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WO2007116870A1 - Procédé de préparation de cellules analogues aux hépatocytes matures - Google Patents

Procédé de préparation de cellules analogues aux hépatocytes matures Download PDF

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WO2007116870A1
WO2007116870A1 PCT/JP2007/057420 JP2007057420W WO2007116870A1 WO 2007116870 A1 WO2007116870 A1 WO 2007116870A1 JP 2007057420 W JP2007057420 W JP 2007057420W WO 2007116870 A1 WO2007116870 A1 WO 2007116870A1
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cells
mesenchymal stem
mature hepatocyte
hepatocyte
cell
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Japanese (ja)
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Takuya Tamatani
Akira Yamauchi
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Effector Cell Institute Inc
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Effector Cell Institute Inc
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—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
    • C12N2501/998—Proteins 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus

Definitions

  • the present invention relates to mature hepatocyte-like cells.
  • Stem cells are cells that have the ability to sort into various types of cells. For example, cells having the ability to differentiate into liver, muscle, nerve, skin, blood cells, etc. are known. For the application to regenerative medicine, a method for efficiently inducing differentiation of cells having specific organ functions with stem cell power is eagerly desired.
  • hepatocyte-like cells In particular, the induction of differentiation into stem cell power hepatocyte-like cells is expected to be applied to the development of biological artificial livers and cell transplantation therapy.
  • Cell transplantation treatment is useful as a treatment method for diseases such as liver failure and hepatitis.
  • hepatocyte-like cells can also be used for drug substitution and toxicity systems and screening systems.
  • Takashima et al. Disclose a method for inducing differentiation of hepatocyte-like cells by culturing fetal-derived immature hepatocytes using oncostatin M (OSM) and dexamethasone.
  • OSM oncostatin M
  • dexamethasone dexamethasone.
  • differentiation into hepatocyte-like cells was observed by culturing immature hepatocytes derived from fetus by adding OSM and darcocorticoid in the presence of extracellular matrix.
  • Teraoka et al Used human growth genes and humans from nucleated cells prepared and separated from human umbilical cord blood using the following growth factors and differentiation inducing factors. It has been confirmed that cultured cells expressing albumin can be obtained. Permissible literature 7). Albumin expression is one of the characteristics of hepatocytes. According to the report by Teraoka et al., The best results were obtained especially in the combination of FGF, HGF, LIF, and SCF.
  • hLIF Human leukemia inhibitory factor
  • hSCF Human stem cell factor
  • FGF Fibroblast growth factor
  • HSHGF Human hepatocyte growth factor
  • ES cells used embryonic stem cells
  • BM hMSC bone marrow derived mesenchyma stem cells
  • ES embryonic stem cells
  • HGF bone marrow derived mesenchyma stem cells
  • FGF essential growth factors
  • Embryonic stem (ES) cells were derived from mice, rats and monkeys, and bone marrow-derived mesenchymal stem cells were derived from human cells.
  • Non-patent Documents 2 and 6 describe a method for inducing differentiation from bone marrow-derived mesenchymal stem cells (BM MSCs) to hepatocyte-like cells using a two-stage differentiation induction method. Disclosure! / In the first stage, HGF was used, and in the second stage maturation medium, OSM was used as the essential factor.
  • Non-patent Document 4 confirmed differentiation induction of hepatocyte-like cells from cord blood stem cells following the method of Lee et al. (Non-patent Document 6).
  • Patent Document 1 Miyajima, et al: JP2000 / 287680
  • Patent Document 2 Miyajima, et al: WO02 / 074937
  • Patent Document 3 Miki, et al: JP2005- 523328
  • Patent Document 4 Ochiya, et al: JP2006-254896
  • Patent Document 5 Tomisawa ,: JP2005-253374
  • Patent Document 6 Lee, et al: US 2005/0233449 Al
  • Patent Document 7 Teraoka, et al: JP2002-360243
  • Non-patent literature l Miyajima, et al: EMBO J. Vol.18, No.8, pp.2127-2136 (1999)
  • Non-patent literature 2 Lee, et al: HEPATOLOGY vol.40: 1275-1284 (2004)
  • Non-Patent Document 3 SE Yang, et al: Cytotherapy Vol. 6, No. 5, 476-486 / 486 (2004)
  • Non-Patent Document 4 SH Hong, et al: Biochemical and Biophysical Research Communicatio ns 330: 1153-1161 (2005)
  • Non-Patent Document 5 K. Teramoto et al .: J Hepatobiliary Pancreat Surg (2005) 12: 196-202
  • Non-Patent Document 6 Lee, et al: Blood vol.103: 1669-1675 (2004)
  • An object of the present invention is to provide a method for producing hepatocyte-like cells with high maturity.
  • an object of the present invention is to provide hepatocyte-like cells that can be produced according to the present invention and uses thereof.
  • hepatocyte growth factor has been considered indispensable for inducing differentiation from stem cells into hepatocyte-like cells.
  • HGF hepatocyte growth factor
  • the present inventor has found that sufficient differentiation into mature hepatocyte-like cells can be induced by utilizing a specific growth differentiation factor even in the absence of HGF. Based on this finding, a method for producing hepatocyte-like cells with high maturity was established, and the present invention was completed. That is, the present invention relates to the following method for producing hepatocyte-like cells, hepatocyte-like cells obtainable by the production method, and various uses thereof.
  • [0011] A step of culturing mesenchymal stem cells in the presence of oncostatin M, dexamethasone or a derivative or salt thereof, and TGF- ⁇ to differentiate the mesenchymal stem cells into mature hepatocyte-like cells.
  • mesenchymal stem cell is a mesenchymal stem cell derived from any tissue selected from cord blood, bone marrow, adipose tissue, placenta, and peripheral blood force A method for producing cell-like cells.
  • a mature hepatocyte-like cell that can be produced by any of the methods described in [1] to [6].
  • a therapeutic agent for liver disease comprising a mature hepatocyte-like cell that can be produced by any of the methods described in [1] to [6].
  • a method for detecting the metabolism of a test compound in the liver comprising the following steps, wherein mature hepatocyte-like cells are present in the presence of oncostatin M, dexamethasone or a derivative or salt thereof, and TGF-
  • a method for detecting hepatotoxicity of a test compound comprising the following steps, wherein mature hepatocyte-like cells are present in the presence of oncostatin M, dexamethasone or a derivative or salt thereof, and TGF-8 A method of mature hepatocyte-like cells differentiated from mesenchymal stem cells by culturing mesenchymal stem cells under;
  • a screening method for a therapeutic agent for liver disease comprising the following steps, wherein the mature hepatocyte-like cell is present in the presence of oncostatin M, dexamethasone or a derivative or salt thereof, and TGF-8.
  • a screening method for an infection inhibitor of hepatitis virus comprising the following steps, wherein the mature hepatocyte-like cell is present in the presence of oncostatin M, dexamethasone or a derivative or salt thereof, and TGF-
  • a screening method for a therapeutic agent for viral hepatitis comprising the following steps, wherein the mature hepatocyte-like cell is present in the presence of oncostatin M, dexamethasone or a derivative or salt thereof, and TGF-8: A method wherein the cells are mature hepatocyte-like cells differentiated from mesenchymal stem cells by culturing mesenchymal stem cells in
  • a method for culturing hepatitis virus comprising a step of infecting hepatitis virus with mature hepatocyte-like cells, wherein the mature hepatocyte-like cells are Oncostatin M, dexamethasone or a derivative or salt thereof, and TGF-
  • culturing hepatitis virus which is a mature hepatocyte-like cell differentiated from mesenchymal stem cells by culturing mesenchymal stem cells in the presence of [15]
  • the present invention provides a method for producing mature hepatocyte-like cells.
  • the method of the present invention can induce the differentiation of mesenchymal stem cell force into mature hepatocyte-like cells in a short time.
  • liver-specific gene expression is used as an indicator of the maturity of hepatocyte-like cells, conventionally, culture of at least several weeks has been required to differentiate stem cells into mature hepatocyte-like cells.
  • gene expression specific to mature hepatocytes can be confirmed within a very short period of 6 days after the start of culture.
  • the hepatocyte-like cells that can be obtained by the present invention have high homology with hepatocytes even though the cells have morphological characteristics. Furthermore, the hepatocyte-like cells obtainable by the present invention also have the characteristics of hepatocytes in terms of cell functions. Specifically, it has been confirmed that the hepatocyte-like cells of the present invention have the following functions characteristic of hepatocytes in a preferred embodiment.
  • the hepatocyte-like cells of the present invention can be infected with hepatitis C virus. This fact confirms that the hepatocyte-like cells that can be obtained by the present invention are highly similar to mature human hepatocytes.
  • FIG. 1 is a photograph showing a morphological change from human umbilical cord blood-derived mesenchymal stem cells to hepatocyte-like cells.
  • A is an undifferentiated human umbilical cord blood-derived mesenchymal stem cell.
  • B is a human umbilical cord blood-derived mesenchymal stem cell on the 5th day after differentiation initiation.
  • C is a human bone marrow-derived mesenchymal stem cell on the second day after differentiation induction.
  • D is a human bone marrow-derived mesenchymal stem cell on the 9th day after the start of induction.
  • FIG. 2 is a photograph showing the morphology of day 5 after human bone marrow-derived mesenchymal stem cells were induced to differentiate into hepatocyte-like cells.
  • FIG. 4 is a photograph showing the results of RT-PCR analysis of hepatocyte-like cells 12 days after induction of differentiation.
  • FIG. 5 is a graph showing an increase in CYP3A4 activity with the passage of differentiation culture days.
  • FIG. 6 is a graph showing CYP3A4 activity of primary human cultured hepatocytes and hepatocyte-like cells after induction of differentiation.
  • FIG. 7 is a graph showing that CYP3A4 activity rises depending on rifampicin concentration in hepatocyte-like cells after induction.
  • FIG. 8 is a graph showing that CYP1A2 activity increases depending on the concentration of omebrazole (OPZ) in hepatocyte-like cells after induction of mitochondrion.
  • OPO omebrazole
  • FIG. 9 shows the results of analysis of glucose metabolism function in hepatocyte-like cells.
  • FIG. 10 shows hepatitis C virus infection experiment of hepatocyte-like cells induced to differentiate from human umbilical cord blood mesenchymal stem cells. The relationship between the time course after inoculation of HCV into hepatocyte-like cells and the amount of HCV-mRNA is shown.
  • the present invention includes a step of culturing mesenchymal stem cells in the presence of oncostatin M, dexamethasone, and TGF- ⁇ , and causing the mesenchymal stem cells to differentiate into mature hepatocyte-like cells.
  • the present invention relates to a method for producing like cells.
  • mesenchymal stem cells derived from mammals including humans are used as the mesenchymal stem cells.
  • a preferred mesenchymal stem cell is a human mesenchymal stem cell.
  • Mesenchymal stem cells MSC
  • mesenchymal stem cells have been reported to have the ability to differentiate into a variety of cells, including adipocytes, chondrocytes, bone cells, cardiomyocytes, and neurons.
  • mesenchymal stem cells which are a type of tissue stem cell, can be isolated from the patient's tissue. Therefore, it is attracting attention as a material for regenerative medicine.
  • the tissue from which the mesenchymal stem cells are derived in the present invention is not limited.
  • Mesenchymal stem cells are cells found as bone marrow stem cells that are divided into cells other than blood cells. Later, it was revealed that mesenchymal stem cells are present not only in bone marrow but also in adipose tissue, placenta, umbilical cord blood, peripheral blood, and tooth tissue. These mesenchymal stem cells derived from tissues other than bone marrow can also be used in the present invention. For example, mesenchymal stem cells derived from human umbilical cord blood and human bone marrow are preferred mesenchymal stem cells in the present invention. Bone marrow can also gain individual strength at any stage of growth, from fetus to adult. Therefore, based on the present invention, mature hepatocyte-like cells for autologous transplantation can be obtained.
  • mesenchymal stem cells can be collected using the cell surface marker (CD271) as an indicator.
  • CD271 cell surface marker
  • a kit for isolating human mesenchymal stem cells using an antibody against CD271 “Mesenchymal stem cell isolation culture box-CD27 1 (LNGFR)” (trade name, manufactured by Miltue Biotech) is also available .
  • LNGFR Mesenchymal stem cell isolation culture box-CD27 1
  • pre-isolated mesenchymal stem cells that are commercially available can also be used in the present invention.
  • the mesenchymal stem cells can be grown not only in the fractional but also in the undigested segment. Therefore, the isolated mesenchymal stem cells can be grown and used in the present invention. In the present invention, it is preferable to proliferate mesenchymal stem cells before sorting.
  • Mesenchymal stem cells are commercially available as a mesenchymal stem cell growth medium, and can be grown using such a medium. Specifically, MF medium (TOYOBO, Japan), MSCG medium (Cambrex, USA), etc. can be used for the proliferation of mesenchymal stem cells. Alternatively, EM medium containing sushi serum can be used for the proliferation of mesenchymal stem cells.
  • Mesenchymal stem cells can be proliferated by culturing under general conditions in cell culture. For example, incubating at 37 ° C in a humidified atmosphere containing about 5% CO
  • mesenchymal stem cells are cultured in the presence of oncostatin M, dexamethasone, and TGF-j8.
  • the concentration of oncostatin M in the medium for culturing mesenchymal stem cells is usually 1 ng / ml to 100 ng / ml, preferably 5 ng / ml to 50 ng / ml.
  • the concentration of dexamethasone is usually 0.1 ⁇ 10 ⁇ 1, preferably 0.
  • TGF-J 8 is usually 0. 2ng / ml ⁇ 20ng / ml, preferably from lng / ml to: a LONG / ml.
  • Oncostatin M is a member of the IL-6 family of site strength. Oncostatin M was identified as a factor that inhibits the growth of human melanoma cell lines (Proc. Natl, Acad. Sci. USA Vol. 83, pp. 9739-9743, December 1986). There are many reports on the involvement of blood cells and stem cells in the sorting of cells. Human oncostatin M is a protein with a molecular weight of about 26 kD and a force of 252 amino acids. It contains a secretion signal, and the 25 amino acid residues at the N-terminal are removed by processing, resulting in a mature protein with a strength of 227 amino acid residues.
  • SEQ ID NO: 13 shows the amino acid sequence of human oncostatin M (GenBank Accession No. AAA36388). SEQ ID NO: 13 amino acid sequence 2 of 52 residues, N-terminal ⁇ J force et al. 26— 227 residues at position 252,! ⁇ MA 26— 196 residues at position 221 force Human Oncostatin ⁇ mature protein It corresponds to.
  • oncostatin ⁇ precursor protein and mature protein both have similar effects on cells. Therefore, oncostatin ⁇ ⁇ ⁇ ⁇ in the present invention can utilize either the precursor protein or the mature protein, or both. However, preferably, oncostatin ⁇ ⁇ ⁇ ⁇ in the present invention is a more physiologically active, mature oncostatin ⁇ (196 amino acids at positions 26 to 221 in SEQ ID NO: 13). In addition, the concentration of oncostatin in the medium exemplified above is the amount used as a precursor protein.
  • the concentration of oncostatin ⁇ ⁇ ⁇ ⁇ in the medium is 0.8 ng / mL to 85 ng / mL, preferably 4.3 ng / mL to 43 ng. / mL or so.
  • the origin of oncostatin M in the present invention is not limited as long as it induces differentiation of mesenchymal stem cells cultured as progenitor cells into mature hepatocyte-like cells.
  • the amino acid sequence of Oncostatin M which has been clarified so far, is exemplified below.
  • Dexamethasone (Dexamethasone; 9-Fluoro-11 ⁇ , 17,2 Trihydroxy-16a-methyl-Predana-1, 1,4-Gen-3, 20-dione; CAS Registration No. 50-02-2) is a synthetic steroid with a darcocorticoid-like physiological action. Like steroid hormones, it is thought to translocate into the nucleus with the receptor and participate in the regulation of transcription. It is a compound that is used clinically as an astringent, analgesic, anti-inflammatory, ophthalmic, anti-itch, and digestive.
  • a dexamethasone derivative having the same action can be used instead of dexamethasone.
  • dexamethasone and acid ester have the same physiological activity as dexamethasone.
  • the following derivatives or salts thereof can be used as dexamethasone in the present invention.
  • Transforming growth factor j8 (transforming growth factor-beta; TGF-j8) is a protein having a dimeric structure, and it is known that three types of isoforms with similar structures exist in mammals. These isoforms are called 1, ⁇ 2 and j83, respectively.
  • TGF-j8 is used as a term including all of these isoforms. Any of these isoforms can be used in the present invention.
  • the amino acid sequence of TGF-8 The amino acid sequence of human TGF-j81 (GenBank Accession No. AAQ18641) is shown in SEQ ID NO: 14.
  • SEQ ID NO: 14 1 12 residues (positions 7 to 118) lacking 6 residues on the N-terminal side are the amino acid sequence of the mature protein of human TGF-jS 1.
  • SEQ ID NO: 14 Ami A protein containing an amino acid sequence of 112 residues on the N-terminal side in the no acid sequence is preferred as TGF-j8 in the present invention.
  • Human TGF- ⁇ 1 GenBank Accession No. AAQ18641, NP—000651, AAA51458, AAL2 7646, AAQ 18642, AAV71148, AAN86616, AAT77144, AAT77143, AAX32228, PO 1137, CAA29283, AAH01180, AAH00125, and AAP35909
  • Human TGF— j8 2 GenBank Accession No. Y00083, M19154
  • Human TGF— j8 3 GenBank Accession No. J03241, X144149
  • Mouse TGF-j8 l GenBank Accession No. NP—001013383, AAH99866, and CAI2 5749
  • Mouse TGF— j8 2 GenBank Accession No. X57413
  • Oncostatin M and TGF- ⁇ are limited in their origin as long as they induce differentiation of mesenchymal stem cells cultured as progenitor cells into mature hepatocyte-like cells.
  • the amino acid sequence of TGF-8 is known to be highly conserved among mammals.
  • 81 the identity of human and mouse amino acid sequences is 99%. Therefore, it is generally considered that the species difference of TGF-8 is almost negligible.
  • TGF-18 when the mature hepatocyte-like cells obtainable by the present invention are used for human medical purposes, it is preferable to use human TGF-18.
  • oncostatin M and TGF-j8 in the present invention natural ones can be used, and genetic recombinants having the same activity as natural molecules can be used.
  • a person skilled in the art can produce a recombinant gene of Oncostatin M or TGF-8 based on the amino acid sequence information or the base sequence information encoding it.
  • oncostatin M or TGF- ⁇ gene recombinants are commercially available.
  • the companies listed below supply genetically engineered products of these proteins. Therefore, these commercially available recombinants can also be used in the present invention.
  • Onyastatin M pre-pro protein
  • Onyastatin M mature protein
  • TGF- ⁇ in humans and mice TGF- ⁇ in humans and mice:
  • the basal medium that can be used in the present invention usually contains inorganic salts, sugars, amino acids, vitamins, and the like. A typical composition of the basal medium is shown below.
  • Nicotinic Acid Amide 0.3-5 mg / L
  • Vitamin B-12 0.01-2 mg / L
  • basal medium used in the present invention a medium composition generally marketed as a basal medium can be used.
  • the following commercially available media can be used as the basic media in the present invention.
  • IMDM medium (Iscove's Modified DMEM) (Sigma, USA)
  • F12K medium F-12 Nutrient Mixture (Ham's F12) Kaighn's Modification) (Invitrogen),
  • HCM medium (Cambrex), etc.
  • the basal medium can further contain additional components in addition to the representative composition.
  • additional components for example, adding albumin or animal serum can improve cell growth support.
  • mesenchymal stem cells are differentiated into mature hepatocyte-like cells by culturing in the presence of oncostatin M, dexamethasone, and TGF-j8.
  • Mesenchymal stem cells can be cultured according to a general animal cell culture method. As specific culture conditions, a temperature of around 37 ° C can be exhibited in an atmosphere of about 5% CO. Furthermore,
  • a culture vessel coated with collagen in order to induce differentiation into mature hepatocyte-like cells.
  • a commercially available collagen coating plate made by Asahi Techno Glass) or the like can be used in the method of the present invention.
  • the number of cells at the start of derivation induction can be appropriately adjusted as long as the survival of the cells is supported and the target derivation can be induced.
  • the number of cells at the start of derivation induction can be appropriately adjusted as long as the survival of the cells is supported and the target derivation can be induced.
  • 5.0 ⁇ 10 3 to 5.0 ⁇ 10 6 cells / culture dish cells can be seeded.
  • mature hepatocyte-like cells are, for example, the following special features: A cell with at least one of features (l)-(4).
  • cells having both (2) and (3) among these indicators are preferred as cells for use in compound screening and regenerative medicine.
  • the cell having the feature (4) is useful as a host cell used for hepatitis virus infection experiments.
  • TAT Tyrosine aminotransferase
  • Cytochrome P450 (CYP1A2, CYP3A4, CYP2D6);
  • TRR Transthyretin
  • Multi-metabolic proteins MRP1, MRP2, MRP3
  • Multidrug resistance-related proteins MDR1, MDR3
  • the above genes can be amplified using primers specific to each gene. Furthermore, it is possible to combine a method for specifically detecting an amplification product by combining a probe that hybridizes to a base sequence characteristic of each gene. For example, ATA C-PCR is a typical method for detecting PCR amplification products using probes.
  • the ability to produce glucose can be confirmed by analyzing the glucose level in the culture supernatant by the glucose oxidase method.
  • the ability to metabolize ammonia can be confirmed by analyzing the ammonia level in the culture medium by the modified indophenol method (Horn DB & Squire CR, Chim. Acta. 14: 185-194. 1966).
  • the ability to produce albumin can be confirmed by analyzing the albumin concentration in the culture solution by a method for measuring the serum albumin concentration.
  • the ability to synthesize urea can be confirmed using, for example, Colorimetric assay (Sigma).
  • hepatocyte-like cells are infected with human hepatitis C virus.
  • hepatitis C virus growth can be detected by RT-PCR using the mRNA recovered from the cells as a cage.
  • Primers capable of amplifying human hepatitis C virus RNA are known (T. Takeuch. Et al. Real-Time Detection System for Quantification of Hepatitis and Virus Genome, astroenterology 1999, 116: 636-642) o
  • hepatocyte-like cells cells that are close to primary normal human cultured hepatocytes and have both morphological characteristics and gene expression profiles are called hepatocyte-like cells.
  • cytochrome P450 CYP
  • MRP multidrug resistance-related protein
  • MDR multidrug resistance
  • the cell having the functional characteristics of the liver described as (3) of the characteristics can be said to be a particularly highly mature cell. Therefore, hepatocyte-like cells having the characteristics (2) to (4) of the liver cells are included in the mature hepatocyte-like cells in the present invention.
  • a cell comprising at least one, preferably two or more, more preferably all of the functional characteristics of (3) characterizing mature hepatocyte-like cells is included in the mature hepatocyte-like cell of the present invention. It is.
  • oncostatin M, dexamethasone, and TGF-j8, which are growth differentiation factors for inducing differentiation from mesenchymal stem cells to mature hepatocyte-like cells are derived from mesenchymal stem cells.
  • the present invention relates to a reagent for inducing differentiation from mesenchymal stem cells to mature hepatocyte-like cells, comprising Oncostatin M, dexamethasone, and TGF-8.
  • oncostatin M, dexamethasone, and TGF- ⁇ are added to the basal medium to induce differentiation from mesenchymal stem cells to mature hepatocyte-like cells.
  • a medium can be prepared.
  • the differentiation-inducing reagent for mature hepatocyte-like cells of the present invention contains Oncostatin ⁇ , dexamethasone, and TGF- ⁇ in an amount necessary to give the concentration of each growth differentiation factor in the induction medium described above. be able to.
  • the formulation for inducing differentiation into mature hepatocyte-like cells of the present invention can be obtained by blending the following amount of growth factor as a prescription per liter of medium.
  • Oncostatin ⁇ 1 ⁇ g to 100 ⁇ g, preferably 5 ⁇ g to 50 ⁇ g
  • Dexamethasone 0. ImM ⁇ : LOmM, preferably 0.5mM ⁇ 5mM
  • TGF- j8 0.2 8 to 20 8, preferably 1 to 10 8
  • the differentiation-inducing reagent for mature hepatocyte-like cells of the present invention may further contain additional components.
  • L-glutamine which is often added calorically to the basal medium, can be added to the separation induction reagent of the present invention.
  • various antibiotics and additional nutrients can be added.
  • the present invention also provides mature hepatocyte-like cells produced by the method of the present invention.
  • the function and morphology of mature hepatocyte-like cells produced by the method of the present invention are more similar to human mature hepatocytes than the function and morphology of hepatocyte-like cells produced by conventional methods.
  • the mature hepatocyte-like cell of the present invention is useful, for example, in the medical field. More specifically, the mature hepatocyte-like cell provided by the present invention is useful as a tool for regenerative medicine.
  • Mature hepatocyte-like cells obtainable by the present invention are seeded in a culture dish for various tests in vitro, for example.
  • liver tissue can be reconstructed by injecting the mature hepatocyte-like cells of the present invention into the body. Due to liver tissue reconstruction Thus, hepatic diseases can be treated.
  • the matured hepatocyte-like cells of the present invention are concentrated by treating the differentiated cells with a solution containing an enzyme, packing them in a test tube, and collecting the cells under mild conditions.
  • a solution containing an enzyme for enzyme treatment of cells, collagenase or dispase can be used.
  • Mild conditions for recovering cells can utilize physical or biochemical procedures such as centrifugation at relatively low speeds (40 x g to 100 x g).
  • the recovered mature liver cell-like cells of the present invention can be seeded in a suitable culture container.
  • a 96-well plate or 24-well plate is used as the culture vessel.
  • the mature hepatocyte-like cells of the present invention can be suspended in an appropriate culture solution or buffer and injected into the living body. Cell suspensions can be administered intravenously or portal vein.
  • the mature hepatocyte-like cells of the present invention can be administered to a living body by subcutaneous administration or intraperitoneal administration.
  • liver diseases can be treated by transplanting mature hepatocyte-like cells embedded in a biocompatible material into a patient. Known materials such as collagen and polyurethane can be used as the biocompatible material.
  • the mature hepatocyte-like cells provided by the present invention can be used as an artificial liver.
  • the artificial liver in the present invention includes those that are transplanted in a living body to supplement the liver function and those that supplement the liver function by contacting with a patient's body fluid in vitro.
  • the artificial liver in the present invention has means for holding mature stem cell-like cells and bringing patient blood into contact with mature stem cell-like cells as necessary.
  • an artificial liver for transplantation into a living body retains mature hepatocyte-like cells in, for example, a serum-permeable cell retention material. Artificial liver transplanted to adults contacts blood serum and metabolizes components in the blood.
  • serum can be brought into contact with mature hepatocyte-like cells by placing mature hepatocyte-like cells in a blood-permeable cell-holding material in the blood circuit. That is, the present invention provides a method for treating liver disease, comprising the step of returning blood taken out of a living body to mature hepatocyte-like cells and then returning the blood to the patient.
  • mature hepatocyte-like cells can be brought into contact with the patient's blood via a serum-permeable membrane such as a dialysis membrane. Serum that permeates through dialysis membrane contacts mature hepatocyte-like cells By doing so, components in serum are metabolized by the action of mature hepatocyte-like cells. The serum after contact is returned to the bloodstream through the dialysis membrane.
  • the present invention also provides use of mature hepatocyte-like cells produced by the above process. That is, the present invention provides a therapeutic agent for liver disease comprising mature hepatocyte-like cells obtainable by the method of the present invention. Also provided is a method for treating liver disease, comprising the step of administering the mature hepatocyte-like cell to a patient having liver disease.
  • the liver diseases of the present invention include all diseases that can be complemented with functions by mature hepatocyte-like cells. Specific examples of such liver diseases include cirrhosis, fulminant hepatitis, biliary atresia, liver cancer, and hepatitis. Hepatitis includes, for example, viral hepatitis or alcoholic hepatitis.
  • the human mature hepatocyte-like cells of the present invention are also useful in, for example, research fields aimed at treating liver diseases.
  • the mature hepatocyte-like cell of the present invention can be used in the research and development of an artificial liver.
  • the mature hepatocyte-like cells of the present invention are useful in the field of development of pharmaceuticals and foods. Specifically, it can be used for the evaluation of test compound metabolism and hepatotoxicity, for screening for hepatic disease therapeutic agents, hepatitis virus infection inhibitors, or viral hepatitis therapeutic agents.
  • the present invention relates to a method for detecting metabolism of a test compound in the liver, comprising the following steps, wherein the mature hepatocyte-like cells are mesenchymal in the presence of oncostatin M, dexamethasone, and TGF-8.
  • a method of mature hepatocyte-like cells differentiated from mesenchymal stem cells is provided.
  • the present invention provides a method for detecting hepatotoxicity of a test compound, comprising the following steps, wherein mature hepatocyte-like cells are interfered in the presence of oncostatin M, dexamethasone, and TGF-8.
  • a method of mature hepatocyte-like cells differentiated from mesenchymal stem cells is provided.
  • the test compound in the present invention is not particularly limited.
  • xenobiotics natural compounds, organic compounds, inorganic compounds, proteins, single compounds such as peptides, compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermentation Examples include microbial products, marine organism extracts, plant extracts, and the like.
  • the xenobiotic includes all xenobiotics that are foreign to the living body. For example, drug and food candidate compounds, and existing drugs and foods can be mentioned.
  • the test compound can usually be brought into contact with mature hepatocyte-like cells by adding the test compound to a medium or a culture solution. In addition, both can be brought into contact by expressing a gene encoding a test compound in mature hepatocyte-like cells. Alternatively, the test compound can be contacted with mature hepatocyte-like cells by co-culture with cells that produce the test compound.
  • Metabolism of the test compound can be measured by methods well known to those skilled in the art. For example, when a metabolite of a test compound is detected, it is determined that the test compound has been metabolized. In addition, when the expression of enzyme genes such as CYP (cytochrome P450), MDR, and MRP is induced by contact with the test compound, or when the activity of these enzymes increases, it is determined that the test compound is metabolized. You can also.
  • Methods for detecting metabolites are known.
  • a test compound or its metabolite can be extracted from a culture of mature hepatocyte-like cells and analyzed by liquid chromatography or mass spectrometry.
  • a metabolite is predicted in advance, the presence of the metabolite can be confirmed by these analysis methods.
  • use of a radiolabeled compound as the test compound will allow metabolism to carbon dioxide or water by tracking radioactivity. It can be confirmed.
  • mRNAs of these genes can be detected. mRNA can be amplified and detected by techniques such as RT-PCR. MRNA of these genes For amplification and detection, for example, a method as described in Examples described later can be used. Alternatively, its expression can be traced using CYP enzyme activity as an index. Reagents for measuring CYP enzyme activity are commercially available.
  • the degree of damage of mature hepatocyte-like cells contacted with a test compound is measured.
  • the degree of damage can be measured using, for example, the survival rate of human mature hepatocyte-like cells and liver damage markers such as GOT and GPT.
  • the test compound determines whether the survival rate of human mature hepatocyte-like cells is reduced by adding the test compound to the culture medium of human mature hepatocyte-like cells. . Conversely, if there is no significant change in survival rate, the test compound is determined not to have liver toxicity. In addition, for example, if GOT or GPT in the culture solution rises after adding the test compound to the culture medium of human mature hepatocyte-like cells, the test compound is determined to have hepatotoxicity. Similarly, if there is no significant change in GOT or GPT, the test compound is determined not to have liver toxicity.
  • hepatic toxicity of the test compound can be quantitatively evaluated by using as a control a compound that is already known to have hepatotoxicity.
  • the mature hepatocyte-like cells obtainable by the present invention can be used for screening of therapeutic agents for liver diseases. That is, the present invention includes the following steps of liver disease: A method for screening therapeutic agents, wherein mature hepatocyte-like cells are differentiated from mesenchymal stem cells by culturing mesenchymal stem cells in the presence of oncostatin M, dexamethasone, and TGF-
  • the test compound when an increase in cell function is observed in mature hepatocyte-like cells contacted with a test compound, the therapeutic effect of the test compound on the liver is detected.
  • the test compound can be brought into contact with mature hepatocyte-like cells by the same operation as the test compound in the evaluation of metabolism and hepatotoxicity described above.
  • the function of mature hepatocyte-like cells in the present invention can be evaluated using, for example, glucose production ability, ammonia metabolism ability, albumin production ability, urea synthesis ability, and activity of enzymes such as CYP as indicators.
  • the ability to produce glucose can be confirmed by analyzing the glucose level in the culture supernatant by the glucose oxidase method.
  • the ability to metabolize ammonia can be confirmed by analyzing the ammonia level in the culture medium using the modified indophenol method (Horn DB & Squire CR, Chim. Acta. 14: 185-194. 1966).
  • the ability to produce albumin can be confirmed by analyzing the albumin concentration in the culture medium by measuring the serum albumin concentration.
  • the ability to synthesize urea can be confirmed using, for example, Colorimetric assay (Sigma).
  • the CYP of the present invention is not particularly limited, and examples thereof include CYP1A1, CYP2C8, CYP2C9, CYP3A4 and the like.
  • a method for measuring CYP activity methods well known to those skilled in the art can be used.
  • any compound for which the liver function improving action should be evaluated can be used as the test compound.
  • a library of natural substances or artificially synthesized compounds can be used as the test compound.
  • Natural materials include force extracted components such as plants, animals, insects, or microorganisms. Or sales These compound libraries can also be screened by the method of the present invention.
  • the mature hepatocyte-like cell of the present invention can be used in a screening method for hepatitis virus infection inhibitors. That is, the present invention provides a method for screening an hepatitis virus infection inhibitor comprising the following steps, wherein the mature hepatocyte-like cells are mesenchymal stem cells in the presence of oncostatin M, dexamethasone, and TGF-8. Is a mature hepatocyte-like cell differentiated from mesenchymal stem cells.
  • infection inhibitors for all viruses that infect the mature hepatocyte-like cells of the present invention can be screened. Specifically, hepatitis C virus, hepatitis A virus, and hepatitis B virus can be targeted. These hepatitis viruses may be established or directly isolated from those infected with hepatitis virus. Further, it may be in a purified state or a crude state (for example, a state of serum obtained from an infected person).
  • the presence or absence of hepatitis virus infection in mature hepatocyte-like cells can be examined using, for example, the amount of hepatitis virus in the cells as an indicator.
  • the amount of hepatitis virus in a cell can be determined using, for example, the amount of hepatitis virus RNA in the cell as an indicator.
  • the amount of hepatitis virus RNA can be measured according to a standard method. Alternatively, the measurement may be performed by a method established by the present inventors (T. Takeuch. Et al. Real-Time Detection System for Wuanthincation of Hepatitis C Viru s Genome. Gastroenterology 1999, 116: 636-642).
  • the mature hepatocyte-like cells obtained by the present invention are also useful for screening for therapeutic agents for viral hepatitis. That is, the present invention includes a viral liver comprising the following steps: A method of screening for a therapeutic agent for inflammation, wherein mature hepatocyte-like cells were differentiated from mesenchymal stem cells by culturing mesenchymal stem cells in the presence of oncostatin M, dexamethasone, and TGF-
  • the compounds that inhibit the growth of the hepatitis virus of the present invention include the following compounds.
  • the growth and disappearance of hepatitis virus can be examined by measuring the amount of hepatitis virus in the cells.
  • test compound in the method for screening an inhibitor of hepatitis virus infection or the method for screening a therapeutic agent for viral hepatitis of the present invention an arbitrary compound whose viral infection inhibitory action or viral hepatitis therapeutic action should be evaluated is used.
  • a library of natural substances or artificially synthesized ichthy compounds can be used as the test compound. Natural substances include components extracted from plants, animals, insects, or microorganisms.
  • a commercially available compound library can be screened by the method of the present invention.
  • hepatitis virus can be cultured using mature hepatocyte-like cells obtainable by the present invention as a host. That is, the present invention relates to a method for culturing hepatitis virus comprising a step of infecting hepatitis virus with mature hepatocyte-like cells, wherein the mature hepatocyte-like cells are present in the presence of oncostatin M, dexamethasone, and TGF-
  • the virus is hepatitis C virus.
  • the method for culturing hepatitis virus of the present invention is useful for passage or amplification of an already isolated virus.
  • hepatitis viruses can also be separated by sample forces derived from the environment and patients.
  • the patient-derived hepatitis virus can be isolated by carrying out the virus culturing method of the present invention using a patient blood sample as a sample.
  • the hepatitis virus is detected by amplification of the gene of the virus.
  • gene amplification even if the virus loses its ability to infect, it will be detected as a virus as long as the gene is present.
  • virus isolation method using virus-sensitive cells, it is possible to isolate a virus that has actually maintained infectivity.
  • Oncostatin M used for the preparation of the culture medium is a recombinant (mature protein having a 227 amino acid residue strength) obtained by expressing human oncostatin M in E. coli.
  • TGF-181 is a human recombinant TGF-
  • OSM Oncostatin M
  • ITS—A Insulin, Transpiercing, Selenium mixture, Sigma, USA
  • PSA penicillin, streptomycin, amphotericin B
  • F12K Invitrogen
  • HCM medium Humidity
  • IMDM in basal medium IDOT medium
  • F12K basal medium FKDOT medium
  • HCM as basal medium HDOT medium
  • the plastic cord force of the human umbilical cord blood-derived mesenchymal stem cells cultured in Example 1 was also peeled off with 0.05% trypsin ⁇ EDTA solution (Sigma, USA). After washing, the cells were suspended in the IDOT medium and seeded on a type I collagen-coated plate (Asahi Techno Glass) at a rate of 30000-60000 per 1 cm 2 . 5% CO in humidified atmosphere
  • Example 3 Morphological observation of hepatocyte-like cells derived from human mesenchymal stem cells
  • FIG. 1 shows the morphological change from human umbilical cord blood-derived and bone marrow-derived mesenchymal stem cells to hepatocyte-like cells.
  • a in FIG. 1 shows the morphology of undifferentiated human umbilical cord blood-derived mesenchymal stem cells, which are in the form of fibroblasts.
  • B shows the morphology of human umbilical cord blood-derived mesenchymal stem cells on the 5th day after the start of the induction of spermatozoa, and it can be seen that the morphology is rounded as a whole.
  • C shows the morphology of human bone marrow-derived mesenchymal stem cells on the second day after the induction of differentiation, and in the case of bone marrow-derived mesenchymal stem cells, the cells have already begun to become rounded on the second day.
  • D shows the morphology of human bone marrow-derived mesenchymal stem cells on the 9th day from the start of differentiation induction, and it can be seen that, like B, it has an overall rounded morphology. It can also be seen that the nucleus is white and the nucleolus is clear.
  • FIG. 2 shows the form of day 5 in which human bone marrow-derived mesenchymal stem cells were induced to differentiate into hepatocyte-like cells.
  • IDOT, HDOT, and FKDOT in Fig. 2 represent differences in basal media supplemented with differentiation factors.
  • the left is a weakly magnified image ( ⁇ 100), and the right is a strongly magnified image (x200). It can be seen that the culture is rounded as a whole on the 5th day from the start of mashing regardless of the culture medium. In the strong image, it is clear that the nucleus is white and the nucleolus is clear.
  • the left is a weakly magnified image ( ⁇ 100)
  • x200 strongly magnified image
  • Anolebumin (ALB);
  • TAT Tyrosine aminotransferase
  • Cytochrome P450 (CYP1A2, CYP3A4, CYP2D6);
  • TTR Transthyretin
  • RT-PCR reverse J j was performed using superscript II Reverse Transcriptase (Invitrogen, USA). All were amplified as single-band cDNA fragments, and the identification of the differential marker was confirmed by PCR using specific primers.
  • the PCR primers used for RT-PCR are as follows. The primer for cytochrome P450 (CYP1A2, CYP3A4, CYP2D6) gene used was a primer for Real Time PCR from Takara Bio.
  • Anolebumin (ALB)
  • Tyrosine aminotransferase (TAT) 5 -TGA GCA GTC TGT CCA CTG CCT-3 '/ SEQ ID NO: 5
  • TRR Transthyretin
  • Figure 3 shows the results of RT-PCR analysis 6 days after the start of culture.
  • lane 1 shows mesenchymal stem cells derived from human umbilical cord blood
  • lane 2 shows the results of analysis of cells after 6 days of differentiation
  • A is 13-actin
  • B is albumin
  • C is TAT
  • D is the RT-PCR analysis result of TD02.
  • albumin, TAT, and TD02 genes which are specific to liver cells, has already been confirmed. Expression of these genes was not confirmed in undifferentiated human umbilical cord blood-derived mesenchymal stem cells.
  • Fig. 4 shows the results of RT-PCR analysis 12 days after the start of culture.
  • lane 1 is mesenchymal stem cells derived from undifferentiated human bone marrow
  • lane 2 is a differentiation of human bone marrow derived mesenchymal stem cells on day 12
  • lane 3 is a human bone marrow of a donor different from lane 2 Origin of mesenchymal stem Cell differentiation Day 12
  • lane 4 is human umbilical cord blood-derived mesenchymal stem cell differentiation Day 12
  • lane 5 is different from lane 4 human umbilical cord blood-derived mesenchymal stem cell differentiation Day 12
  • Lane 6 shows the results of RT-PCR analysis of undifferentiated human umbilical cord blood-derived mesenchymal stem cells.
  • albumin TD02, a liver cell-specific molecule
  • TTR a liver cell-specific molecule
  • CYP3A4 expression induction and CYP2D6 expression enhancement were observed in differentiation-inducing cells.
  • the CYP activity of cells cultured in a medium containing dexamethasone, oncostatin M (OSM), and TGF-j81 was measured by the GLO method (Promega).
  • the CYPs whose activity was measured are CYP3A4 and CYP1A2.
  • HCM medium containing 50 ⁇ L of luminescent substrate was added to floating 1 ⁇ 10 5 cells.
  • Cells that were not subjected to induction were cultured on a plate, the medium in the plate was aspirated, washed once with HCM medium, and then HCM medium containing a luminescent substrate was added.
  • the HCM medium containing a luminescent substrate was added with 200 ⁇ L for a 24-well plate and 50 ⁇ L for a 96-well plate.
  • the luminescent substrate used for the measurement is as follows.
  • the plate was incubated at 37 ° C for 4 hours, and after the reaction was completed, a part (50 L) of the cell culture supernatant was transferred to a white multi-well plate for measuring luminescence (Luminescence). Furthermore, an equal amount of luciferin detection reagent was added and stirred for 10 seconds. After reacting at room temperature for 20 minutes, the relative luminescence (RLU) was measured with a plate reader and recorded. Na When measuring the activity, the luminescent substrate incubated at 37 ° C for 4 hours was also measured in the same manner as the sample, and the measured value was corrected using this as a knock ground value in the absence of cells.
  • a CYP inducer (CYP 1A2: Omeprazole CYP3A4: Rifampicin) was added to a plate containing culture medium inoculated with sputum cells and cultured for 1-3 days. This was done by measuring the activity.
  • FIG. 6 shows a comparison of CYP3A4 activity with primary human cultured liver cells.
  • Lane A is the differentiation of human bone marrow-derived mesenchymal stem cells on day 34
  • Lane B is human primary cultured hepatocytes (Daiichi Kagaku; # 77)
  • Lane C is human cord blood-derived mesenchymal stem cells Day 23 of differentiation
  • Lane D is human umbilical cord blood-derived mesenchymal stem cell differentiation Day 20
  • lane E is different from lanes C and D Donor human umbilical cord blood-derived mesenchymal stem cell differentiation Day 20
  • the CYP3A4 activity of the human B bone marrow-derived mesenchymal stem cells and the C human cord blood-derived mesenchymal stem cells are almost the same as the primary human cultured liver cells. It was.
  • Fig. 7 shows the results of a study of CYP3A4 inducibility.
  • Hepatocyte-like cells derived from human umbilical cord blood-derived mesenchymal stem cells by the method of [22] are treated with a medium containing rifampicin 1, 3, 10 / z M for 2 to 3 days, CYP activity was measured by the method of [5-2].
  • Fig. 7A shows the cell activity on the 62nd day of differentiation induction and B shows the cell activity on the 17th day.
  • CYP3A4 activity increased in a rifampicin concentration-dependent manner. When rifampicin was exposed to 10 ⁇ for 3 days, it increased approximately 2.3 times compared to the case without induction (Fig. 7 ⁇ ).
  • FIG. 8 shows the results of an examination of the CYP1A2 inducibility.
  • hepatocyte-like cells obtained by inducing differentiation of human bone marrow-derived mesenchymal stem cells by the method [2-2] (differentiation induction day 25), omebrazo (OPZ) 5, 15, 50 / z M was treated for 2 days, and CYP activity was measured by the method [5-2].
  • omebrazo (OPZ) 5 5, 15, 50 / z M was treated for 2 days, and CYP activity was measured by the method [5-2].
  • the activity of CYP1A2 increased depending on the concentration of omebrazole (OPZ).
  • human hepatocyte-like cells obtained by the method of the present invention induced human CYP450 enzyme in the same manner as primary normal human cultured hepatocytes, and human hepatocyte-like cells of the present invention. This suggests that it can be used for screening of cell force drugs.
  • Example 6 Mesenchymal stem cell strength derived from human umbilical cord blood derived from mitochondrial analysis of glucose metabolism function of the obtained hepatocyte-like cells
  • the obtained hepatocyte-like cells are seeded in a 96-well plate at a rate of 2.0X10 4 / well, washed approximately 24 hours later with PBS, then each concentration of insulin is added, and the glucose concentration in the culture supernatant 48 hours later was measured.
  • glucose concentration glucose Cn-test for glucose measurement (Mutarotase 'GOD method) (Wako Pure Chemical Industries, Ltd.) was used and the resulting force glucose concentration was determined.
  • Example 7 Hepatitis C virus infection experiment of hepatocyte-like cells obtained by inducing differentiation from human umbilical cord blood-derived mesenchymal stem cells
  • Hepatocyte-like cells obtained by inducing differentiation from human umbilical cord blood-derived mesenchymal stem cells were seeded in 12-well and 6-well cell culture plates coated with collagen or Matrigel. After cells were fully implanted, they were washed once with Williams E medium. The cells were inoculated with the sera of HCV-infected persons who were confirmed to have infectious HCV. The amount of HCV inoculated was 0.5 and 1.0 copies, respectively, as the amount of HCV gene per cell. 37 degree CO in The virus was adsorbed to the cells for 3 hours in a incubator and then washed 3 times with Williams E medium to remove unadsorbed HCV.
  • Figure 10 shows the relationship between the time after hepatitis C virus (HCV) infection and the amount of HCV-RNA.
  • HCV infection culture using hepatocyte-like cells derived from human umbilical cord blood-derived mesenchymal stem cells virus infection and proliferation were confirmed. From this fact, it was confirmed that mature hepatocyte-like cells obtained by the method of the present invention can be used for screening for HCV infection inhibitors and HCV growth inhibitors.
  • the present invention provides a method for inducing differentiation into mesenchymal stem cell power hepatocyte-like cells in vitro.
  • the present invention is useful as a tool for elucidating the differentiation mechanism of stem cells into hepatocytes at the molecular level.
  • the present invention also provides hepatocyte-like cells with high maturity. Mature hepatocyte-like cells that can be obtained by the present invention can be used to evaluate the effects of compounds on the liver.
  • research (experiment) on the effects of compounds on living organisms was often conducted using living organisms. For example, laboratory animals are used in carcinogenesis experiments or safety assessment tests such as food additives and anticancer agents.
  • the mature hepatocyte-like cells of the present invention can be used as a new accessory model in place of experimental animals in these test methods.
  • the human hepatocyte-like cells obtained in the present invention can be infected with hepatitis virus and maintained in the infected state.
  • the mature hepatocyte-like cells of the present invention can be used for research on prevention or treatment of diseases caused by hepatitis virus.
  • hepatitis C virus is highly species-specific to humans.
  • no experimental animals have been identified that can be infected with human hepatitis C.
  • Chimpanzees have been designated as endangered international rare wildlife species by the Washington Convention.
  • migration for academic purposes has been recognized, it remains rare, and cell lines have been established that enable efficient culture of hepatitis C virus in vitro. In other words, the infection model and culture method necessary for the research of hepatitis C virus are not sufficient at present. It has become your Keru major obstacle to the prevention and treatment of research.
  • Mature hepatocyte-like cells obtained by the present invention were confirmed to be infected with hepatitis C virus. Therefore, the mature hepatocyte-like cells of the present invention can be used for proliferation and infection experiments of hepatitis C virus in an in vitro system, that is, at the cellular level. Therefore, the mature hepatocyte-like cell of the present invention contributes to the research of human hepatitis C virus.
  • the mature hepatocyte-like cell of the present invention highly reproduces the function of the liver cell. Therefore, the mature hepatocyte-like cell of the present invention can be used as an artificial organ that substitutes for liver function.
  • an artificial liver can be obtained by filling an osmotic membrane with the mature hepatocyte-like cells of the present invention and circulating blood. In artificial dialysis and cardiopulmonary bypass, Osmotic membranes with low antigenicity against humans have already been put into practical use.
  • the treatment of patient blood in the blood circuit and returning it to the patient is routinely performed. By contacting the mature hepatocyte-like cells of the present invention with patient blood in such a blood circuit, the metabolic function of the liver can be substituted.

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Abstract

L'invention concerne un procédé de préparation de cellules analogues aux hépatocytes matures, comprenant la mise en culture de cellules souches mésenchymateuses en présence d'oncostatine M, de dexaméthasone et de TGF-β afin d'induire ainsi leur différenciation en cellules analogues aux hépatocytes matures. Les cellules analogues aux hépatocytes matures ainsi obtenues possèdent les fonctions cellulaires et les caractéristiques morphologiques des hépatocytes. Il a également été confirmé que les cellules précitées sont infectées par des hépatovirus tels que le VHC. Les cellules analogues aux hépatocytes matures décrites ci-dessus sont utiles pour évaluer le métabolisme et l'hépatotoxicité d'un composé de test. En outre, les cellules analogues aux hépatocytes matures décrites ci-dessus peuvent être utilisées pour cribler un médicament destiné à traiter des maladies hépatiques, un inhibiteur d'infection par un hépatovirus, un médicament destiné à traiter une hépatite virale, ou autres.
PCT/JP2007/057420 2006-04-03 2007-04-03 Procédé de préparation de cellules analogues aux hépatocytes matures Ceased WO2007116870A1 (fr)

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WO2016029267A1 (fr) * 2014-08-27 2016-03-03 Prince Henry's Institute Of Medical Research Trading As The Hudson Institute Of Medical Research Procédé de culture de cellules souches mésenchymateuses
CN108823148A (zh) * 2018-07-23 2018-11-16 广东唯泰生物科技有限公司 一种脂肪间充质干细胞诱导分化为肝脏样细胞的方法
CN111073843A (zh) * 2018-10-22 2020-04-28 立沃生物科技（深圳）有限公司 一种肝样细胞成熟与扩增的方法
CN115025121A (zh) * 2022-06-23 2022-09-09 深圳凯兰赛尔健康管理有限公司 一种复合细胞制剂及其制备方法与应用
CN115025121B (zh) * 2022-06-23 2023-10-03 深圳凯兰赛尔健康管理有限公司 一种复合细胞制剂及其制备方法与应用
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