とヒト肝前駆細胞との共培養)
(1)マウス胎仔肝の消化・溶血処理
胎齢14.5日のマウス胎仔(C57BL/6マウス、日本エスエルシー株式会社)から、小型バサミとピンセットを用いて外科的に肝臓を摘出した。摘出した肝組織を小型バサミにより細切した。肝組織を10~20 mlのLPM(Thermo Fisher Scientific) に移し、37℃の温浴にて5分間静置した。その後、Liver Digest Medium(Thermo Fisher Scientific)に液交換し、37℃の温浴にて5分間攪拌した。その後、ディスポーザブルピペットを用いてピペッティングにより機械的に肝組織を消化した。1800 rpm、3分間の遠心分離を行い上清除去後、10% FBSを添加したDMEMで懸濁した。70 μmセルストレイナーに細胞懸濁液を通し、1800 rpm、3分間の遠心分離を行い上清除去後、得られた細胞を5 mlの塩化アンモニウム溶液に懸濁し、4℃、6分間反応させ、溶血処理を行った。10% FBSを添加したDMEMを添加し、70 μmセルストレイナーに通し、1800 rpm、3分間遠心分離を行い上清除去後、細胞を回収した。
溶血処理を行った細胞を1% BSA/PBSで懸濁し、マウスFcR(抗体(1:100希釈)を添加し4℃、15分間ブロッキングを行った。次に一次抗体(1:100希釈)を 30分間反応させた。一次抗体はビオチン標識あるいはFITC標識されたStab2抗体、ビオチン標識されたMsln抗体、APC標識されたp75NTR抗体を用いた。一次抗体を1% BSA/PBSで除去後、同様に二次抗体(1:100希釈)を20分間反応させた。二次抗体はSA-APC抗体あるいはSA-PE抗体を用いた。二次抗体を1% BSA/PBS で除去後、MACS Running Buffer(Miltenyi Biotec K.K.)で20倍に希釈された磁気ビーズ(Miltenyi Biotec K.K.)を10分間反応させた。その後auto MACS (Miltenyi Biotec K.K.)を用いて磁気細胞分離を行った。単細胞分離を行う場合はさらにMoflo(Beckman Coulter)を用いて分離を行った。
分離したマウス胎仔肝非実質細胞は、コラーゲンTypeIC(Nitta Gelatin)及びコラーゲンTypeIA(Nitta Gelatin)上のプレートに2% B27(Life Technology)、50 ng/ml rhVEGF(Peprotech)、10 μg/ml Y27632(Wako)、 0.5 μM A83-01(Tocris) を添加したEGM+MSC(1:1、LONZA)にて 37℃、5% CO2 、湿度 95% 条件下にて培養を行った。
コラーゲンTypeIC(Nitta Gelatin)及びコラーゲンTypeIA(Nitta Gelatin)上のプレートに播種した。培地は50 ng/ml rhVEGF(Peprotech)、10 μg/ml Y27632(Wako)、0.5μM A83-01(Tocris)が添加されたEGM(LONZA)を用いた。37℃、5% CO2 、湿度95% 条件下にて培養を行った。
コラーゲンTypeIC(Nitta Gelatin)及びコラーゲンTypeIA(Nitta Gelatin)上のプレートに播種した。培地は2% B27(Life Technology)、50 ng/ml rhVEGF(Peprotech)、10 μg/ml Y27632(Wako)、 0.5 μM A83-01(Tocris) を添加したEGM+MSC(1:1、LONZA)を用いた。37℃、5% CO2 、湿度95% 条件下にて培養を行った。
コラーゲンTypeIV(Nitta Gelatin)及びコラーゲンTypeIA(Nitta Gelatin)上のプレートに播種した。培地は10% FBS、50 nmol/L mercaptoethanol、10 ng/ml oncostatin M (0SM)(Peprotech)、10 ng/ml bFGF(Peprotech)が添加されたαMEMを用いた。37℃、5% CO2、湿度95% 条件下にて培養を行った。培地交換は1日おきに行った。
コラーゲン1ゲル(Nitta Gelatin)を48-wellプレート1 wellあたり200 μl添加し30分間、インキュベーター内でゲル化させた。当該ゲルに上記実施例9(3)のマウス由来非実質細胞を播種し、培養後、これをフィーダー細胞として、実施例1で調製された肝前駆細胞と共に、肝前駆細胞が維持・増殖可能な維持培地及び肝前駆細胞を成熟肝細胞へ誘導する分化培地中で共培養した。なお、維持培地として、実施例1(2)において肝前駆細胞の培養に用いたDMEM-F12培地を使用した。分化培地として、20 ng/ml rhOSM(Peprotech)、1% ITS premix(Life Technology)、0.5 μM デキサメタゾン、0.5 μM A83-01(Tocris)を添加した HCM SingleQuots Kit(LONZA、rhEGFのみ非添加)を用いた。また、肝前駆細胞を上記維持培地及び分化培地中で単独培養した。
96-well-EZsphere(IWAKI)に5×104 cells/wellの密度で培養した。上記実施例9(4)~(6)の肝類洞内皮細胞(3×104 cells/well)、肝星細胞(3×104 cells/well)又は肝中皮細胞(3×103 cells/well)を、上記実施例1のヒトiPS細胞由来肝前駆細胞と同時に播種した。上記の分化培地を使用し、培養二日目に半量培地交換した。37℃、5% CO2、湿度95%条件下にて培養を行った。三次元培養した場合において、スフェロイド形成が認められた。マーカーの発現解析結果を図35に示す。HNF4AとCEBPAの発現量は、肝星細胞と共培養を行った際に特に上昇することが示された。また、アルブミン発現量における平面培養と三次元培養との違いを示す結果を図36に示す。平面培養4日目、三次元培養2日目、三次元培養4日目において比較すると、三次元培養の条件下において、効率的に成熟化が促進されたことが示された。
ヒトB型肝炎疾患モデルの肝細胞の調製
実施例1(1)で調製されたヒト肝前駆細胞及び実施例2(2)で調製されたヒト肝細胞にHBVを感染させた。HBV感染後16日目で培養上清および細胞を回収した。
病原体の感染に必要な物質の産生の分析
実施例2(1)で調製されたヒト肝細胞(以下で「CPM+-Hep」とも呼ぶ)と、実施例1(1)でiPS細胞から肝前駆細胞へと分化誘導後、CPM陽性を指標として肝前駆細胞を分取する工程を経ずに、実施例2(1)と同様の手順で分化誘導をして調製された肝細胞(以下で「iPSCs-Hep」とも呼ぶ)とにおいて、B型肝炎ウィルス、C型肝炎ウィルス及びマラリア原虫が細胞に感染するために必要とされる物質が産生されているか否かを定量RT-PCRを用いて分析した。結果を図40に示す。
マウス胎仔肝細胞の分析(マウス肝前駆細胞)
胎齢 12.5 日マウス胎仔(C57BL/6マウス)の肝臓を回収した。当該肝臓を細断し、 Liver Digestion Medium (Life Technologies, California, US)で 15分間消化した。消化した胎仔マウス肝臓細胞を、 40μmセルストレイナー (BD bioscience, New Jersey, US)に通し、単一細胞懸濁液を得た。この細胞をFcブロック試薬でブロッキングし、PE標識抗CPM抗体及びFITC標識抗DLK1抗体とインキュベートした。PE及びFITC標識抗アイソタイプコントロールをネガティブコントロールとして用いた。CPM陽性(CPM+)及びCPM陰性(CPM-)細胞をMoFlo XDP セルソーター(Beckman Coulter, Inc, California, US)で単離した。
マウス胎仔肝細胞の分析(マウス肝類洞内皮前駆細胞)
胎齢12.5日マウス胎仔(C57BL/6マウス)の肝臓を回収した。当該肝臓を細断し、Liver Digestion Medium (Life Technologies, California, US)で 10分間消化した。消化した胎仔マウス肝臓細胞を溶血後、 70μmセルストレイナー (BD bioscience, New Jersey, US)に通した。この細胞をFcRブロック試薬で20分間ブロッキングした後、FITC標識抗CD31抗体 (BD Biosciences)、PE標識抗F1k1抗体 (eBioscience, SanDiego, USA)、ビオチン標識抗CD34抗体(eBioscience)、BV395標識抗CD45抗体(BD bioscience)で30分間標識した。細胞を洗浄後、ストレプトアビジンAPC (BD Biosciences) と抗FITCマイクロビーズ仰(Miltenyi Biotech)で 20分間標識した。CD31+細胞をautoMACS Pro セパレーター(Miltenyi Biotech)で濃縮後、MoFlo XDP セルソーター(Beckman Coulter, Inc, California, US)を用いてCD45-CD31+F1k1+CD34+/-細胞を単離した。
Claims (43)
- 肝前駆細胞を含む細胞画分から、CPM陽性の表現型を指標として肝前駆細胞を分取する工程を含む、肝前駆細胞の調製方法。
- 胚体内胚葉細胞又は肝内胚葉細胞を分化誘導して肝前駆細胞を含む前記細胞画分を調製する工程をさらに含む、請求項1に記載の方法。
- 多能性幹細胞を分化誘導して前記胚体内胚葉細胞又は前記肝内胚葉細胞を調製する工程をさらに含む、請求項1に記載の方法。
- 前記多能性幹細胞がヒトiPS細胞である、請求項2又は3に記載の方法。
- 請求項1~4のいずれか1項に記載の方法で調製され得、CPM陽性の表現型を有し、増殖能を有し、肝細胞又は胆管上皮細胞への分化能を有する、肝前駆細胞。
- 凍結保存し、解凍した後における前記肝前駆細胞の増殖能が、凍結保存前における肝前駆細胞の増殖能と比較して低下しない、請求項5に記載の肝前駆細胞。
- 全細胞に対して90%以上の肝前駆細胞を含む細胞画分であって、前記肝前駆細胞はCPM陽性の表現型を有し、増殖能を有し、肝細胞又は胆管上皮細胞への分化能を有する、細胞画分。
- 請求項5又は6に記載の肝前駆細胞を肝細胞へと分化誘導する工程を含む、肝細胞の調製方法。
- 請求項8に記載の方法で調製され得る、増殖能を有する肝細胞。
- 請求項5又は6に記載の肝前駆細胞を胆管上皮細胞へと分化誘導する工程を含む、胆管上皮細胞の調製方法。
- 請求項10に記載の方法で調製され得る、増殖能を有する胆管上皮細胞。
- 肝類洞内皮前駆細胞を含む細胞画分から、FLK1陽性、CD34陽性及びCD31陽性の表現型を指標として肝類洞内皮前駆細胞を分取する工程を含む、肝類洞内皮前駆細胞の調製方法。
- 中胚葉細胞を分化誘導して肝類洞内皮前駆細胞を含む前記細胞画分を調製する工程をさらに含む、請求項12に記載の方法。
- 多能性幹細胞を分化誘導して前記中胚葉細胞を調製する工程をさらに含む、請求項13に記載の方法。
- 前記多能性幹細胞がヒトiPS細胞である、請求項14に記載の方法。
- 請求項12~15のいずれか1項に記載の方法で調製され得、FLK1陽性、CD34陽性及びCD31陽性の表現型を有し、増殖能を有し、肝類洞内皮細胞への分化能を有する、肝類洞内皮前駆細胞。
- 全細胞に対して90%以上の肝類洞内皮前駆細胞を含む細胞画分であって、前記肝類洞内皮前駆細胞はFLK1陽性、CD34陽性及びCD31陽性の表現型を有し、増殖能を有し、肝類洞内皮細胞への分化能を有する、細胞画分。
- (1)TGF-β阻害剤を用いて請求項16に記載の肝類洞内皮前駆細胞を分化誘導して、肝類洞内皮細胞を含む細胞画分を調製する工程、及び
(2)肝類洞内皮細胞を含む前記細胞画分からCD31陽性およびFcγR II陽性の表現型を指標として肝類洞内皮細胞を分取する工程
を含む、肝類洞内皮細胞の調製方法。 - 請求項18に記載の方法で調製され得、CD31陽性およびFcγR II陽性の表現型を有し、増殖能を有する、肝類洞内皮細胞。
- 肝星前駆細胞を含む細胞画分からALCAM陽性の表現型を指標として肝星前駆細胞を分取する工程を含む、肝星前駆細胞の調製方法。
- 多能性幹細胞を分化誘導して肝星前駆細胞を含む前記細胞画分を調製する工程をさらに含む、肝星前駆細胞の調製方法。
- 前記多能性幹細胞がヒトiPS細胞である、請求項21に記載の方法。
- 請求項20~22のいずれか1項に記載の方法で調製され得、ALCAM陽性の表現型を有し、増殖能を有し、肝星細胞への分化能を有する、肝星前駆細胞。
- 全細胞に対して90%以上の肝星前駆細胞を含む細胞画分であって、前記肝星前駆細胞はALCAM陽性の表現型を有し、増殖能を有し、肝星細胞への分化能を有する、細胞画分。
- Rock阻害剤を用いて、肝星前駆細胞を肝星細胞へと分化誘導する工程を含む、肝星細胞の調製方法。
- 前記肝星前駆細胞が、請求項23に記載の肝星前駆細胞である、請求項25に記載の方法。
- 請求項25又は26に記載の方法で調製され得、増殖能を有する、肝星細胞。
- 請求項5若しくは6に記載の肝前駆細胞、又は請求項7に記載の細胞画分を、肝類洞内皮細胞、肝星細胞及び肝中皮細胞から成る群から選択される少なくとも1つの肝非実質細胞と共培養する工程を含む、肝細胞組織モデルの調製方法。
- 前記肝類洞内皮細胞が、請求項19に記載の肝類洞内皮細胞である、請求項28に記載の方法。
- 前記肝星細胞が、請求項27に記載の肝星細胞である、請求項28又は29に記載の方法。
- 肝細胞と、肝類洞内皮細胞、肝星細胞及び肝中皮細胞から成る群から選択される少なくとも1つの肝非実質細胞とを含み、請求項28~30のいずれか1項に記載の方法で調製され得る、肝細胞組織モデル。
- 請求項5又は6に記載の肝前駆細胞、請求項7に記載の細胞画分、請求項9に記載の肝細胞、請求項11に記載の胆管上皮細胞、請求項16に記載の肝類洞内皮前駆細胞、請求項17に記載の細胞画分、請求項19に記載の肝類洞内皮細胞、請求項23に記載の肝星前駆細胞、請求項24に記載の細胞画分、請求項27に記載の肝星細胞、或いは請求項31に記載の肝細胞組織モデルを含む、医薬組成物。
- 請求項9に記載の肝細胞又は請求項31に記載の肝細胞組織モデルに肝疾患治療剤の候補品を投与することを含む、肝疾患治療剤のスクリーニング方法。
- 請求項9に記載の肝細胞又は請求項31に記載の肝細胞組織モデルに薬剤を投与することを含む、薬剤の肝毒性の評価方法。
- (1)請求項5若しくは6に記載の肝前駆細胞、又は請求項7に記載の細胞画分中の肝前駆細胞に病原体を感染させる工程、
(2)病原体を感染した前記肝前駆細胞を分化誘導して病原体感染疾患モデルの肝細胞を調製すること
を含む、感染性疾患モデルの肝細胞の調製方法。 - 請求項9に記載の肝細胞に病原体を感染させる工程を含む、感染性疾患モデルの肝細胞の調製方法。
- 前記病原体が、肝炎ウィルス又はマラリア原虫である、請求項35又は36に記載の調製方法。
- 請求項35~37に記載の方法によって調製され得る、感染性疾患モデルの肝細胞。
- (1)請求項5又は6に記載の肝前駆細胞、又は請求項7に記載の細胞画分中の肝前駆細胞に病原体を感染させる工程、
(2)病原体を感染した前記肝前駆細胞を、肝類洞内皮細胞、肝星細胞及び肝中皮細胞から成る群から選択される少なくとも1つの肝非実質細胞と共培養する工程
を含む、感染性疾患モデル組織の調製方法。 - 前記病原体が、肝炎ウィルス又はマラリア原虫である、請求項39に記載の調製方法。
- 請求項39又は40に記載の方法によって調製され得る、感染性疾患モデル組織。
- 請求項38に記載の感染性疾患モデルの肝細胞又は請求項41に記載の感染性疾患モデル組織に感染性肝疾患治療剤の候補品を投与することを含む、感染性肝疾患治療剤のスクリーニング方法。
- 請求項5又は6に記載の肝前駆細胞、請求項7に記載の細胞画分、請求項9に記載の肝細胞、請求項11に記載の胆管上皮細胞、請求項16に記載の肝類洞内皮前駆細胞、請求項17に記載の細胞画分、請求項19に記載の肝類洞内皮細胞、請求項23に記載の肝星前駆細胞、請求項24に記載の細胞画分、請求項27に記載の肝星細胞、或いは請求項31に記載の肝細胞組織モデルを含む、キット。
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15/558,879 US20180147242A1 (en) | 2015-03-18 | 2016-03-16 | Hepatocytes and hepatic non-parenchymal cells, and methods for preparation thereof |
EP16765045.6A EP3272855B1 (en) | 2015-03-18 | 2016-03-16 | Liver cells and liver non-parenchymal cells, and methods for preparation thereof |
EP20160937.7A EP3708652A3 (en) | 2015-03-18 | 2016-03-16 | Liver sinusoidal endothelial progenitor cells, and methods for preparation thereof |
JP2017506598A JP6812003B2 (ja) | 2015-03-18 | 2016-03-16 | 肝細胞及び肝非実質細胞、並びにそれらの調製方法 |
US16/685,761 US20200188444A1 (en) | 2015-03-18 | 2019-11-15 | Hepatocytes and hepatic non-parenchymal cells, and methods for preparation thereof |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201562134899P | 2015-03-18 | 2015-03-18 | |
US62/134,899 | 2015-03-18 | ||
US201662287933P | 2016-01-28 | 2016-01-28 | |
US62/287,933 | 2016-01-28 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/558,879 A-371-Of-International US20180147242A1 (en) | 2015-03-18 | 2016-03-16 | Hepatocytes and hepatic non-parenchymal cells, and methods for preparation thereof |
US16/685,761 Continuation US20200188444A1 (en) | 2015-03-18 | 2019-11-15 | Hepatocytes and hepatic non-parenchymal cells, and methods for preparation thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2016148216A1 true WO2016148216A1 (ja) | 2016-09-22 |
Family
ID=56919165
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2016/058411 WO2016148216A1 (ja) | 2015-03-18 | 2016-03-16 | 肝細胞及び肝非実質細胞、並びにそれらの調製方法 |
Country Status (4)
Country | Link |
---|---|
US (2) | US20180147242A1 (ja) |
EP (2) | EP3708652A3 (ja) |
JP (1) | JP6812003B2 (ja) |
WO (1) | WO2016148216A1 (ja) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018097127A1 (ja) * | 2016-11-22 | 2018-05-31 | 国立大学法人京都大学 | 肝芽細胞から胆管上皮前駆細胞への段階的誘導方法 |
WO2019078357A1 (ja) * | 2017-10-20 | 2019-04-25 | 中外製薬株式会社 | 細胞への分子の取り込みを測定する方法 |
WO2019131938A1 (ja) * | 2017-12-27 | 2019-07-04 | 国立研究開発法人国立成育医療研究センター | 機能的肝前駆細胞もしくは肝細胞または機能的小腸上皮前駆細胞もしくは小腸上皮細胞を調製する方法 |
WO2020166726A1 (ja) * | 2019-02-15 | 2020-08-20 | 国立大学法人 東京大学 | 静止期肝星細胞の調製方法と活性化評価モデル |
CN112342182A (zh) * | 2020-10-29 | 2021-02-09 | 浙江大学 | 一种高效分离小鼠肝脏胆管细胞的方法 |
WO2021085649A1 (ja) * | 2019-10-31 | 2021-05-06 | 公立大学法人横浜市立大学 | 細胞評価方法、細胞評価システム、及びプログラム |
JPWO2021100709A1 (ja) * | 2019-11-19 | 2021-05-27 | ||
WO2022230919A1 (ja) * | 2021-04-28 | 2022-11-03 | 国立大学法人 東京医科歯科大学 | 細胞の製造方法 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1424391A1 (en) * | 2001-08-08 | 2004-06-02 | Ajinomoto Co., Inc. | Gene panel participating in liver astrocyte activation |
JP2006254896A (ja) * | 2005-01-07 | 2006-09-28 | Effector Cell Institute Inc | ヒト肝細胞様細胞およびその利用 |
WO2007140243A2 (en) * | 2006-05-26 | 2007-12-06 | University Of North Carolina At Chapel Hill | Hepatic stellate cell precursors and methods of isolating same |
WO2011154552A1 (en) * | 2010-06-11 | 2011-12-15 | Cellartis Ab | 3-dimensional scaffolds for improved differentiation of pluripotent stem cells to hepatocytes |
WO2011158125A2 (en) * | 2010-06-17 | 2011-12-22 | Katholieke Universiteit Leuven | Methods for differentiating cells into hepatic stellate cells and hepatic sinusoidal endothelial cells, cells produced by the methods, and methods for using the cells |
EP2671944A1 (en) * | 2011-01-31 | 2013-12-11 | National Center for Global Health and Medicine | Highly functional liver cells derived from pluripotent stem cells, method for producing same, and method for testing metabolism/toxicity of drug |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB201111244D0 (en) * | 2011-06-30 | 2011-08-17 | Konink Nl Akademie Van Wetenschappen Knaw | Culture media for stem cells |
-
2016
- 2016-03-16 US US15/558,879 patent/US20180147242A1/en not_active Abandoned
- 2016-03-16 JP JP2017506598A patent/JP6812003B2/ja active Active
- 2016-03-16 EP EP20160937.7A patent/EP3708652A3/en not_active Withdrawn
- 2016-03-16 WO PCT/JP2016/058411 patent/WO2016148216A1/ja active Application Filing
- 2016-03-16 EP EP16765045.6A patent/EP3272855B1/en active Active
-
2019
- 2019-11-15 US US16/685,761 patent/US20200188444A1/en active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1424391A1 (en) * | 2001-08-08 | 2004-06-02 | Ajinomoto Co., Inc. | Gene panel participating in liver astrocyte activation |
JP2006254896A (ja) * | 2005-01-07 | 2006-09-28 | Effector Cell Institute Inc | ヒト肝細胞様細胞およびその利用 |
WO2007140243A2 (en) * | 2006-05-26 | 2007-12-06 | University Of North Carolina At Chapel Hill | Hepatic stellate cell precursors and methods of isolating same |
WO2011154552A1 (en) * | 2010-06-11 | 2011-12-15 | Cellartis Ab | 3-dimensional scaffolds for improved differentiation of pluripotent stem cells to hepatocytes |
WO2011158125A2 (en) * | 2010-06-17 | 2011-12-22 | Katholieke Universiteit Leuven | Methods for differentiating cells into hepatic stellate cells and hepatic sinusoidal endothelial cells, cells produced by the methods, and methods for using the cells |
EP2671944A1 (en) * | 2011-01-31 | 2013-12-11 | National Center for Global Health and Medicine | Highly functional liver cells derived from pluripotent stem cells, method for producing same, and method for testing metabolism/toxicity of drug |
Non-Patent Citations (7)
Title |
---|
ASAHINA, KINJI ET AL.: "Mesenchymal origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liver development", HEPATOLOGY, vol. 49, no. 3, 2009, pages 998 - 1011, XP055238050 * |
HRISTOV, MIHAIL ET AL.: "Endothelial Progenitor CellsMobilization, Differentiation, and Homing", ARTERIOSCLEROSIS, THROMBOSIS, AND VASCULAR BIOLOGY, vol. 23, 2003, pages 1185 - 1189, XP055312016 * |
IKEDA, HITOSHI ET AL.: "Involvement of Rho/Rho kinase pathway in regulation of apoptosis in rat hepatic stellate cells", AM J PHYSIOL GASTROINTEST LIVER PHYSIOL, vol. 285, 2003, pages G880 - 886, XP009167419 * |
MOUSAVI, SEYED ALI ET AL.: "Receptor-Mediated Endocytosis of Immune Complexesin Rat Liver Sinusoidal Endothelial Cells Is Mediatedby FcyRIIb2", HEPATOLOGY, vol. 46, no. 3, 2007, pages 871 - 884, XP055312018 * |
SEIICHI ISHIDA: "Development of Hepatocyte Co- culture System with Liver Nonparenchymal Cells", ADVANCES IN PHARMACEUTICAL SCIENCES KENKYU SEIKA HOKOKUSHU, vol. 29, 1 March 2013 (2013-03-01), pages 51 - 54, XP009505959 * |
WANG, LIN ET AL.: "Liver sinusoidal endothelial cell progenitor cells promote liver regeneration in rats", JOURNAL OF CLINICAL INVESTIGATION, vol. 122, no. 4, 2012, pages 1567 - 1573, XP055312012 * |
YOSHIDA, MASAYUKI ET AL.: "Involvement of signaling of VEGF and TGF-beta in differentiation of sinusoidal endothelial cells during culture of fetal rat liver cells", CELL AND TISSUE RESEARCH, vol. 329, no. 2, 2007, pages 273 - 282, XP019517944 * |
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP7148134B2 (ja) | 2016-11-22 | 2022-10-05 | 国立大学法人京都大学 | 肝芽細胞から胆管上皮前駆細胞への段階的誘導方法 |
JPWO2018097127A1 (ja) * | 2016-11-22 | 2019-10-17 | 国立大学法人京都大学 | 肝芽細胞から胆管上皮前駆細胞への段階的誘導方法 |
WO2018097127A1 (ja) * | 2016-11-22 | 2018-05-31 | 国立大学法人京都大学 | 肝芽細胞から胆管上皮前駆細胞への段階的誘導方法 |
WO2019078357A1 (ja) * | 2017-10-20 | 2019-04-25 | 中外製薬株式会社 | 細胞への分子の取り込みを測定する方法 |
WO2019131938A1 (ja) * | 2017-12-27 | 2019-07-04 | 国立研究開発法人国立成育医療研究センター | 機能的肝前駆細胞もしくは肝細胞または機能的小腸上皮前駆細胞もしくは小腸上皮細胞を調製する方法 |
CN111727239A (zh) * | 2017-12-27 | 2020-09-29 | 梅泽明弘 | 一种制备功能性肝前体细胞或肝细胞或者功能性小肠上皮前体细胞或小肠上皮细胞的方法 |
CN111727239B (zh) * | 2017-12-27 | 2024-04-19 | 梅泽明弘 | 一种制备功能性肝前体细胞或肝细胞或者功能性小肠上皮前体细胞或小肠上皮细胞的方法 |
JP7191041B2 (ja) | 2017-12-27 | 2022-12-16 | 明弘 梅澤 | 機能的肝前駆細胞もしくは肝細胞または機能的小腸上皮前駆細胞もしくは小腸上皮細胞を調製する方法 |
JPWO2019131938A1 (ja) * | 2017-12-27 | 2021-08-26 | 明弘 梅澤 | 機能的肝前駆細胞もしくは肝細胞または機能的小腸上皮前駆細胞もしくは小腸上皮細胞を調製する方法 |
WO2020166726A1 (ja) * | 2019-02-15 | 2020-08-20 | 国立大学法人 東京大学 | 静止期肝星細胞の調製方法と活性化評価モデル |
WO2021085649A1 (ja) * | 2019-10-31 | 2021-05-06 | 公立大学法人横浜市立大学 | 細胞評価方法、細胞評価システム、及びプログラム |
JPWO2021100709A1 (ja) * | 2019-11-19 | 2021-05-27 | ||
WO2021100709A1 (ja) * | 2019-11-19 | 2021-05-27 | 凸版印刷株式会社 | 細胞構造体及びその製造方法並びに被験物質の肝毒性の評価方法 |
CN112342182B (zh) * | 2020-10-29 | 2022-12-20 | 浙江大学 | 一种高效分离小鼠肝脏胆管细胞的方法 |
CN112342182A (zh) * | 2020-10-29 | 2021-02-09 | 浙江大学 | 一种高效分离小鼠肝脏胆管细胞的方法 |
WO2022230919A1 (ja) * | 2021-04-28 | 2022-11-03 | 国立大学法人 東京医科歯科大学 | 細胞の製造方法 |
Also Published As
Publication number | Publication date |
---|---|
EP3708652A2 (en) | 2020-09-16 |
JP6812003B2 (ja) | 2021-01-13 |
EP3272855B1 (en) | 2020-04-22 |
JPWO2016148216A1 (ja) | 2018-02-08 |
US20200188444A1 (en) | 2020-06-18 |
EP3708652A3 (en) | 2020-12-16 |
EP3272855A1 (en) | 2018-01-24 |
EP3272855A4 (en) | 2018-09-12 |
US20180147242A1 (en) | 2018-05-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2016148216A1 (ja) | 肝細胞及び肝非実質細胞、並びにそれらの調製方法 | |
KR102101060B1 (ko) | 성체 간 전구 세포의 제조 방법 | |
KR102453717B1 (ko) | 원시 장 내배엽 세포 및 그 제작 방법 | |
JP6450311B2 (ja) | 腎前駆細胞の製造方法及び腎前駆細胞を含む医薬 | |
KR101987395B1 (ko) | Cd82 양성 심근 전구세포 | |
US9677085B2 (en) | Engineering a heterogeneous tissue from pluripotent stem cells | |
JP5597129B2 (ja) | 胎盤由来の幹細胞および前駆体細胞の単離方法 | |
US20110151554A1 (en) | Method for culturing and subculturing primate embryonic stem cell, as well as method for inducing differentiation thereof | |
JP6691921B2 (ja) | 動脈内皮細胞集団の生成 | |
WO2018079714A1 (ja) | ヒト肝前駆細胞の調製方法 | |
JP6789572B2 (ja) | 多能性幹細胞を減少させる方法、多能性幹細胞を減少させた細胞集団の製造方法 | |
Okada et al. | Prospective isolation and characterization of bipotent progenitor cells in early mouse liver development | |
US20230374462A1 (en) | Method for producing myocardial stem/progenitor cell and method for suppressing myocardial fibrosis | |
US20190062699A1 (en) | Highly functional hepatocyte and use thereof | |
JP2020162551A (ja) | 肝前駆細胞を含む細胞集団を製造する方法 | |
JPWO2019189324A1 (ja) | 細胞塊融合法 | |
US20230139291A1 (en) | Expansion and maintenance of adult primary human hepatocytes in culture | |
WO2023022111A1 (ja) | 嚢胞様構造を有する三次元肝細胞培養物およびその製造方法 | |
Jahan | Multi-stage endothelial differentiation and expansion of human pluripotent stem cells | |
TW202216989A (zh) | 周邊血衍生小多能細胞 | |
JP2013201943A (ja) | 成熟肝細胞の製造方法 |
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: 16765045 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2017506598 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15558879 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2016765045 Country of ref document: EP |