WO2007116870A1 - Method of preparing mature hepatocyte-like cell - Google Patents
Method of preparing mature hepatocyte-like cell Download PDFInfo
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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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- 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
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2501/39—Steroid hormones
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- C12N2501/998—Proteins not provided for elsewhere
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
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- 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.
Abstract
It is intended to provide a method of preparing a mature hepatocyte-like cell which comprises culturing mesenchymal stem cells in the presence of oncostatin M, dexamethasone and TGF-β and thus inducing the differentiation thereof into mature hepatocyte-like cells. The mature hepatocyte-like cell thus obtained has the cellular functions and morphological characteristics of hepatocytes. It has been also confirmed that the above cell is infected with hepatitis viruses such as HCV. The mature hepatocyte-like cell as described above is useful in evaluating the metabolism and hepatotoxicity of a test compound. Moreover, the mature hepatocyte-like cell as described above is usable in screening a remedy for liver diseases, a hepatitis virus-infection inhibitor, a remedy for viral hepatitis and so on.
Description
明 細 書 Specification
成熟肝細胞様細胞の製造方法 Method for producing mature hepatocyte-like cells
技術分野 Technical field
[0001] 本発明は、成熟肝細胞様細胞 (mature hepatocyte-like cells)に関する。 [0001] The present invention relates to mature hepatocyte-like cells.
背景技術 Background art
[0002] 幹細胞は、さまざまなタイプの細胞に分ィ匕する能力を有する細胞である。例えば、 肝臓、筋肉、神経、皮膚、あるいは血液細胞などに分化する能力を有する細胞が知 られている。再生医療への応用のために、幹細胞力 特定の臓器機能を持つ細胞を 効率的に分化誘導する方法が切望されている。 [0002] 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.
特に幹細胞力 肝細胞様細胞 (hepatocyte-like cells)への分化誘導は生物学的な 人工肝臓( biological artificial liver)の開発や、細胞移植治療 (Cell Transplantation t herapy)への応用などが期待される。細胞移植治療は、肝不全、肝炎などの疾患に対 する治療方法として有用である。また創薬においても、肝細胞様細胞は、薬物の代 謝や毒性のアツセィ系やスクリーニング系に利用することができる。 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. In drug discovery, hepatocyte-like cells can also be used for drug substitution and toxicity systems and screening systems.
[0003] 幹細胞力 肝細胞を分ィ匕誘導する方法としては、負傷した動物の再生能力を利用 する方法や、細胞の共培養を利用する方法が知られている。しかし、機能的肝細胞 を多量に生産するためには、 in vitroにおいて、幹細胞から肝細胞への分化を誘導で きる培養系が望ましい。 in vitroにおける肝細胞への分化について、種々の試みが報 告されている。 [0003] Stem cell power As methods for inducing hepatocyte differentiation, methods utilizing the regenerative ability of injured animals and methods utilizing cell co-culture are known. However, in order to produce large amounts of functional hepatocytes, a culture system that can induce differentiation from stem cells to hepatocytes in vitro is desirable. Various attempts have been reported regarding differentiation into hepatocytes in vitro.
[0004] 宫島らは、胎児由来の未熟肝細胞をオンコスタチン M(oncostatin M; OSM)および デキサメタゾンを用いて培養することによって、肝細胞様細胞と分化を誘導する方法 を開示している。あるいは、細胞外マトリクス存在下で OSMおよびダルココルチコイド を加えて胎児由来の未熟肝細胞を培養することによって、肝細胞様細胞への分化が 観察された (非特許文献 1、特許文献 1— 2)。 [0004] 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. Alternatively, 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 (Non-patent Document 1, Patent Document 1-2) .
[0005] また、寺岡らは、次のような増殖因子 (growth factor)や分化誘導因子 (differentiation inducing factor)を使って、ヒト臍帯血から分離'調製した有核細胞から、ヒトアルブミ ン遺伝子およびヒトアルブミンを発現する培養細胞が得られることを確認している (特
許文献 7)。アルブミンの発現は、肝細胞の特徴の一つである。寺岡らの報告におい ては、特に、 FGF、 HGF、 LIF、および SCFの組み合わせにおいて、最も良好な成績 が得られた。 [0005] In addition, 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.
ヒト曰血病抑制因子 (human leukemia inhibitory factor; hLIF)、 Human leukemia inhibitory factor (hLIF),
ヒト幹細胞因子 (human Stem cell factor; hSCF)、 Human stem cell factor (hSCF),
繊維芽細胞成長因子 (fibroblast growth factor; FGF)、 Fibroblast growth factor (FGF),
ヒト肝細胞成長因子 (human Hepatocyte growth factor; hHGF)、 Human hepatocyte growth factor (hHGF),
OSM,および OSM, and
デキサメタゾン等 Dexamethasone, etc.
[0006] あるいは、落谷らは、胚性幹細胞 (embryonic stem cell;ES cell),あるいは骨髄由来 間葉系幹糸田胞 (bone marrow derived mesenchyma stem cells; BM hMSC)力ら、 糸田 胞様細胞を分化誘導する方法を開示している。この報告においては、 OSM、 HGF、 および FGFが、必須の分ィ匕増殖因子として用いられた (特許文献 4)。胚性幹 (ES)細 胞はマウス、ラット、およびサル由来の細胞が、また骨髄由来間葉系幹細胞にはヒト 由来の細胞が用いられた。 [0006] Alternatively, Ochiya et al. Used embryonic stem cells (ES cells) or bone marrow derived mesenchyma stem cells (BM hMSC), A method for inducing differentiation is disclosed. In this report, OSM, HGF, and FGF were used as essential growth factors (Patent Document 4). Embryonic stem (ES) cells were derived from mice, rats and monkeys, and bone marrow-derived mesenchymal stem cells were derived from human cells.
[0007] Leeら (非特許文献 2、特許文献 6)は、 2段階の分ィ匕誘導方法によって、骨髄由来 間葉系幹細胞 (BM MSC)から肝細胞様細胞への分化を誘導する方法を開示して!/、 る。第 1段階では HGFを、そして第 2段階の成熟用培地においては OSMを必須因子 とする培地がそれぞれ用いられた。 [0007] Lee et al. (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.
また、 Hong et al.ら (非特許文献 4)は Leeらの方法 (非特許文献 6)にならつて臍帯 血幹細胞からの肝細胞様細胞の分化誘導を確認している。 Hong et al. (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).
[0008] 特許文献 1 : Miyajima, et al: JP2000/287680 [0008] Patent Document 1: Miyajima, et al: JP2000 / 287680
特許文献 2 : Miyajima, et al: WO02/074937 Patent Document 2: Miyajima, et al: WO02 / 074937
特許文献 3 : Miki, et al: JP2005- 523328 Patent Document 3: Miki, et al: JP2005- 523328
特許文献 4 : Ochiya, et al: JP2006- 254896 Patent Document 4: Ochiya, et al: JP2006-254896
特許文献 5 : Tomisawa,: JP2005-253374 Patent Document 5: Tomisawa ,: JP2005-253374
特許文献 6 : Lee, et al: US 2005/0233449 Al Patent Document 6: Lee, et al: US 2005/0233449 Al
特許文献 7 : Teraoka, et al: JP2002- 360243
非特許文献 l : Miyajima, et al: EMBO J. Vol.18, No.8, pp.2127- 2136(1999) 非特許文献 2 : Lee, et al: HEPATOLOGY vol.40 : 1275-1284 (2004) 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)
非特許文献 3 : S-E Yang, et al: Cytotherapy Vol. 6, No. 5, 476-486 /486(2004) 非特許文献 4 : S.H. Hong, et al: Biochemical and Biophysical Research Communicatio ns 330: 1153-1161 (2005) 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)
非特許文献 5 : K.Teramoto et al.: J Hepatobiliary Pancreat Surg (2005) 12:196-202 非特許文献 6 : Lee, et al: Blood vol.103 : 1669- 1675 (2004) 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)
発明の開示 Disclosure of the invention
発明が解決しょうとする課題 Problems to be solved by the invention
[0009] 本発明は、成熟度 (maturity)の高 、肝細胞様細胞の製造方法の提供を課題とする 。あるいは本発明は、本発明によって製造することができる肝細胞様細胞と、その用 途の提供を課題とする。 [0009] An object of the present invention is to provide a method for producing hepatocyte-like cells with high maturity. Alternatively, an object of the present invention is to provide hepatocyte-like cells that can be produced according to the present invention and uses thereof.
課題を解決するための手段 Means for solving the problem
[0010] 従来は、幹細胞からの肝細胞様細胞への分化誘導には、肝細胞成長因子 (HGF) が不可欠であると考えられてきた。しかし、本発明者は、 HGFの不存在下においても 、特定の増殖分化因子を利用することによって成熟肝細胞様細胞への充分な分ィ匕 が誘導できることを見出した。そして、この知見に基づいて、成熟度の高い肝細胞様 細胞の製造方法を確立し、本発明を完成した。すなわち本発明は、以下の肝細胞様 細胞の製造方法、並びに当該製造方法によって得ることができる肝細胞様細胞と、 その各種の用途に関する。 [0010] Conventionally, hepatocyte growth factor (HGF) has been considered indispensable for inducing differentiation from stem cells into hepatocyte-like cells. However, 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] 〔1〕オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩、および TGF- βの存在下で間葉系幹細胞を培養し、間葉系幹細胞を成熟肝細胞様細胞に分化さ せる工程を含む、成熟肝細胞様細胞の製造方法。 [0011] [1] A step of culturing mesenchymal stem cells in the presence of oncostatin M, dexamethasone or a derivative or salt thereof, and TGF-β to differentiate the mesenchymal stem cells into mature hepatocyte-like cells. A method for producing a mature hepatocyte-like cell.
〔2〕間葉系幹細胞が、臍帯血、骨髄、脂肪組織、胎盤、および抹消血力 なる群力 選択されるいずれかの組織に由来する間葉系幹細胞である〔1〕に記載の成熟肝細 胞様細胞の製造方法。 [2] The mature liver according to [1], wherein the 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.
〔3〕間葉系幹細胞が、臍帯血に由来する間葉系幹細胞である〔2〕に記載の成熟肝 細胞様細胞の製造方法。
〔4〕間葉系幹細胞が、骨髄に由来する間葉系幹細胞である〔2〕に記載の成熟肝細 胞様細胞の製造方法。 [3] The method for producing a mature hepatocyte-like cell according to [2], wherein the mesenchymal stem cell is a mesenchymal stem cell derived from umbilical cord blood. [4] The method for producing a mature hepatocyte-like cell according to [2], wherein the mesenchymal stem cell is a mesenchymal stem cell derived from bone marrow.
[5]間葉系幹細胞が、少なくとも 6日間培養される〔1〕に記載の成熟肝細胞様細胞の 製造方法。 [5] The method for producing mature hepatocyte-like cells according to [1], wherein the mesenchymal stem cells are cultured for at least 6 days.
〔6〕オンコスタチン M、デキサメタゾン、および TGF- |8の間葉系幹細胞を培養するた めの培地における濃度力、それぞれ l〜100ng/mL、 0. 1〜: LO M、そして 0. 2〜2 Ong/mLである〔1〕に記載の成熟肝細胞様細胞の製造方法。 [6] Concentration in medium for culturing oncostatin M, dexamethasone, and TGF- | 8 mesenchymal stem cells, 1 to 100 ng / mL, 0.1 to: LO M, and 0.2 to The method for producing a mature hepatocyte-like cell according to [1], which is 2 Ong / mL.
〔7〕〔1〕〜〔6〕に記載のいずれかの方法によって製造することができる成熟肝細胞様 細胞。 [7] A mature hepatocyte-like cell that can be produced by any of the methods described in [1] to [6].
〔8〕〔1〕〜〔6〕に記載のいずれかの方法によって製造することができる成熟肝細胞様 細胞を含む、肝臓疾患の治療剤。 [8] 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].
〔9〕次の工程を含む、被験化合物の肝における代謝を検出する方法であって、成熟 肝細胞様細胞が、オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩 、および TGF- |8の存在下で間葉系幹細胞を培養することによって、間葉系幹細胞 から分化した成熟肝細胞様細胞である方法; [9] 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- | 8: A method of mature hepatocyte-like cells differentiated from mesenchymal stem cells by culturing mesenchymal stem cells in
(1)成熟肝細胞様細胞に被験化合物を接触させる工程、および (1) contacting the test compound with mature hepatocyte-like cells, and
(2)成熟肝細胞様細胞における被験化合物の代謝を検出する工程。 (2) A step of detecting metabolism of a test compound in mature hepatocyte-like cells.
〔10〕次の工程を含む、被験化合物の肝毒性を検出する方法であって、成熟肝細胞 様細胞が、オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩、およ び TGF- |8の存在下で間葉系幹細胞を培養することによって、間葉系幹細胞から分 化した成熟肝細胞様細胞である方法; [10] 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;
(1)成熟肝細胞様細胞に被験化合物を接触させる工程、および (1) contacting the test compound with mature hepatocyte-like cells, and
(2)成熟肝細胞様細胞の障害が検出されたときに被験化合物の肝毒性が検出され る工程。 (2) A step in which hepatotoxicity of a test compound is detected when a disorder of mature hepatocyte-like cells is detected.
〔11〕次の工程を含む、肝疾患の治療剤のスクリーニング方法であって、成熟肝細胞 様細胞が、オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩、およ び TGF- |8の存在下で間葉系幹細胞を培養することによって、間葉系幹細胞から分 化した成熟肝細胞様細胞である方法;
(1)成熟肝細胞様細胞に被験化合物を接触させる工程、 [11] 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 mature hepatocyte-like cell differentiated from mesenchymal stem cells by culturing mesenchymal stem cells in (1) contacting the test compound with mature hepatocyte-like cells,
(2)被験化合物を接触させた成熟肝細胞様細胞の機能を測定する工程;および (2) measuring the function of mature hepatocyte-like cells contacted with the test compound; and
(3)対照と比較して、成熟肝細胞様細胞の機能を亢進させる作用を有する化合物 を選択する工程。 (3) A step of selecting a compound having an action of enhancing the function of mature hepatocyte-like cells as compared with the control.
〔12〕次の工程を含む、肝炎ウィルスの感染阻害剤のスクリーニング方法であって、 成熟肝細胞様細胞が、オンコスタチン M、デキサメタゾンまたはその誘導体もしくはそ の塩、および TGF- |8の存在下で間葉系幹細胞を培養することによって、間葉系幹 細胞から分化した成熟肝細胞様細胞である方法; [12] 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- | 8: A mature hepatocyte-like cell differentiated from mesenchymal stem cells by culturing mesenchymal stem cells in
(1)成熟肝細胞様細胞に被験化合物の存在下で肝炎ウィルスを接触させるか、ま たは成熟肝細胞様細胞に肝炎ウィルスを接触させた後に被験化合物を接触させる 工程、 (1) contacting the hepatitis virus with mature hepatocyte-like cells in the presence of the test compound, or contacting the test compound after contacting hepatitis virus with mature hepatocyte-like cells;
(2)成熟肝細胞様細胞への肝炎ウィルスの感染のレベルを測定する工程;および (2) measuring the level of hepatitis virus infection of mature hepatocyte-like cells; and
(3)対照と比較して、成熟肝細胞様細胞への肝炎ウィルスの感染レベルが低い化 合物を選択する工程。 (3) A step of selecting a compound having a lower infection level of hepatitis virus to mature hepatocyte-like cells than the control.
〔13〕次の工程を含む、ウィルス性肝炎の治療剤のスクリーニング方法であって、成 熟肝細胞様細胞が、オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその 塩、および TGF- |8の存在下で間葉系幹細胞を培養することによって、間葉系幹細 胞から分化した成熟肝細胞様細胞である方法; [13] 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
(1)肝炎ウィルスを感染させた成熟肝細胞様細胞に被験化合物を接触させる工程 (1) A step of bringing a test compound into contact with a mature hepatocyte-like cell infected with hepatitis virus
(2)成熟肝細胞様細胞における肝炎ウィルスの増殖を測定する工程;および(2) measuring the growth of hepatitis virus in mature hepatocyte-like cells; and
(3)対照と比較して、肝炎ウィルスの増殖の阻害作用が検出された化合物を選択す る工程。 (3) A step of selecting a compound in which an inhibitory effect on the growth of hepatitis virus is detected compared to the control.
〔14〕成熟肝細胞様細胞に肝炎ウィルスを感染させる工程を含む肝炎ウィルスの培 養方法であって、成熟肝細胞様細胞がオンコスタチン M、デキサメタゾンまたはその 誘導体もしくはその塩、および TGF- |8の存在下で間葉系幹細胞を培養することによ つて、間葉系幹細胞から分化した成熟肝細胞様細胞である肝炎ウィルスの培養方法
〔15〕オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩、および TGF - ι8を含む、間葉系幹細胞から成熟肝細胞様細胞への分化誘導試薬。 [14] 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- | 8 For 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] A differentiation-inducing reagent from mesenchymal stem cells to mature hepatocyte-like cells, comprising oncostatin M, dexamethasone or a derivative or salt thereof, and TGF-ι8.
〔16〕オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩、および TGF - j8を含む、培地。 [16] A medium containing oncostatin M, dexamethasone or a derivative or salt thereof, and TGF-j8.
〔17〕間葉系幹細胞を成熟肝細胞様細胞に分化させるための、〔16〕に記載の培地。 〔18〕オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩、および TGF - j8の培地における濃度力 それぞれ 1〜: LOOng/mL、 0. 1〜: LO /z M、そして 0. 2〜 20ng/mLである〔 16〕又は〔 17〕に記載の培地。 [17] The medium according to [16] for differentiating mesenchymal stem cells into mature hepatocyte-like cells. [18] Concentration power of Oncostatin M, dexamethasone or a derivative thereof or a salt thereof, and TGF-j8 in the medium 1 to: LOOng / mL, 0.1 to: LO / z M, and 0.2 to 20 ng / mL, respectively The medium according to [16] or [17].
発明の効果 The invention's effect
[0012] 本発明によって、成熟肝細胞様細胞を製造する方法が提供された。本発明の方法 は、間葉系幹細胞力 成熟肝細胞様細胞への分ィ匕を短時間のうちに誘導することが できる。たとえば肝臓特異的な遺伝子の発現を肝細胞様細胞の成熟度の指標とした 場合、従来、幹細胞を成熟肝細胞様細胞に分化させるためには、少なくとも数週間 の培養が必要とされていた。ところが驚くべきことに、本発明の方法においては、培養 開始後 6日間という非常に短期間の内に成熟肝細胞特異的な遺伝子発現を確認す ることがでさる。 [0012] 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. For example, when 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. However, surprisingly, in the method of the present invention, gene expression specific to mature hepatocytes can be confirmed within a very short period of 6 days after the start of culture.
[0013] 本発明によって得ることができる肝細胞様細胞は、細胞の形態的な特徴にっ 、ても 、肝細胞との高い相同性を有する。更に、本発明によって得ることができる肝細胞様 細胞は、細胞機能の点においても、肝細胞の特徴を備える。具体的には、本発明の 肝細胞様細胞は、好ましい態様において、肝細胞に特徴的な次のような機能を有す ることが確認されている。 [0013] 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.
肝細胞に特異的な遺伝子が高く発現している、 Highly expressed genes specific to hepatocytes,
糖代謝、 Sugar metabolism,
アルブミン産生、および Albumin production, and
CYP活性 CYP activity
カロえて、好ましい態様において、本発明の肝細胞様細胞には、 C型肝炎ウィルスを 感染させることができる。この事実は、本発明によって得ることができる肝細胞様細胞 の成熟度が高ぐヒト成熟肝細胞に高度に類似した細胞であることを裏付けている。
図面の簡単な説明 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. Brief Description of Drawings
[0014] [図 1]ヒト臍帯血由来間葉系幹細胞から肝細胞様細胞への形態変化を示す写真であ る。 Aは未分化のヒト臍帯血由来間葉系幹細胞。 Bは分化誘導開始 5日目のヒト臍帯 血由来間葉系幹細胞。 Cは分化誘導開始 2日目のヒト骨髄由来間葉系幹細胞。 Dは 分ィ匕誘導開始 9日目のヒト骨髄由来間葉系幹細胞。 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.
[図 2]ヒト骨髄由来間葉系幹細胞を肝細胞様細胞へ分化誘導させた 5日目の形態を 示す写真である。 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.
[図 3]未分化のヒト臍帯血由来間葉系幹細胞と、分化誘導 6日後の肝細胞様細胞の R T-PCR解析結果 (1がヒト臍帯血由来間葉系幹細胞、 2が分化誘導 6日後の肝細胞様 細胞)を示す写真である。 [Figure 3] RT-PCR analysis results of undifferentiated human umbilical cord blood-derived mesenchymal stem cells and hepatocyte-like cells 6 days after induction of differentiation (1 is human cord blood-derived mesenchymal stem cells, 2 is differentiation-induced 6 It is a photograph showing hepatocyte-like cells after day).
[図 4]分化誘導 12日後の肝細胞様細胞の RT-PCR解析結果を示す写真である。 FIG. 4 is a photograph showing the results of RT-PCR analysis of hepatocyte-like cells 12 days after induction of differentiation.
[図 5]分化培養日数の経過に伴う CYP3A4活性の上昇を示す図である。 FIG. 5 is a graph showing an increase in CYP3A4 activity with the passage of differentiation culture days.
[図 6]ヒト初代培養肝臓細胞と分ィ匕誘導後の肝細胞様細胞との CYP3A4活性を示す 図である。 FIG. 6 is a graph showing CYP3A4 activity of primary human cultured hepatocytes and hepatocyte-like cells after induction of differentiation.
[図 7]分ィ匕誘導後の肝細胞様細胞において CYP3A4活性がリファンピシン濃度に依 存して上昇することを示す図である。 FIG. 7 is a graph showing that CYP3A4 activity rises depending on rifampicin concentration in hepatocyte-like cells after induction.
[図 8]分ィ匕誘導後の肝細胞様細胞において CYP1A2活性がオメブラゾール (OPZ)濃 度に依存して上昇することを示す図である。 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.
[図 9]肝細胞様細胞のグルコース代謝機能分析結果を示す図である。 FIG. 9 shows the results of analysis of glucose metabolism function in hepatocyte-like cells.
[図 10]ヒト臍帯血間葉系幹細胞由来から分化誘導した肝細胞様細胞の C型肝炎ウイ ルス感染実験を示す図である。 HCVを肝細胞様細胞に接種後の時間経過と HCV-m RNA量の関係を示す。 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.
発明を実施するための形態 BEST MODE FOR CARRYING OUT THE INVENTION
[0015] 本発明は、オンコスタチン M、デキサメタゾン、および TGF- βの存在下で間葉系幹 細胞を培養し、間葉系幹細胞を成熟肝細胞様細胞に分化させる工程を含む、成熟 肝細胞様細胞の製造方法に関する。 [0015] The present invention includes a step of culturing mesenchymal stem cells in the presence of oncostatin M, dexamethasone, and TGF-β, and causing the mesenchymal stem cells to differentiate into mature hepatocyte-like cells. The present invention relates to a method for producing like cells.
[0016] 本発明において、間葉系幹細胞は、ヒトを含む哺乳動物由来の間葉系幹細胞が用 いられる。好ましい間葉系幹細胞は、ヒト間葉系幹細胞である。間葉系幹細胞 (MSC)
は、脂肪細胞をはじめとして、軟骨細胞、骨細胞、心筋細胞、ニューロンといった多 様な細胞への分化能を有して 、ることが報告されて 、る。胎児にしかな 、胚性幹細 胞と異なり,組織幹細胞の一種である間葉系幹細胞は、患者の組織から分離するこ とができる。そのため、再生医療の素材として注目されている。 In the present invention, 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) Have been reported to have the ability to differentiate into a variety of cells, including adipocytes, chondrocytes, bone cells, cardiomyocytes, and neurons. Unlike embryonic stem cells, 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.
[0017] 本発明における間葉系幹細胞が由来する組織は限定されない。間葉系幹細胞は、 血液細胞以外の細胞に分ィ匕する骨髄幹細胞として見出された細胞である。その後、 骨髄の他、脂肪組織、胎盤、臍帯血、末梢血、そして歯牙組織にも間葉系幹細胞が 存在することが明らかにされた。これらの、骨髄以外の組織に由来する間葉系幹細 胞も、本発明に利用することができる。たとえば、ヒト臍帯血、およびヒト骨髄由来の間 葉系幹細胞は、本発明における好ましい間葉系幹細胞である。骨髄は、胎児から成 体にいたる任意の生育ステージの個体力も得ることができる。したがって、本発明に 基づ 、て、自家移植用の成熟肝細胞様細胞を得ることができる。 [0017] 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.
[0018] 間葉系幹細胞を分離する方法は公知である。たとえば、間葉系幹細胞を細胞表面 マーカー (CD271)を指標として回収することができる。 CD271に対する抗体を利用し て、ヒト間葉系幹細胞を分離するためのキット「間葉系幹細胞'分離培養 Box - CD27 1(LNGFR)」 (ミルテューバイオテク社製、商品名)も市販されている。あるいは、巿販 の、予め分離された間葉系幹細胞を本発明に利用することもできる。 [0018] Methods for isolating mesenchymal stem cells are known. For example, mesenchymal stem cells can be collected using the cell surface marker (CD271) as an indicator. 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 . Alternatively, pre-isolated mesenchymal stem cells that are commercially available can also be used in the present invention.
[0019] 更に、間葉系幹細胞は、分ィ匕のみならず、未分ィ匕のまま増殖させることができる。し たがって、分離した間葉系幹細胞を増殖させて本発明に利用することができる。本発 明において、分ィ匕前に間葉系幹細胞を増殖させることは好ましい。間葉系幹細胞は 、間葉系幹細胞増殖用培地として市販されて 、る培地を使って増殖させることができ る。具体的には、 MF培地(TOYOBO社、日本)、 MSCG培地(Cambrex社、米国)な どを、間葉系幹細胞の増殖に利用することができる。あるいは、ゥシ血清を含む EM培地を間葉系幹細胞の増殖に用いることもできる。間葉系幹細胞は、細胞培養 において一般的な条件で培養することによって増殖させることができる。例えば、約 5 %程度の COを含有する加湿大気中 37°Cにおけるインキュベーションを一般的な培養 [0019] Furthermore, 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
2 2
条件として示すことができる。間葉系幹細胞の増幅を目的とする培養には、コラーゲ ンのコーティングの無い培養容器を用いるのが一般的である。
[0020] 本発明の成熟肝細胞様細胞の製造方法にお!、て、間葉系幹細胞は、オンコスタチ ンM、デキサメタゾン、および TGF- j8の存在下で培養される。本発明において、間 葉系幹細胞を培養するための培地におけるオンコスタチン Mの濃度は、通常 lng/ml 〜100ng/ml、好ましくは 5ng/ml〜50ng/mlである。同様に、デキサメタゾンの濃度は 、通常 0. 1 \1〜10 \1、好ましくは0. 5 iu M〜5 iu Mでぁる。そしてTGF—J8の濃度 は、通常 0. 2ng/ml〜20ng/ml、好ましくは lng/ml〜: LOng/mlである。 It can be shown as a condition. A culture vessel without a collagen coating is generally used for the culture for the purpose of amplifying mesenchymal stem cells. [0020] In the method for producing mature hepatocyte-like cells of the present invention, mesenchymal stem cells are cultured in the presence of oncostatin M, dexamethasone, and TGF-j8. In the present invention, 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. Similarly, the concentration of dexamethasone is usually 0.1 \ 10 \ 1, preferably 0. 5 i u M~5 i u M Dearu. And the concentration of TGF-J 8 is usually 0. 2ng / ml~20ng / ml, preferably from lng / ml to: a LONG / ml.
[0021] オンコスタチン M(Oncostatin M)は、 IL- 6ファミリーのサイト力インの一種である。ヒト のメラノーマ(黒色腫)細胞株の増殖を抑制する因子としてオンコスタチン Mが同定さ れた (Proc. Natl, Acad. Sci. USA Vol. 83, pp. 9739-9743, December 1986)。血液細 胞ゃ幹細胞の分ィ匕への関与についても多くの報告がある。ヒトのオンコスタチン Mは 、 252アミノ酸力 なる分子量約 26kDのタンパク質である。分泌シグナルを含み、プ ロセシングにより N末端の 25アミノ酸残基が除去されて 227アミノ酸残基力もなる成熟 タンパク質となる。更に C末端側の 31アミノ酸残基が除去された、 196アミノ酸残基か らなる分子量約 22kDaの成熟タンパク質の存在も知られて 、る。これらの成熟タンパ ク質の生理活性は、前駆タンパク質よりも 5〜60倍高いとされている (Linsley, et al., 1 990, Mol. Cell. Biol. 10:1882 - 1890)。配列番号: 13にヒトオンコスタチン Mのァミノ 酸配列 (GenBank Accession No. AAA36388)を示す。配列番号: 13のアミノ酸配列 2 52残基中、 N末端佃 J力ら 26— 252位の 227残基、ある!/ヽ ίま 26— 221位の 196残基 力 ヒトオンコスタチン Μの成熟タンパク質に相当する。 [0021] Oncostatin M (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. It is also known that there is a mature protein consisting of 196 amino acid residues and having a molecular weight of about 22 kDa from which 31 amino acid residues on the C-terminal side have been removed. The physiological activity of these mature proteins is said to be 5 to 60 times higher than that of the precursor protein (Linsley, et al., 1 990, Mol. Cell. Biol. 10: 1882-1890). 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 佃 J force et al. 26— 227 residues at position 252,! ΊMA 26— 196 residues at position 221 force Human Oncostatin Μ mature protein It corresponds to.
[0022] オンコスタチン Μの前駆タンパク質と成熟タンパク質は、いずれも細胞に対して同様 の作用を持つ。したがって、本発明におけるオンコスタチン Μは、前駆タンパク質お よび成熟タンパク質のいずれ力、あるいは両方を利用することができる。しかし本発 明における好ま 、オンコスタチン Μは、より生理活性の高 、成熟型のオンコスタチ ン Μ (配列番号: 13における 26— 221位の 196アミノ酸)である。なお先に例示したォ ンコスタチン Μの培地中の濃度は、前駆タンパク質としての使用量である。したがつ て、もしも前駆タンパク質に代えて成熟タンパク質を用いる場合には、培地中におけ るオンコスタチン Μの濃度は、 0. 8ng/mL〜85ng/mL、好ましくは 4. 3ng/mL〜43ng /mL程度とすることができる。
[0023] 本発明におけるオンコスタチン Mは、前駆細胞として培養される間葉系幹細胞の成 熟肝細胞様細胞への分化を誘導する限り、その由来は限定されない。以下に、現在 までに明らかにされているオンコスタチン Mのアミノ酸配列を例示する。 [0022] Oncostatin Μ precursor protein and mature protein both have similar effects on cells. Therefore, oncostatin に お け る in the present invention can utilize either the precursor protein or the mature protein, or both. However, preferably, oncostatin に お け る in the present invention is a more physiologically active, mature oncostatin Μ (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. Therefore, if mature protein is used instead of precursor protein, the concentration of oncostatin ス タ チ ン in the medium is 0.8 ng / mL to 85 ng / mL, preferably 4.3 ng / mL to 43 ng. / mL or so. [0023] 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.
ヒトのオンコスタチン M : GenBank Accession No. AAA36388、 AAB21666, AAD3143 5、 NP— 003990 Human oncostatin M: GenBank Accession No. AAA36388, AAB21666, AAD3143 5, NP—003990
マウスのオンコスタチン M : GenBank Accession No. NP— 035707、 AAH13738 [0024] デキサメタゾン (Dexamethasone; 9-フルォロ- 11 β , 17,2 トリヒドロキシ- 16 a -メチル -プレダナ- 1 ,4-ジェン -3,20-ジオン; CAS Registration No. 50-02-2)は、ダルココル チコイド様の生理作用を有する合成ステロイドである。ステロイドホルモンと同様に、 受容体とともに核内に移行し、転写の調節に関与すると考えられている。収れん薬、 鎮痛薬、消炎薬、眼科用薬、かゆみ止め、消化器作用薬などとして臨床的に用いら れる化合物である。 Mouse Oncostatin M: GenBank Accession No. NP— 035707, AAH13738 [0024] Dexamethasone (Dexamethasone; 9-Fluoro-11 β, 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.
[0025] 本発明においては、デキサメタゾンに代えて、同様の作用を有するデキサメタゾン の誘導体を利用することもできる。たとえば、デキサメタゾンと酸のエステル力 デキサ メタゾンと同様の生理活性を持つことが知られている。たとえば次に示す誘導体また はその塩は、本発明におけるデキサメタゾンとして利用することができる。 In the present invention, a dexamethasone derivative having the same action can be used instead of dexamethasone. For example, it is known that dexamethasone and acid ester have the same physiological activity as dexamethasone. For example, the following derivatives or salts thereof can be used as dexamethasone in the present invention.
— 21-酢酸デキサメタゾン(CAS Registration No. 1177-87-3); — 21-dexamethasone acetate (CAS Registration No. 1177-87-3);
プロピオン酸デキサメタゾン(CAS Registration No.55541- 30- 5); Dexamethasone propionate (CAS Registration No.55541-30-5);
—リン酸デキサメタゾンナトリウム(CAS Registration No. 2392-39-4); —Dexamethasone Sodium Phosphate (CAS Registration No. 2392-39-4);
[0026] トランスフォーミング増殖因子 j8 (transforming growth factor- beta;TGF- j8 )は、二 量体構造を持つタンパク質で、哺乳類では構造の類似した 3種類のァイソフォームの 存在が知られている。これらのァイソフォームは、それぞれ 1、 β 2、および j8 3と呼 ばれている。以下、特に断らない限り、「TGF- j8」は、これらのァイソフォームの全て を含む用語として用いる。これらのァイソフォームは、いずれも本発明に利用すること ができる。以下に、現在までに明らかにされている TGF- |8のアミノ酸配列を例示する 。ヒト TGF- j8 1のアミノ酸配列 (GenBank Accession No. AAQ18641)を、配列番号: 14 に示した。配列番号: 14のアミノ酸配列中、 N末端側の 6残基を欠いた 1 12残基(7— 118位)がヒト TGF- jS 1の成熟タンパク質のアミノ酸配列である。配列番号: 14のアミ
ノ酸配列中、 N末端側 112残基のアミノ酸配列を含むタンパク質は、本発明における TGF- j8として好ましい。 [0026] 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, β 2 and j83, respectively. Hereinafter, unless otherwise specified, “TGF-j8” is used as a term including all of these isoforms. Any of these isoforms can be used in the present invention. In the following, 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. In the amino acid sequence of 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.
[0027] ヒト TGF- β 1 : GenBank Accession No. AAQ18641, NP— 000651、 AAA51458, AAL2 7646、 AAQ 18642, AAV71148, AAN86616、 AAT77144, AAT77143, AAX32228, PO 1137、 CAA29283, AAH01180、 AAH00125,および AAP35909 [0027] Human TGF-β 1: GenBank Accession No. AAQ18641, NP—000651, AAA51458, AAL2 7646, AAQ 18642, AAV71148, AAN86616, AAT77144, AAT77143, AAX32228, PO 1137, CAA29283, AAH01180, AAH00125, and AAP35909
ヒト TGF— j8 2 : GenBank Accession No. Y00083, M19154 Human TGF— j8 2: GenBank Accession No. Y00083, M19154
ヒト TGF— j8 3 : GenBank Accession No. J03241, X144149 Human TGF— j8 3: GenBank Accession No. J03241, X144149
マウス TGF- j8 l : GenBank Accession No. NP— 001013383、 AAH99866、および CAI2 5749 Mouse TGF-j8 l: GenBank Accession No. NP—001013383, AAH99866, and CAI2 5749
マウス TGF— j8 2 : GenBank Accession No. X57413 Mouse TGF— j8 2: GenBank Accession No. X57413
マウス TGF— j8 3 : GenBank Accession No. M32745 Mouse TGF— j8 3: GenBank Accession No. M32745
[0028] 本発明にお!/、て、オンコスタチン M並びに TGF- βは、前駆細胞として培養される 間葉系幹細胞の成熟肝細胞様細胞への分化を誘導する限り、それらの由来は限定 されない。特に TGF- |8のアミノ酸配列は、哺乳動物の間で高度に保存されているこ とが知られている。たとえば TGF- |8 1の場合、ヒトとマウスのアミノ酸配列の同一性は 99%である。そのため、一般に、 TGF- |8の種差はほとんど無視できると考えられて いる。ただし、本発明によって得ることができる成熟肝細胞様細胞をヒトの医療目的に 用いる場合には、ヒト TGF- 18を利用するのが好ましい。 [0028] In the present invention, Oncostatin M and TGF-β are limited in their origin as long as they induce differentiation of mesenchymal stem cells cultured as progenitor cells into mature hepatocyte-like cells. Not. In particular, the amino acid sequence of TGF-8 is known to be highly conserved among mammals. For example, in the case of TGF- | 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. However, 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.
[0029] 本発明におけるオンコスタチン M、並びに TGF- j8は、天然のものを用いることもで きるし、天然の分子と同様の活性を有する遺伝子組み換え体を利用することもできる 。当業者は、前記のアミノ酸配列情報、あるいはそれをコードする塩基配列情報に基 づいて、オンコスタチン Mや TGF- |8の遺伝子組み換え体を製造することができる。 [0029] For 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.
[0030] あるいは、オンコスタチン Mあるいは TGF- βの遺伝子組み換え体は、商業的にも 供給されている。たとえば、以下に示す企業は、これらのタンパク質の遺伝子組み換 え体を商業的に供給している。したがって、これらの市販の遺伝子組み換え体を本 発明に利用することもできる。 [0030] Alternatively, oncostatin M or TGF-β gene recombinants are commercially available. For example, the companies listed below supply genetically engineered products of these proteins. Therefore, these commercially available recombinants can also be used in the present invention.
ヒトゃマウスのオンコスタチン M (pre-proタンパク質) Onyastatin M (pre-pro protein) from humans and mice
United States Biological社、
ProSpec- Tany TechnoGene Ltd社など United States Biological, ProSpec-Tany TechnoGene Ltd etc.
ヒトゃマウスのオンコスタチン M (成熟タンパク質) Onyastatin M (mature protein) from humans and mice
R&D Systems Inc.社等 R & D Systems Inc.
ヒトゃマウスの TGF- β: TGF-β in humans and mice:
CHEMICON International, Inc社、 CHEMICON International, Inc,
Fitzgerald industries Intl.社の RDI Divison RDI Divison from Fitzgerald industries Intl.
これらの増殖分ィ匕因子は、一般に、間葉系幹細胞の分ィ匕に利用されている動物細 胞用の培地に加えることができる。本発明に利用することができる基礎培地は、通常 、無機塩類、糖類、アミノ酸類、およびビタミン類等を含む。基礎培地の代表的な組 成を以下に示す。 These growth factor can generally be added to the medium for animal cells used for the differentiation of mesenchymal stem cells. 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.
無機塩類: Inorganic salts:
Sodium Chloride 4000-8000 mg/L Sodium Chloride 4000-8000 mg / L
Potassium Chloride 200- 330 mg/L Potassium Chloride 200- 330 mg / L
Potassium Nitrate 0— 0.1 mg/L Potassium Nitrate 0— 0.1 mg / L
Sodium Selenite 0- 0.02 mg/L Sodium Selenite 0- 0.02 mg / L
Calcium Chloride, anhyd. 20— 165 mg/L Calcium Chloride, anhyd. 20— 165 mg / L
Magnesium Sulfate, anhyd. 0- 100 mg/L Magnesium Sulfate, anhyd. 0-100 mg / L
Sodium Phosphate, dibasic anhyd. 100— 250 mg/L Sodium Phosphate, dibasic anhyd. 100— 250 mg / L
糖類: Sugars:
Glucose (Dextrose) 1200-4500 mg/L Glucose (Dextrose) 1200-4500 mg / L
アミノ酸: Amino acids:
L- Alanine 5- 25 mg/L L- Alanine 5-25 mg / L
L-Arginine HC1 84- 450 mg/L L-Arginine HC1 84- 450 mg / L
L-Asparagine · H20 15- 30 mg/L L-AsparagineH20 15-30 mg / L
L- Aspartic Acid 10- 30 mg/L L- Aspartic Acid 10-30 mg / L
L- Cystine 2 Na salt 0- 90 mg/L L- Cystine 2 Na salt 0- 90 mg / L
L- Cysteine H20 0- 40 mg/L L- Cysteine H20 0- 40 mg / L
L— Glutamic Acid 10— 80 mg/L
Glycine 5- 30 mg/L L— Glutamic Acid 10— 80 mg / L Glycine 5-30 mg / L
L-Histidine HC1-H20 20— 50 mg/L L-Histidine HC1-H20 20— 50 mg / L
L- Isoleucine 2- 110 mg/L L- Isoleucine 2- 110 mg / L
L- Leucine 10- 110 mg/L L- Leucine 10- 110 mg / L
L- Lysine HCl 30- 150 mg/L L- Lysine HCl 30- 150 mg / L
L- Methionine 3- 30 mg/L L- Methionine 3- 30 mg / L
L- Phenylalanine 4- 80 mg/L L- Phenylalanine 4- 80 mg / L
L- Proline 30- 70 mg/L L- Proline 30- 70 mg / L
L- Serine 10- 50 mg/L L- Serine 10-50 mg / L
L-Threonine 10- 100 mg/L L-Threonine 10-100 mg / L
L- Tryptophan 2- 20 mg/L L- Tryptophan 2- 20 mg / L
L- Tyrosine 2 Na-2H20 5- 110 mg/L L- Tyrosine 2 Na-2H20 5- 110 mg / L
L- Valine 10- 100 mg/L L-Valine 10-100 mg / L
L-Glutamine 140- 600 mg/L ビタミン類など: L-Glutamine 140- 600 mg / L Vitamins:
Folic Acid 1- 5 mg/L Folic Acid 1-5 mg / L
Inositol 7- 20 mg/L Inositol 7-20 mg / L
Nicotinic Acid Amide 0.3- 5 mg/L Nicotinic Acid Amide 0.3-5 mg / L
Riboflavin 0.02— 0.4 mg/L Riboflavin 0.02— 0.4 mg / L
Thiamine HCl 0.3- 5 mg/L Thiamine HCl 0.3-5 mg / L
d-Biotin 0.007—0.08 mg/L d-Biotin 0.007—0.08 mg / L
Pantothenic Acid, Ca salt 0.2— 5 mg/L Pantothenic Acid, Ca salt 0.2— 5 mg / L
Pyruvic Acid, Na salt 100— 250 mg/LPyruvic Acid, Na salt 100— 250 mg / L
Vitamin B-12 0.01-2 mg/L Vitamin B-12 0.01-2 mg / L
Pyridoxine HCl 0.05—5 mg/L Pyridoxine HCl 0.05—5 mg / L
Choline Chloride 4- 15 mg/L 緩衝液など: Choline Chloride 4-15 mg / L buffer etc:
Phenol Red, Na salt 0— 15 mg/L
HEPES, acid form 0-6000 mg/L Phenol Red, Na salt 0— 15 mg / L HEPES, acid form 0-6000 mg / L
HEPES, 1 Na salt 0- 680 mg/L HEPES, 1 Na salt 0- 680 mg / L
Sodium Bicarbonate 1000-3500 mg/L Sodium Bicarbonate 1000-3500 mg / L
[0032] 本発明に利用する基礎培地には、一般的に基礎培地として市販されている培地組 成物を利用することができる。たとえば、次のような市販の培地を、本発明における基 礎培地に利用することができる。 [0032] As the basal medium used in the present invention, a medium composition generally marketed as a basal medium can be used. For example, the following commercially available media can be used as the basic media in the present invention.
IMDM培地(Iscove's Modified DMEM) (Sigma社,米国) IMDM medium (Iscove's Modified DMEM) (Sigma, USA)
F12K培地(F— 12 Nutrient Mixture (Ham's F12) Kaighn's Modification) (Invitroge n社)、 F12K medium (F-12 Nutrient Mixture (Ham's F12) Kaighn's Modification) (Invitrogen),
HCM培地(Cambrex社)等 HCM medium (Cambrex), etc.
基礎培地は、上記代表的な組成に加えて、更に付加的な成分を含むことができる。 たとえば、アルブミンや動物血清を加えることによって、細胞の発育支持能を改善す ることがでさる。 The basal medium can further contain additional components in addition to the representative composition. For example, adding albumin or animal serum can improve cell growth support.
[0033] 本発明にお!/、て、間葉系幹細胞は、オンコスタチン M、デキサメタゾン、および TGF - j8の存在下で培養することによって、成熟肝細胞様細胞に分化する。間葉系幹細 胞は、一般的な動物細胞の培養方法に従って培養することができる。具体的な培養 条件として、約 5%の COの雰囲気下、 37°C前後の温度を示すことができる。更に、 [0033] In the present invention, 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,
2 2
本発明においては、成熟肝細胞様細胞への分化を誘導するために、コラーゲンコー トされた培養容器を利用するのが有利である。市販のコラーゲンコーティングプレート (アサヒテクノガラス製)等を、本発明の方法に利用することができる。 In the present invention, it is advantageous to use 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.
[0034] 本発明にお 、て、分ィ匕誘導開始時の細胞数は、細胞の生存が支持され、かつ目的 とする分ィ匕を誘導できる範囲で適宜調節することができる。具体的には、たとえば、 5. 0 X 103〜5.0 X 106細胞/培養皿の細胞を播種することができる。 [0034] In 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. Specifically, for example, 5.0 × 10 3 to 5.0 × 10 6 cells / culture dish cells can be seeded.
[0035] このような条件で、たとえば 3日以上、通常 5日以上、好ましくは 6日以上、あるいは 10日以上培養することによって、間葉系幹細胞の成熟肝細胞様細胞への分化が誘 導される。成熟肝細胞の特徴とすることができる種々のマーカーが知られている。こ れらのマーカーを観察することによって、成熟肝細胞様細胞に分化している細胞を 確認することができる。本発明において、成熟肝細胞様細胞とは、たとえば以下の特
徴 (l)-(4)の少なくとも 1つを備える細胞を言う。特に、これらの指標のうち (2)および (3) の両方を有する細胞は、化合物のスクリーニングや、再生医療に利用するための細 胞として好ましい。あるいは、特徴 (4)を備える細胞は、肝炎ウィルスの感染実験に利 用する宿主細胞として有用である。 [0035] Under such conditions, for example, culturing for 3 days or more, usually 5 days or more, preferably 6 days or more, or 10 days or more induces differentiation of mesenchymal stem cells into mature hepatocyte-like cells. Is done. Various markers are known that can be characteristic of mature hepatocytes. By observing these markers, cells differentiated into mature hepatocyte-like cells can be confirmed. In the present invention, mature hepatocyte-like cells are, for example, the following special features: A cell with at least one of features (l)-(4). In particular, cells having both (2) and (3) among these indicators are preferred as cells for use in compound screening and regenerative medicine. Alternatively, the cell having the feature (4) is useful as a host cell used for hepatitis virus infection experiments.
[0036] (1)形態学的特徴: [0036] (1) Morphological features:
全体的に丸みを帯びた形態を有する; Has a generally rounded form;
細胞質に顆粒を含む; Containing granules in the cytoplasm;
明るい核と明瞭な核小体を有する Has bright nuclei and distinct nucleoli
上記の形態学的な特徴は、顕微鏡観察によって確認することができる。 The above morphological characteristics can be confirmed by microscopic observation.
[0037] (2)肝細胞に特徴的な遺伝子発現 [0037] (2) Gene expression characteristic of hepatocytes
(少なくとも 1種、好ましくは複数種類、望ましくは全て): (At least one, preferably multiple, preferably all):
アルブミン (ALB) ; Albumin (ALB);
チロシンアミノトランスフェラーゼ (TAT); Tyrosine aminotransferase (TAT);
トリプトファン 2, 3-ジォキシゲナーゼ(TD02) ; Tryptophan 2,3-dioxygenase (TD02);
シトクローム P450(CYP1A2, CYP3A4, CYP2D6); Cytochrome P450 (CYP1A2, CYP3A4, CYP2D6);
トランスサイレチン (TTR); Transthyretin (TTR);
多剤而性関連タンパク質 (MRP1、 MRP2、 MRP3) Multi-metabolic proteins (MRP1, MRP2, MRP3)
多剤耐性関連タンパク質 (MDR1、 MDR3) Multidrug resistance-related proteins (MDR1, MDR3)
上記の遺伝子は、各遺伝子に特異的なプライマーを利用して増幅することができる 。更に、各遺伝子に特徴的な塩基配列にハイブリダィズするプローブを組み合わせ て、増幅産物を特異的に検出する方法を組み合わせることができる。たとえば、 ATA C-PCRは、 PCRの増幅産物をプローブを使って検出するための代表的な方法である 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.
[0038] (3)機能的な特徴: [0038] (3) Functional features:
(少なくとも 1種、好ましくは複数種類、望ましくは全て): (At least one, preferably multiple, preferably all):
グルコース産生能; Glucose production ability;
アンモニア代 f會 ; Ammonia cost f 會;
アルブミン生産能;
尿素合成能 Albumin producing ability; Urea synthesis capacity
上記の各機能を確認するための方法は公知である。たとえば、グルコース生産能は 、グルコースォキシダーゼ法によって培養上清中のグルコースレベルを分析すること で確認できる。アンモニア代謝能は、改変インドフエノール法(Horn DB & Squire CR, Chim. Acta. 14: 185-194. 1966)によって、培養培地中のアンモニアレベルを分析 することで確認できる。アルブミン生産能は、血清アルブミン濃度を測定する方法に より、培養液中のアルブミン濃度を分析することで確認できる。また、尿素合成能は、 例えば Colorimetric assay (シグマ社)を使用して確認できる。 Methods for confirming each of the above functions are known. For example, 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).
[0039] (4)ウィルス感受性: [0039] (4) Virus susceptibility:
ヒト C型肝炎ウィルス感受性 Human hepatitis C virus susceptibility
肝細胞様細胞が、ヒト C型肝炎ウィルスに感染したことは、たとえば実施例に示す方 法によって確認することができる。すなわち細胞から回収された mRNAを铸型とする R T-PCRにより C型肝炎ウィルスの増殖を検出することができる。ヒト C型肝炎ウィルス の RNAを増幅することができるプライマーは公知である(T. Takeuch. et al. Real-Tim e Detection System for Quantification of Hepatitisし Virus Genome, astroenterolo gy 1999, 116:636-642) o It can be confirmed, for example, by the method shown in the Examples that hepatocyte-like cells are infected with human hepatitis C virus. In other words, 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
[0040] 一般に、初代正常ヒト培養肝細胞に近!、形態学的特徴および遺伝子発現プロファ ィルの両方を有する細胞は、肝細胞様細胞と呼ばれる。具体的には、たとえば、チト クロム P450 (CYP)、多剤耐性関連蛋白質 (MRP)、および多剤耐性 (MDR)蛋白質を発 現する細胞は、肝細胞様細胞 (hepatocyte-like cells)に含まれる。肝細胞様細胞の中 で、更に初代正常ヒト培養肝細胞に近い特徴を有する細胞は、特に成熟度の高い肝 細胞様細胞である。 [0040] In general, cells that are close to primary normal human cultured hepatocytes and have both morphological characteristics and gene expression profiles are called hepatocyte-like cells. Specifically, for example, cells expressing cytochrome P450 (CYP), multidrug resistance-related protein (MRP), and multidrug resistance (MDR) protein are included in hepatocyte-like cells. It is. Among the hepatocyte-like cells, cells having characteristics similar to those of primary normal human cultured hepatocytes are hepatocyte-like cells having particularly high maturity.
たとえば、前記特徴の (3)として記載した、肝臓の機能的な特徴を備えた細胞は、特 に成熟度の高い細胞と言うことができる。したがって、前記肝臓細胞の特徴 (2)-(4)を 備える肝細胞様細胞は、本発明における成熟肝細胞様細胞 (mature hepatocyte-like cells)に含まれる。本発明において、成熟肝細胞様細胞を特徴付ける (3)の機能的な 特徴を、少なくとも 1つ、好ましくは 2つ以上、より好ましくは全てを備える細胞は、本 発明における成熟肝細胞様細胞に含まれる。
[0041] 本発明にお 、て、間葉系幹細胞から成熟肝細胞様細胞への分化を誘導するため の増殖分化因子であるオンコスタチン M、デキサメタゾン、および TGF- j8は、間葉系 幹細胞から成熟肝細胞様細胞への分化を誘導するための試薬として有用である。す なわち本発明は、オンコスタチン M、デキサメタゾン、および TGF- |8を含む、間葉系 幹細胞から成熟肝細胞様細胞への分化誘導試薬に関する。 For example, 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. 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. In the present invention, 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. It is useful as a reagent for inducing differentiation into mature hepatocyte-like cells. That is, 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.
[0042] 本発明にお!/、て、オンコスタチン M、デキサメタゾン、および TGF- βは、基礎培地 に添加することによって、間葉系幹細胞から成熟肝細胞様細胞への分化を誘導する ための培地を調製することができる。本発明の成熟肝細胞様細胞への分化誘導試薬 には、先に記載した誘導培地における各増殖分化因子の濃度を与えるのに必要な 量のオンコスタチン Μ、デキサメタゾン、および TGF- βを配合することができる。たと えば培地 1Lあたりの処方として、次の量の増殖分ィ匕因子を配合することによって、本 発明の成熟肝細胞様細胞への分化誘導試薬とすることができる。 [0042] In the present invention, oncostatin M, dexamethasone, and TGF-β 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 Μ, dexamethasone, and TGF-β in an amount necessary to give the concentration of each growth differentiation factor in the induction medium described above. be able to. For example, 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.
オンコスタチン Μ: 1 μ g〜100 μ g、好ましくは 5 μ g〜50 μ g Oncostatin Μ: 1 μg to 100 μg, preferably 5 μg to 50 μg
デキサメタゾン: 0. ImM〜: LOmM、好ましくは 0. 5mM〜5mM Dexamethasone: 0. ImM ~: LOmM, preferably 0.5mM ~ 5mM
TGF- j8 : 0. 2 8〜20 8、好ましく【ま1 8〜10 8 TGF- j8: 0.2 8 to 20 8, preferably 1 to 10 8
[0043] 本発明の成熟肝細胞様細胞への分化誘導試薬には、更に付加的な成分を配合す ることもできる。たとえば、基礎培地に対して付カロ的に配合されることが多い L-グルタ ミンを本発明の分ィ匕誘導試薬に配合しておくことができる。その他、各種の抗生物質 や、付加的な栄養素などを配合することもできる。 [0043] The differentiation-inducing reagent for mature hepatocyte-like cells of the present invention may further contain additional components. For example, L-glutamine, which is often added calorically to the basal medium, can be added to the separation induction reagent of the present invention. In addition, various antibiotics and additional nutrients can be added.
[0044] また、本発明は、本発明の方法により製造された成熟肝細胞様細胞を提供する。 [0044] The present invention also provides mature hepatocyte-like cells produced by the method of the present invention.
本発明の方法により製造された成熟肝細胞様細胞の機能や形態は、従来の方法に より製造された肝細胞様細胞の機能や形態と比較して、ヒト成熟肝細胞に、より近い という特徴を有する。したがって、本発明の成熟肝細胞様細胞は、例えば医療分野 において有用である。より具体的には、本発明によって提供される成熟肝細胞様細 胞は、再生医療のためのツールとして有用である。 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. Have Therefore, 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.
[0045] 本発明によって得ることができる成熟肝細胞様細胞は、たとえば生体外での各種の 試験のために培養皿に播種される。あるいは、本発明の成熟肝細胞様細胞を体内に 注入することによって、肝臓組織を再構築することができる。肝臓組織の再構築によ
つて、肝疾患を治療することができる。 [0045] Mature hepatocyte-like cells obtainable by the present invention are seeded in a culture dish for various tests in vitro, for example. Alternatively, 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.
具体的には、分化後の細胞を酵素を含む溶液にて処理して試験管に詰め、穏やか な条件で細胞を回収することによって、本発明の成熟肝細胞様細胞が濃縮される。 細胞の酵素処理には、コラゲナーゼゃデイスパーゼなどを利用することができる。細 胞を回収するための穏やかな条件としては、比較的低速 (40x g〜100x g)の遠心分 離のような、物理的な、あるいは生化学的な操作を利用することができる。 Specifically, 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. 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).
各種の試験を目的として細胞を培養する場合には、回収された本発明の成熟肝細 胞様細胞を、適当な培養容器に撒くことができる。培養容器としては、 96穴プレート や 24穴プレートを利用される。あるいは生体中に細胞を投与する場合には、適当な 培養液や緩衝液に本発明の成熟肝細胞様細胞を浮遊して生体内に注入することが できる。細胞浮遊液は、経静脈的に、または経門脈的に投与することができる。ある いは、皮下投与や腹腔内投与によって、本発明の成熟肝細胞様細胞を生体に投与 することもできる。更に、生体親和的材料に包埋した成熟肝細胞様細胞を患者に移 植することにより、肝疾患を治療することもできる。生体親和性材料には、コラーゲン、 ポリウレタン等の公知の素材を利用することができる。 When cells are cultured for various tests, 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. Alternatively, when administering cells into a living body, 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. Alternatively, the mature hepatocyte-like cells of the present invention can be administered to a living body by subcutaneous administration or intraperitoneal administration. Furthermore, 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.
[0046] あるいは、本発明によって提供された成熟肝細胞様細胞を、人工肝臓として利用す ることができる。本発明における人工肝臓とは、生体中に移植して肝機能を補うものと 、生体外において患者体液と接触させることによって肝機能を補うものを含む。本発 明における人工肝臓は、成熟幹細胞様細胞を保持し、必要に応じて患者血液を成 熟幹細胞様細胞に接触させるための手段を備える。たとえば生体中に移植するため の人工肝臓は、たとえば血清透過性の細胞保持材料中に成熟肝細胞様細胞を保持 する。成体に移植された人工肝臓は、血清と接触して血液中の成分を代謝する。 [0046] Alternatively, 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. For example, 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.
[0047] あるいは、血液回路中に血清透過性の細胞保持材料中に成熟肝細胞様細胞を配 置することによって、成熟肝細胞様細胞に血清を接触させることもできる。すなわち本 発明は、生体外に取り出した血液を成熟肝細胞様細胞に接触後に、当該血液を患 者に戻す工程を含む、肝疾患の治療方法を提供する。本発明の治療方法において 、成熟肝細胞様細胞は、たとえば透析膜のような血清透過性の膜を介して、患者の 血液と接触させることができる。透析膜を透過した血清が成熟肝細胞様細胞と接触
することにより、血清中の成分は成熟肝細胞様細胞の作用によって代謝される。接触 後の血清は、透析膜を介して再び血流中に戻される。 Alternatively, 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. In the treatment method of the present invention, 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.
[0048] このように、本発明は上記工程により製造された成熟肝細胞様細胞の用途もまた提 供する。すなわち本発明は、本発明の方法によって得ることができる成熟肝細胞様 細胞を含む肝疾患の治療剤を提供する。また、当該成熟肝細胞様細胞を肝疾患を 有する患者に投与する工程を含む、肝疾患の治療方法を提供する。本発明の肝疾 患としては、成熟肝細胞様細胞による機能の補完が可能なあらゆる疾患が含まれる。 このような肝疾患としては、具体的には、肝硬変、劇症肝炎、胆道閉鎖症、肝癌、肝 炎が挙げられる。肝炎には、例えばウィルス性肝炎またはアルコール性肝炎などが 含まれる。 [0048] Thus, 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.
[0049] また本発明のヒト成熟肝細胞様細胞は、例えば肝疾患の治療を目的とした研究分 野においても有用である。例えば、人工肝臓の研究開発において、本発明の成熟肝 細胞様細胞を用いることができる。さらに、以下に述べるように、本発明の成熟肝細 胞様細胞は、医薬品や食品等の開発の分野においても有用である。具体的には、被 験化合物の代謝や肝毒性の評価、肝疾患治療剤、肝炎ウィルス感染阻害剤、また はウィルス性肝炎治療剤のスクリーニングに利用できる。 [0049] The human mature hepatocyte-like cells of the present invention are also useful in, for example, research fields aimed at treating liver diseases. For example, in the research and development of an artificial liver, the mature hepatocyte-like cell of the present invention can be used. Furthermore, as described below, 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.
[0050] 本発明の方法により製造されたヒト成熟肝細胞様細胞を利用することで、被験化合 物の代謝や肝毒性を評価することができる。すなわち本発明は、次の工程を含む、 被験化合物の肝における代謝を検出する方法であって、成熟肝細胞様細胞が、オン コスタチン M、デキサメタゾン、および TGF- |8の存在下で間葉系幹細胞を培養する ことによって、間葉系幹細胞から分化した成熟肝細胞様細胞である方法を提供する。 [0050] By using the human mature hepatocyte-like cells produced by the method of the present invention, the metabolism and hepatotoxicity of the test compound can be evaluated. That is, 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. By culturing stem cells, a method of mature hepatocyte-like cells differentiated from mesenchymal stem cells is provided.
(1)成熟肝細胞様細胞に被験化合物を接触させる工程、および (1) contacting the test compound with mature hepatocyte-like cells, and
(2)成熟肝細胞様細胞における被験化合物の代謝を検出する工程。 (2) A step of detecting metabolism of a test compound in mature hepatocyte-like cells.
[0051] あるいは本発明は、次の工程を含む、被験化合物の肝毒性を検出する方法であつ て、成熟肝細胞様細胞が、オンコスタチン M、デキサメタゾン、および TGF- |8の存在 下で間葉系幹細胞を培養することによって、間葉系幹細胞から分化した成熟肝細胞 様細胞である方法を提供する。 [0051] Alternatively, 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. By culturing mesenchymal stem cells, a method of mature hepatocyte-like cells differentiated from mesenchymal stem cells is provided.
(1)成熟肝細胞様細胞に被験化合物を接触させる工程、および
(2)成熟肝細胞様細胞の障害が検出されたときに被験化合物の肝毒性が検出され る工程。 (1) contacting the test compound with mature hepatocyte-like cells, and (2) A step in which hepatotoxicity of a test compound is detected when a disorder of mature hepatocyte-like cells is detected.
[0052] 本発明における被験化合物には、特に制限はない。例えば、生体異物、天然化合 物、有機化合物、無機化合物、タンパク質、ペプチドなどの単一化合物、並びに、化 合物ライブラリー、遺伝子ライブラリーの発現産物、細胞抽出物、細胞培養上清、発 酵微生物産生物、海洋生物抽出物、植物抽出物等が挙げられる。生体異物には、 生体にとって異物であるあらゆる生体異物が含まれる。例えば薬剤や食品の候補ィ匕 合物、既存の薬剤や食品が挙げられる。 [0052] The test compound in the present invention is not particularly limited. For example, 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.
[0053] 本発明にお 、て、被験化合物は、通常、培地や培養液に被験化合物を添加するこ とによって成熟肝細胞様細胞と接触させることができる。その他、成熟肝細胞様細胞 内で被験化合物をコードする遺伝子を発現させることによって、両者を接触させること ができる。あるいは、被験化合物を産生する細胞との共培養によって、被験化合物を 成熟肝細胞様細胞に接触させることもできる。 In the present invention, 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.
[0054] 被験化合物の代謝は、当業者に周知の方法で測定することが可能である。例えば 被験化合物の代謝産物が検出された場合に、被験化合物が代謝されたと判定され る。また、被験化合物の接触により、 CYP (チトクロム P450)、 MDR、 MRP等の酵素遺 伝子の発現が誘導された場合や、これら酵素の活性が上昇した場合に、被験化合物 が代謝されたと判定することもできる。 [0054] 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.
[0055] 代謝産物を検出するための方法は公知である。たとえば、成熟肝細胞様細胞の培 養物から被験化合物やその代謝産物を抽出し、液体クロマトグラフィーや質量分析 などによって分析することができる。予め代謝産物が予測されている場合には、当該 代謝産物の存在を、これらの分析方法によって確認することができる。あるいは、炭 酸ガスや水などへの代謝が予測される場合には、被験化合物として放射標識したィ匕 合物を利用すれば、放射活性を追跡することによって、炭酸ガスや水への代謝を確 認することができる。 [0055] Methods for detecting metabolites are known. For example, 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. When a metabolite is predicted in advance, the presence of the metabolite can be confirmed by these analysis methods. Alternatively, if metabolism to carbon dioxide gas or water is predicted, use of a radiolabeled compound as the test compound will allow metabolism to carbon dioxide or water by tracking radioactivity. It can be confirmed.
[0056] あるいは、 CYP、 MDR、並びに MRPなどの、薬物代謝に関連する遺伝子の発現を 指標にする場合には、これらの遺伝子の mRNAを検出することができる。 mRNAは、 R T-PCRなどの手法によって増幅し、検出することができる。これらの遺伝子の mRNA
の増幅と検出には、たとえば後に述べる実施例に記載されたような方法を利用するこ とができる。あるいは、 CYPの酵素活性を指標として、その発現を追跡することもでき る。 CYPの酵素活性を測定するための試薬が市販されて ヽる。 [0056] Alternatively, when the expression of genes related to drug metabolism such as CYP, MDR, and MRP is used as an index, 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.
[0057] 一方、肝毒性の評価にお!、ては、被験化合物を接触させた成熟肝細胞様細胞の 障害の程度を測定する。障害の程度は、例えばヒト成熟肝細胞様細胞の生存率や G OTや GPTなどの肝障害マーカーを指標に測定できる。 [0057] On the other hand, for evaluation of hepatotoxicity, 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.
[0058] 例えば、ヒト成熟肝細胞様細胞の培養液に被験化合物を添加することにより、ヒト成 熟肝細胞様細胞の生存率が低下する場合、該被験化合物は肝毒性を有すると判定 される。逆に、生存率に有意な変化がない場合、該被験化合物は肝毒性を有さない と判定される。また、例えば、ヒト成熟肝細胞様細胞の培養液に被験化合物を添加後 、培養液中の GOTや GPTが上昇する場合、該被験化合物は肝毒性を有すると判定 される。同様に、 GOTや GPTに有意な変化がない場合、該被験化合物は肝毒性を有 さないと判定される。 [0058] For example, if 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, the test compound is determined to have hepatotoxicity. . 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.
なお、すでに肝毒性の有無が判明している化合物を対照として用いることで、被験 化合物の肝毒性を定量的に評価することができる。 In addition, hepatic toxicity of the test compound can be quantitatively evaluated by using as a control a compound that is already known to have hepatotoxicity.
[0059] 被験化合物の代謝や肝毒性の評価には、従来、動物モデル等が用いられて!/、た 力 一度に評価できる被験化合物の数に制限があった。また動物モデル等で得られ た評価を、そのままヒトに適用できないという問題があった。そのため、ヒト肝がん細胞 株や初代正常ヒト培養肝細胞を用いる評価方法が採用されつつある。し力しながら、 ヒト肝がん細胞株はがん細胞であるため、ヒト肝がん細胞株で得られた評価が、ヒト正 常肝細胞に適用できないという可能性が残る。また、初代正常ヒト培養肝細胞は安定 供給やコストの面での問題がある。また、初代正常ヒト培養肝細胞を不死化した細胞 株は、不死化していない場合と比較して、 CYP3A4の活性が低下していることが示さ れて ヽる (International Journal of Molecular Medicine 14: 663—668, 2004, Akiyama I . et al.) 0本発明の方法により製造された成熟肝細胞様細胞を利用することで、この ような問題を解決しうる。 [0059] Conventionally, animal models have been used to evaluate the metabolism and hepatotoxicity of test compounds! /, Force There was a limit to the number of test compounds that could be evaluated at one time. In addition, there is a problem that the evaluation obtained with an animal model or the like cannot be applied to humans as it is. Therefore, evaluation methods using human hepatoma cell lines and primary normal human cultured hepatocytes are being adopted. However, since human liver cancer cell lines are cancer cells, there is a possibility that the evaluation obtained with human liver cancer cell lines cannot be applied to human normal hepatocytes. In addition, primary normal human cultured hepatocytes have problems in terms of stable supply and cost. In addition, cell lines in which primary normal human cultured hepatocytes have been immortalized have been shown to have reduced CYP3A4 activity compared to those that have not been immortalized (International Journal of Molecular Medicine 14: 663 —668, 2004, Akiyama I. et al.) 0 This problem can be solved by using mature hepatocyte-like cells produced by the method of the present invention.
[0060] また、本発明によって得ることができる成熟肝細胞様細胞は、肝疾患治療剤のスク リーニングに利用することができる。すなわち本発明は、次の工程を含む、肝疾患の
治療剤のスクリーニング方法であって、成熟肝細胞様細胞が、オンコスタチン M、デ キサメタゾン、および TGF- |8の存在下で間葉系幹細胞を培養することによって、間 葉系幹細胞から分化した成熟肝細胞様細胞である方法を提供する。 [0060] Moreover, 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- | 8. Methods are provided that are hepatocyte-like cells.
(1)成熟肝細胞様細胞に被験化合物を接触させる工程、 (1) contacting the test compound with mature hepatocyte-like cells,
(2)被験化合物を接触させた成熟肝細胞様細胞の機能を測定する工程;および (2) measuring the function of mature hepatocyte-like cells contacted with the test compound; and
(3)対照と比較して、成熟肝細胞様細胞の機能を亢進させる作用を有する化合物 を選択する工程。 (3) A step of selecting a compound having an action of enhancing the function of mature hepatocyte-like cells as compared with the control.
[0061] 本発明にお 、ては、被験化合物を接触させた成熟肝細胞様細胞にお!、て、細胞 機能の亢進が見られた場合に、被験化合物の肝臓に対する治療効果が検出される 。本発明のスクリーニング方法において、被験化合物は、先に記載した代謝や肝毒 性の評価における被験化合物と同様の操作によって、成熟肝細胞様細胞に接触さ せることができる。 [0061] In the present invention, 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. . In the screening method of the present invention, 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.
本発明における成熟肝細胞様細胞の機能は、例えば、グルコース産生能、アンモ ニァ代謝能、アルブミン生産能、尿素合成能、 CYP等の酵素の活性を指標に評価す ることがでさる。 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.
[0062] グルコース生産能は、グルコースォキシダーゼ法によって培養上清中のグルコース レベルを分析することで確認できる。アンモニア代謝能は、改変インドフエノール法( Horn DB & Squire CR, Chim. Acta. 14: 185-194. 1966)によって、培養培地中のァ ンモユアレベルを分析することで確認できる。アルブミン生産能は、血清アルブミン濃 度を測定する方法により、培養液中のアルブミン濃度を分析することで確認できる。 また、尿素合成能は、例えば Colorimetric assay (シグマ社)を使用して確認できる。 本発明の CYPは特に制限はないが、例えば CYP1A1、 CYP2C8、 CYP2C9、 CYP3A4 などが挙げられる。 CYPの活性測定方法は、当業者に周知の方法を使用することが できる。 [0062] 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. As a method for measuring CYP activity, methods well known to those skilled in the art can be used.
[0063] 本発明のスクリーニング方法において、被験化合物には、肝機能の改善作用を評 価すべき任意の化合物を用いることができる。具体的には、天然物質や人工的に合 成された化合物のライブラリーを被験化合物とすることができる。天然物質には、植 物、動物、昆虫、あるいは微生物など力 抽出された成分が含まれる。あるいは巿販
の化合物ライブラリーを、本発明の方法によってスクリーニングすることもできる。 [0063] In the screening method of the present invention, any compound for which the liver function improving action should be evaluated can be used as the test compound. Specifically, 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.
[0064] 本発明の方法により製造された成熟肝細胞様細胞の機能や形態は、成熟肝細胞 により近いため、肝炎ウィルスに感染しうる。したがって本発明の成熟肝細胞様細胞 は、肝炎ウィルス感染阻害剤のスクリーニング方法に利用することができる。すなわち 本発明は、次の工程を含む、肝炎ウィルスの感染阻害剤のスクリーニング方法であつ て、成熟肝細胞様細胞が、オンコスタチン M、デキサメタゾン、および TGF- |8の存在 下で間葉系幹細胞を培養することによって、間葉系幹細胞から分化した成熟肝細胞 様細胞である方法を提供する。 [0064] Since the function and morphology of mature hepatocyte-like cells produced by the method of the present invention are closer to mature hepatocytes, they can be infected with hepatitis virus. Therefore, 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.
(1)成熟肝細胞様細胞に被験化合物の存在下で肝炎ウィルスを接触させるか、ま たは成熟肝細胞様細胞に肝炎ウィルスを接触させた後に被験化合物を接触させる 工程、 (1) contacting the hepatitis virus with mature hepatocyte-like cells in the presence of the test compound, or contacting the test compound after contacting hepatitis virus with mature hepatocyte-like cells;
(2)成熟肝細胞様細胞への肝炎ウィルスの感染のレベルを測定する工程;および (2) measuring the level of hepatitis virus infection of mature hepatocyte-like cells; and
(3)対照と比較して、成熟肝細胞様細胞への肝炎ウィルスの感染レベルが低い化 合物を選択する工程。 (3) A step of selecting a compound having a lower infection level of hepatitis virus to mature hepatocyte-like cells than the control.
[0065] 本発明のスクリーニング方法によって、本発明の成熟肝細胞様細胞に感染するあら ゆるウィルスの感染阻害剤をスクリーニングすることができる。具体的には、 C型肝炎 ウィルス、 A型肝炎ウィルス、 B型肝炎ウィルスを対象とすることができる。これら肝炎 ウィルスは、株化されたものであってもよいし、肝炎ウィルス感染者カゝら直接単離され たものでもよい。また、精製された状態であってもよいし、クルードな状態 (例えば感 染者から得られた血清の状態)であってもよ 、。 [0065] By the screening method of the present invention, 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).
[0066] 成熟肝細胞様細胞における肝炎ウィルスの感染の有無は、例えば細胞中の肝炎ゥ ィルス量を指標に検査することができる。細胞中の肝炎ウィルス量は、例えば細胞中 の肝炎ウィルスの RNA量を指標に判定できる。肝炎ウィルスの RNA量は、定法に従つ て測定することができる。また、本発明者らが確立した方法によって測定してもよい (T . Takeuch. et al. Real-Time Detection System for wuantincation of Hepatitis C Viru s Genome. Gastroenterology 1999, 116:636-642)。 [0066] 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).
[0067] あるいは本発明によって得られる成熟肝細胞様細胞は、ウィルス性肝炎治療剤の スクリーニングにも有用である。すなわち本発明は、次の工程を含む、ウィルス性肝
炎の治療剤のスクリーニング方法であって、成熟肝細胞様細胞が、オンコスタチン M 、デキサメタゾン、および TGF- |8の存在下で間葉系幹細胞を培養することによって、 間葉系幹細胞から分化した成熟肝細胞様細胞である方法を提供する。 [0067] Alternatively, 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- | 8 Methods are provided that are mature hepatocyte-like cells.
(1)肝炎ウィルスを感染させた成熟肝細胞様細胞に被験化合物を接触させる工程 (1) A step of bringing a test compound into contact with a mature hepatocyte-like cell infected with hepatitis virus
(2)成熟肝細胞様細胞における肝炎ウィルスの増殖を測定する工程;および(2) measuring the growth of hepatitis virus in mature hepatocyte-like cells; and
(3)対照と比較して、肝炎ウィルスの増殖の阻害作用が検出された化合物を選択す る工程。 (3) A step of selecting a compound in which an inhibitory effect on the growth of hepatitis virus is detected compared to the control.
[0068] 本発明の肝炎ウィルスの増殖を阻害する化合物には、次のような化合物が含まれ る。 [0068] The compounds that inhibit the growth of the hepatitis virus of the present invention include the following compounds.
1)被験化合物を接触させてな 、場合と比較して、肝炎ウィルスの増殖を阻害するィ匕 合物、 1) a compound that inhibits the growth of hepatitis virus compared to the case without contact with the test compound,
2)肝炎ウィルスの増殖を完全に阻害する化合物、および 2) a compound that completely inhibits the growth of hepatitis virus, and
3)肝炎ウィルスを消失させる化合物 3) Compounds that eliminate hepatitis virus
肝炎ウィルスの増殖や消失は、細胞中の肝炎ウィルス量を測定することで検査する ことができる。 The growth and disappearance of hepatitis virus can be examined by measuring the amount of hepatitis virus in the cells.
[0069] 本発明の肝炎ウィルスの感染阻害剤のスクリーニング方法、あるいはウィルス性肝 炎治療剤のスクリーニング方法における被験化合物には、ウィルス感染阻害作用や ウィルス性肝炎治療作用を評価すべき任意の化合物を用いることができる。具体的 には、天然物質や人工的に合成されたィヒ合物のライブラリーを被験化合物とすること ができる。天然物質には、植物、動物、昆虫、あるいは微生物など力も抽出された成 分が含まれる。あるいは市販の化合物ライブラリーを、本発明の方法によってスクリー ニングすることちできる。 [0069] As a 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. Can be used. Specifically, 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. Alternatively, a commercially available compound library can be screened by the method of the present invention.
[0070] 先に述べたとおり、本発明によって得ることができる成熟肝細胞様細胞を宿主とし て、肝炎ウィルスを培養することができる。すなわち本発明は、成熟肝細胞様細胞に 肝炎ウィルスを感染させる工程を含む肝炎ウィルスの培養方法であって、成熟肝細 胞様細胞がオンコスタチン M、デキサメタゾン、および TGF- |8の存在下で間葉系幹 細胞を培養することによって、間葉系幹細胞力 分ィ匕した成熟肝細胞様細胞である
肝炎ウィルスの培養方法を提供する。本発明に基づく肝炎ウィルスの培養方法にお[0070] As described above, 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- | 8. It is a mature hepatocyte-like cell that has been mesenchymal stem cell cultivated by culturing mesenchymal stem cells A method for culturing hepatitis virus is provided. In the method for culturing hepatitis virus according to the present invention,
V、て、特に好まし 、ウィルスは C型肝炎ウィルスである。 V. Especially preferred, the virus is hepatitis C virus.
[0071] 本発明の肝炎ウィルスの培養方法は、既に単離されたウィルスの継代や増幅に有 用である。あるいは本発明を利用して、環境や患者に由来するサンプル力も肝炎ウイ ルスを分離することができる。たとえば、患者の血液試料をサンプルとして本発明のゥ ィルスの培養法を実施することによって、患者由来の肝炎ウィルスを分離することが できる。 [0071] The method for culturing hepatitis virus of the present invention is useful for passage or amplification of an already isolated virus. Alternatively, using the present invention, hepatitis viruses can also be separated by sample forces derived from the environment and patients. For example, 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.
[0072] 現在、肝炎ウィルスは、ウィルスの遺伝子の増幅によって検出されている。しかし遺 伝子増幅法では、ウィルスが感染能力を失っていても、遺伝子が存在している限り、 ウィルスとして検出されてしまう。一方、ウィルス感受性細胞を利用するウィルスの分 離方法にぉ ヽては、実際に感染性を維持したウィルスを分離することができる。 [0072] Currently, the hepatitis virus is detected by amplification of the gene of the virus. However, with 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. On the other hand, using a virus isolation method using virus-sensitive cells, it is possible to isolate a virus that has actually maintained infectivity.
[0073] これまでに報告された、 in vitroにおける C型肝炎ウィルスの感染モデルは、感染実 験に用いるには不十分であった。そのため、 in vitroで肝細胞を用いて、 C型肝炎ウイ ルスの感染阻害剤や肝炎治療剤を開発することは現実的ではなかった。また好適な ウィルスの感染モデルが確立されて ヽな 、ために、 C型肝炎ウィルスのライフサイク ルの研究が進展していないという現状があった。これに対し、本発明者らは、本発明 の成熟肝細胞様細胞が C型肝炎ウィルスに感染すること、その感染効率が非常に高 いことを見出した。この結果は、本発明の成熟肝細胞様細胞を用いることで、 C型肝 炎ウィルスの感染阻害剤や肝炎治療剤をスクリーニングできること、 C型肝炎ウィルス のライフサイクルの解明ができることを示して 、る。 [0073] The in vitro hepatitis C virus infection model reported so far has been insufficient for use in infection experiments. Therefore, it has not been practical to develop hepatitis C virus infection inhibitors or hepatitis treatment agents using hepatocytes in vitro. In addition, since a suitable viral infection model has been established, there has been no progress in research on the life cycle of hepatitis C virus. In contrast, the present inventors have found that the mature hepatocyte-like cells of the present invention are infected with the hepatitis C virus, and the infection efficiency is very high. This result indicates that by using the mature hepatocyte-like cells of the present invention, it is possible to screen for infection inhibitors and hepatitis treatment agents for hepatitis C virus, and to elucidate the life cycle of hepatitis C virus. .
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に 組み入れられる。 All prior art documents cited in the present specification are incorporated herein by reference.
実施例 Example
[0074] 〔実施例 1〕ヒト間葉系幹細胞の培養 [Example 1] Culture of human mesenchymal stem cells
Medipost Biomedical Research Institute社(韓国)より入手したヒト臍帯血由来間葉 系幹細胞を、 MF培地(TOYOBO社、日本)にて、加湿大気中 5%CO含有下、 37°C Human umbilical cord blood-derived mesenchymal stem cells obtained from Medipost Biomedical Research Institute (Korea) in MF medium (TOYOBO, Japan) containing 5% CO in a humidified atmosphere at 37 ° C
2 2
で組織培養用プラスチック皿上で培養し増殖させた。細胞は 2〜3日間に 1度の割合 で継代培養した。
一方、ヒト骨髄由来間葉系幹細胞 (Cambrex社、米国)は MSCG培地 (Cambrex社、 米国)を用いて上記と同様に培養し増殖させた。 And grown on a tissue culture plastic dish. Cells were subcultured once every 2-3 days. On the other hand, human bone marrow-derived mesenchymal stem cells (Cambrex, USA) were cultured and expanded in the same manner as described above using MSCG medium (Cambrex, USA).
[0075] 〔実施例 2〕ヒト間葉系幹細胞の分化誘導 [Example 2] Differentiation induction of human mesenchymal stem cells
〔2— 1〕培地の調製 [2-1] Preparation of medium
IMDM培地 (Sigma社,米国)に、次の成分をカ卩えて培地を調製した。培地の調製に 用いたオンコスタチン Mは、ヒトオンコスタチン Mを大腸菌で発現させて得られた組み 換え体(227アミノ酸残基力もなる成熟タンパク質)である。一方、 TGF- 18 1は、 CHO 細胞で発現させて得られたヒト組み換え TGF- |8 1 ( 112アミノ酸残基力もなる成熟タン パク質)である。 The following components were prepared in IMDM medium (Sigma, USA) to prepare a medium. 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. On the other hand, TGF-181 is a human recombinant TGF- | 81 (mature protein having a force of 112 amino acid residues) obtained by expressing in CHO cells.
1 μ Μデキサメタゾン(Dexamethasone) {Sigma社,米国), 1 μΜ Dexamethasone (Sigma, USA),
10ng/mlオンコスタチン M(OSM) {Peprotech社,米国), 10ng / ml Oncostatin M (OSM) (Peprotech, USA),
2ng/ml TGF- j8 1 {R&D社,米国 } , 2ng / ml TGF-j8 1 {R & D, USA},
ITS— A (Insulin, Transpiercing, Seleniumの混合液, Sigma社,米国), ITS—A (Insulin, Transpiercing, Selenium mixture, Sigma, USA),
L-glutamine (Sigma社,米国) , L-glutamine (Sigma, USA),
PSA (.penicillin, streptomycin, amphotericin Bの |&合揿, Invitrogen社,未国) 基礎培地として、上記 IMDM培地以外に、 F12K (Invitrogen社)あるいは HCM培地( Cambrex社)を使用しても分ィ匕誘導において同様な結果を得ることができる。以下、こ れらの培地を基礎培地に上記成分を加えた培地を、それぞれ次のように記載する。 PSA (.penicillin, streptomycin, amphotericin B | & go, Invitrogen, uncountry) In addition to the above IMDM medium, F12K (Invitrogen) or HCM medium (Cambrex) can also be used as the basal medium. Similar results can be obtained in wrinkle induction. Hereinafter, the media obtained by adding these components to the basal media are described as follows.
IMDMを基礎培地にしたもの: IDOT培地; IMDM in basal medium: IDOT medium;
F12Kを基礎培地にしたもの: FKDOT培地; F12K basal medium: FKDOT medium;
HCMを基礎培地にしたもの: HDOT培地 HCM as basal medium: HDOT medium
[0076] 〔2— 2〕分化誘導 [0076] [2-2] Differentiation induction
実施例 1にて培養したヒト臍帯血由来間葉系幹細胞を、 0.05%トリプシン · EDTA溶 液 (Sigma社,米国)によりプラスチック皿力も剥離した。細胞を洗浄の後、上記 IDOT培 地に懸濁し、 1cm2あたり 30000〜60000個の割合で I型コラーゲンコーティングプレート (アサヒテクノガラス社)上に播種した。プレートに播種した細胞を加湿大気中 5%CO 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
2 含有下、 37°Cで培養することにより細胞の分ィ匕を誘導した。なお、 IDOT培地の代わり に HDOT培地を用いても同様の結果を得ることができる。
実施例 1にて培養したヒト骨髄由来間葉系幹細胞にっ 、ても、 HDOT培地を用いて 上記と同様に分化を誘導した。なお、 HDOT培地の代わりに FKDOT培地又は IDOT 培地を用いても同様の結果を得ることができる。 2 Cell culture was induced by culturing at 37 ° C in the presence of 2. Similar results can be obtained by using HDOT medium instead of IDOT medium. For human bone marrow-derived mesenchymal stem cells cultured in Example 1, differentiation was induced in the same manner as described above using HDOT medium. Similar results can be obtained by using FKDOT medium or IDOT medium instead of HDOT medium.
[0077] 〔実施例 3〕ヒト間葉系幹細胞由来肝臓細胞様細胞の形態学的観察 [Example 3] Morphological observation of hepatocyte-like cells derived from human mesenchymal stem cells
ヒト臍帯血由来間葉系幹細胞、およびヒト骨髄由来間葉系幹細胞を分化させて得ら れた細胞を顕微鏡観察した。未分化なヒト間葉系幹細胞や分化誘導因子を入れて ヽ ない培地で培養した細胞は、線維芽細胞様の細長い形態を示した。一方、デキサメ タゾン(Dexamethasone)、オンコスタチン M(OSM)、および TGF- j8 1を入れた培地で 培養した細胞は、培養開始後 2日で既に全体的に丸みを帯びた形態を示す傾向を 示した。培養開始後 1週間までに細胞質に顆粒を伴い、明るい核と明瞭な核小体を もつ肝細胞様形態となり、その後顆粒は経時的に増カロしていった。このことは分化誘 導因子を入れた培地で培養した細胞は形態上、高!ヽ効率で肝細胞様に分化して 、 ることを示して ヽる。 Cells obtained by differentiating human umbilical cord blood-derived mesenchymal stem cells and human bone marrow-derived mesenchymal stem cells were observed under a microscope. Undifferentiated human mesenchymal stem cells and cells cultured in a medium containing no differentiation inducer showed a fibroblast-like elongated shape. On the other hand, cells cultured in a medium containing dexamethasone (Dexamethasone), oncostatin M (OSM), and TGF-j81 showed a tendency to be completely rounded 2 days after the start of culture. It was. By 1 week after the start of the culture, granules in the cytoplasm became hepatocyte-like with bright nuclei and clear nucleolus, and then the granules increased over time. This indicates that cells cultured in a medium containing a differentiation-inducing factor differentiate in a hepatocyte-like manner with high morphological efficiency.
[0078] 図 1において、ヒト臍帯血由来及び骨髄由来の間葉系幹細胞から肝細胞様細胞へ の形態変化を示す。 FIG. 1 shows the morphological change from human umbilical cord blood-derived and bone marrow-derived mesenchymal stem cells to hepatocyte-like cells.
図 1の Aは未分ィ匕のヒト臍帯血由来間葉系幹細胞の形態を示しており、線維芽細 胞様の形態をしている。 Bは分ィ匕誘導開始 5日目のヒト臍帯血由来間葉系幹細胞の 形態を示しており、全体的に丸みを帯びた形態をしていることが分かる。 Cは分化誘 導開始 2日目のヒト骨髄由来間葉系幹細胞の形態を示しており、骨髄由来の間葉系 幹の場合も 2日目で既に細胞は丸みを帯び始めていることが分かる。 Dは分化誘導 開始 9日目のヒト骨髄由来間葉系幹細胞の形態を示しており、 Bと同様に全体的に丸 みを帯びた形態をしていることが分かる。また核が白く抜け、核小体がはっきりしてい るのが分かる。 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.
[0079] 図 2において、ヒト骨髄由来間葉系幹細胞を肝細胞様細胞へ分化誘導させた 5日 目の形態を示す。図 2の IDOT、 HDOT,および FKDOTは、分化因子を添カ卩した基礎 培地の違いを表している。左が弱拡像 (χ100)、右が強拡像 (x200)である。いずれの培 地で培養した場合も、分ィ匕開始 5日目で全体的に丸みを帯びた形態をしていること が分かる。また強拡像において、核が白く抜け、核小体がはっきりしているのが分か
る。 [0079] 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 (χ100), 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
[0080] 〔実施例 4〕遺伝子発現解析 [Example 4] Gene expression analysis
〔4 1〕遺伝子発現解析の実施対象 [4 1] Target of gene expression analysis
ヒト間葉系幹細胞をデキサメタゾン (Dexamethasone)、オンコスタチン M(OSM)、およ び TGF- iS 1を添加した培地で培養した細胞の遺伝子発現を、 PCR法で解析した。発 現を解析した遺伝子は次のとおりである。 Gene expression of cells cultured in human mesenchymal stem cells cultured in a medium supplemented with dexamethasone (Dexamethasone), oncostatin M (OSM), and TGF-iS 1 was analyzed by PCR. The genes analyzed for expression are as follows.
ァノレブミン (ALB) ; Anolebumin (ALB);
チロシンアミノトランスフェラーゼ (TAT); Tyrosine aminotransferase (TAT);
トリプトファン 2,3-ジォキシゲナーゼ(TD02) ; Tryptophan 2,3-dioxygenase (TD02);
シトクローム P450(CYP1A2, CYP3A4, CYP2D6) ; Cytochrome P450 (CYP1A2, CYP3A4, CYP2D6);
トランスサイレチン (TTR);および Transthyretin (TTR); and
β -ァクチン β-actin
[0081] 〔4 2〕解析方法 [0081] [4 2] Analysis method
まず、未分化のヒト臍帯血由来間葉系幹細胞およびヒト臍帯血由来間葉系幹細胞 から分化誘導して得られた肝細胞様細胞から、 ISOGEN溶液 (二ツボンジーン、 日本) を用いて単離された総 RNAを、 DNasel (増幅グレード試薬;タカラ、京都、 日本)で処 理した。 RT- PCR反 J j は、 superscript II Reverse Transcriptase (Invitrogen,米国)を 用いて行った。全て単一バンドの cDNA断片として増幅し、分ィ匕マーカーの同定は特 異プライマーを用いて PCR法より確認した。 RT-PCRに使用した PCRプライマーは次 の通りである。またシトクローム P450(CYP1A2, CYP3A4, CYP2D6)遺伝子のプライマ 一は、タカラバイオ社の Rea卜 time PCR用のプライマーを使用した。 First, it was isolated from undifferentiated human umbilical cord blood-derived mesenchymal stem cells and hepatocyte-like cells obtained by induction of differentiation from human umbilical cord blood-derived mesenchymal stem cells using an ISOGEN solution (Nitsubon Gene, Japan). The total RNA was treated with DNasel (amplification grade reagent; Takara, Kyoto, Japan). 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.
[0082] ァノレブミン (ALB) [0082] Anolebumin (ALB)
5 -GTC ACC AAA TGC TGC ACA GA- 3'/配列番号: 1 5 -GTC ACC AAA TGC TGC ACA GA-3 '/ SEQ ID NO: 1
5 -ACG AGC TCA ACA AGT GCA GT- 3'/配列番号: 2 5 -ACG AGC TCA ACA AGT GCA GT-3 '/ SEQ ID NO: 2
トリプトファン 2,3-ジォキシゲナーゼ (TD02) Tryptophan 2,3-dioxygenase (TD02)
5 -CTG AAG AAA AAG AGG AAC AG- 3'/配列番号: 3 5 -CTG AAG AAA AAG AGG AAC AG-3 '/ SEQ ID NO: 3
5 -TCT GTG CAC CAT GCA CAC AT- 3'/配列番号: 4 5 -TCT GTG CAC CAT GCA CAC AT-3 '/ SEQ ID NO: 4
チロシン アミノトランスフェラーゼ (TAT)
5 -TGA GCA GTC TGT CCA CTG CCT- 3'/配列番号: 5 Tyrosine aminotransferase (TAT) 5 -TGA GCA GTC TGT CCA CTG CCT-3 '/ SEQ ID NO: 5
5 -ATG TGA ATG AGG AGG ATC TGA G- 3'/配列番号: 6 5 -ATG TGA ATG AGG AGG ATC TGA G-3 '/ SEQ ID NO: 6
トランスサイレチン (TTR) (プレアルブミン、アミロイド一シスタイプ 1) Transthyretin (TTR) (prealbumin, amyloid-cis type 1)
5 -TAC TGG AAG GCA CTT GGC AT- 3'/配列番号: 7 5 -TAC TGG AAG GCA CTT GGC AT-3 '/ SEQ ID NO: 7
5 -TTC CTT GGG ATT GGT GAC GA- 3'/配列番号: 8 5 -TTC CTT GGG ATT GGT GAC GA-3 '/ SEQ ID NO: 8
P450 7A1 P450 7A1
5 -AGG ACG GTT CCT ACA ACA TC- 3'/配列番号: 9 5 -AGG ACG GTT CCT ACA ACA TC-3 '/ SEQ ID NO: 9
5 -CGA TCC AAA GGG CAT GTA GT- 3'/配列番号: 10 5 -CGA TCC AAA GGG CAT GTA GT-3 '/ SEQ ID NO: 10
βァクチン β-actin
5 -CAA GAG ATG GCC ACG GCT GCT- 3'/配列番号: 11 5 -CAA GAG ATG GCC ACG GCT GCT-3 '/ SEQ ID NO: 11
5 -TCC TTC TGC ATC CTG TCG GCA- 3'/配列番号: 12 5 -TCC TTC TGC ATC CTG TCG GCA-3 '/ SEQ ID NO: 12
[0083] [4- 3]解析結果 [0083] [4- 3] Analysis results
ヒト臍帯血由来間葉系幹細胞をデキサメタゾン(Dexamethasone) ,才ンコスタチン M (OSM), TGF- |8 1存在下で分ィ匕培養することにより、培養開始後 6日で既に肝細胞特 異的遺伝子 (アルブミン、 TD02, TAT )の発現が観られることを見出している。一方、 デキサメタゾン(Dexamethasone) ,オンコスタチン M(OSM), TGF- j8 1陰性培地で処 置した細胞においては、 TD02, TAT遺伝子発現は出現しなかった。このことはデキ サメタゾン(Dexamethasone) ,オンコスタチン M(OSM), TGF- j8 1存在下で培養した細 胞は遺伝子発現レベルでも高 、効率で肝細胞様に分ィ匕して 、ることを示して 、る。 培養開始後 6日後の RT-PCRの解析結果を図 3に示す。図 3において、レーン 1はヒ ト臍帯血由来の間葉系幹細胞、レーン 2は分化 6日後の細胞の解析結果を示し、そ れぞれ Aが 13 -ァクチン、 Bがアルブミン、 Cが TAT, Dが TD02の RT-PCRの解析結 果である。分ィ匕誘導 6日目の細胞において、すでに肝臓細胞特異的な分子であるァ ルブミン、 TAT, TD02遺伝子発現が確認された。未分化のヒト臍帯血由来の間葉 系幹細胞では、それら遺伝子の発現は確認されなかった。 By culturing human cord blood-derived mesenchymal stem cells in the presence of dexamethasone (Dexamethasone), aged ncostatin M (OSM), TGF- | 8 1 It has been found that expression of (albumin, TD02, TAT) is observed. On the other hand, TD02 and TAT gene expression did not appear in cells treated with dexamethasone (Dexamethasone), oncostatin M (OSM), or TGF-j81-negative medium. This indicates that cells cultured in the presence of dexamethasone (Dexamethasone), oncostatin M (OSM), and TGF-j81 are highly expressed at high gene expression levels and are efficiently separated into hepatocytes. And Figure 3 shows the results of RT-PCR analysis 6 days after the start of culture. In Fig. 3, 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. In the cells on day 6 after induction, the expression of 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.
[0084] 培養開始後 12日後の RT-PCRの解析結果を図 4に示す。図 4において、レーン 1は 未分化のヒト骨髄由来の間葉系幹細胞、レーン 2はヒト骨髄由来の間葉系幹細胞の 分化 12日目の細胞、レーン 3はレーン 2とは異なるドナーのヒト骨髄由来の間葉系幹
細胞の分化 12日目、レーン 4はヒト臍帯血由来の間葉系幹細胞の分化 12日目、レ ーン 5はレーン 4とは異なるドナーのヒト臍帯血由来の間葉系幹細胞の分化 12日目、 レーン 6は未分化のヒト臍帯血由来の間葉系幹細胞のそれぞれ RT-PCRの解析結果 である。 [0084] Fig. 4 shows the results of RT-PCR analysis 12 days after the start of culture. In FIG. 4, 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.
ヒト骨髄由来の間葉系幹細胞においても分ィ匕誘導 12日目で、肝臓細胞特異的な 分子であるアルブミン、 TD02の遺伝子発現が確認された。ドナーが異なるヒト臍帯 血由来の間葉系幹細胞力 分ィ匕誘導された、どちらの肝細胞様細胞においてもアル ブミンの発現増強及び肝臓細胞特異的な分子である TTRの遺伝子発現が確認され た。また分化誘導細胞において CYP3A4の発現誘導、 CYP2D6の発現増強が認めら れた。 In human bone marrow-derived mesenchymal stem cells, expression of albumin, TD02, a liver cell-specific molecule, was confirmed on the 12th day after induction of differentiation. Increased expression of albumin and gene expression of TTR, a liver cell-specific molecule, were observed in both hepatocyte-like cells induced by mesenchymal stem cells from different human umbilical cord blood donors . Furthermore, CYP3A4 expression induction and CYP2D6 expression enhancement were observed in differentiation-inducing cells.
[0085] 〔実施例 5〕分化誘導した幹細胞の CYP活性測定 [0085] [Example 5] Measurement of CYP activity of differentiation-induced stem cells
[5- 1] CYP活性の測定 [5-1] Measurement of CYP activity
ヒト間葉系幹細胞をデキサメタゾン(Dexamethasone) ,オンコスタチン M(OSM), TGF - j8 1を入れた培地で培養した細胞の CYP活性を GLO法 (プロメガ社)により測定した 。活性を測定した CYPは、 CYP3A4、および CYP1A2である。 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.
[0086] 〔5— 2〕測定方法 [0086] [5-2] Measuring method
分ィ匕誘導を行って得られた細胞をコラゲナーゼ /ディスパーゼを用いてプレートより 剥がし、浮遊状態の 1 X 105細胞に対して 50 μ Lの発光基質を含む HCM培地を加え た。また分ィ匕誘導を行わない細胞はプレート上で培養し、プレート中の培地を吸引し 、 HCM培地で 1回洗浄した後、発光基質を含む HCM培地を加えた。発光基質を含む HCM培地は、 24 well plateの場合 200 μ Lを、また 96 well plateの場合 50 μ Lを加え た。測定に用いた発光基質は次のとおりである。 Cells obtained by induction were separated from the plate using collagenase / dispase, and HCM medium containing 50 μL 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 μL for a 24-well plate and 50 μL for a 96-well plate. The luminescent substrate used for the measurement is as follows.
P450-Glo™ CYP1A2 Assay (Pr omega Cat.# V8772)、または P450-Glo ™ CYP1A2 Assay (Pr omega Cat. # V8772), or
P450-Glo™ CYP3A4 Assay (Pr omega Cat.# V8801) P450-Glo ™ CYP3A4 Assay (Pr omega Cat. # V8801)
プレートを 37°Cで 4時間インキュベートし、反応終了後、細胞培養上清の一部(50 L)を発光(Luminescence)測定用の white multi-well plateに移した。さらに、等量のル シフェリン検出試薬(luciferin detection reagent)を加え、 10秒間撹拌した。室温で 20 分間反応させた後、相対発光量(RLU)を、プレートリーダーにより測定 '記録した。な
お、活性測定をする際にはサンプルと同様に 37°C, 4時間インキュベートした発光基 質も測定し、これを細胞非存在下のノックグラウンド値として測定値の補正を行った。 測定実験は、分ィ匕細胞を播種した培養用培地入りのプレートに CYPの誘導剤 (CYP 1A2: Omeprazole CYP3A4: Rifampicin)を添カ卩し、 1—3日培養後、上記の方法にて CYP活性を測定することにより実施した。 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. In the measurement experiment, 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.
[0087] 〔5— 3〕測定結果 [0087] [5-3] Measurement results
ヒト臍帯血由来間葉系幹細胞を〔2— 2〕の方法で分ィ匕誘導を行い、経時間的に CY P3A4の活性を測定した。図 5に結果を示す。図 5において、分化誘導日数に依存的 に CYP3A4活性の上昇が確認された。 Human cord blood-derived mesenchymal stem cells were induced by the method [2-2] and the activity of CYP3A4 was measured over time. Figure 5 shows the results. In FIG. 5, an increase in CYP3A4 activity was confirmed depending on the number of days of differentiation induction.
図 6は、 CYP3A4活性のヒト初代培養肝臓細胞との比較を示す。レーン Aはヒト骨髄 由来間葉系幹細胞の分化 34日目の細胞、レーン Bはヒト初代培養肝臓細胞 (第一化 学薬品; # 77)、レーン Cはヒト臍帯血由来の間葉系幹細胞の分化 23日目、レーン D はヒト臍帯血由来の間葉系幹細胞の分化 20日目、レーン Eはレーン C, Dとは異なる ドナーのヒト臍帯血由来の間葉系幹細胞の分化 20日目のそれぞれ CYP3A4の活性 を示す。 Aのヒト骨髄由来間葉系幹細胞の分ィ匕細胞、 Cのヒト臍帯血由来の間葉系 幹細胞の分ィ匕細胞の CYP3A4活性は、ヒト初代培養肝臓細胞とほぼ同等の活性を有 していた。 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 Each shows CYP3A4 activity. 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.
[0088] 次にヒト臍帯血由来間葉系幹細胞を〔2— 2〕の方法で分ィ匕誘導した細胞の CYPの 誘導能を検討した。 CYP3A4の誘導剤としてリファンピシン (RIF)を使用した(M. Yueh , M. Kawahara, J. Raucy, Drug Metab. Dispos. 33, 38—48 (2005》。 [0088] Next, the CYP induction ability of cells obtained by separating human cord blood-derived mesenchymal stem cells by the method [2-2] was examined. Rifampicin (RIF) was used as a CYP3A4 inducer (M. Yueh, M. Kawahara, J. Raucy, Drug Metab. Dispos. 33, 38-48 (2005)).
図 7に CYP3A4の誘導能の検討の結果を示す。ヒト臍帯血由来間葉系幹細胞を〔2 2〕の方法で分ィ匕誘導した肝細胞様細胞について、リファンピシン 1, 3, 10 /z Mを含 む培地により 2日間〜 3日間処理を行い、〔5— 2〕の方法にて CYPの活性を測定した 。図 7の Aは分化誘導 62日目、 Bは 17日目の細胞の活性を示す。図 7から明らかな ように、 CYP3A4の活性はリファンピシンの濃度依存的に増大した。リファンピシン 10 μ Μで 3日間曝露した場合には未誘導時に比べ約 2.3倍に増大した (図 7Β)。 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. As is apparent from FIG. 7, CYP3A4 activity increased in a rifampicin concentration-dependent manner. When rifampicin was exposed to 10 μΜ for 3 days, it increased approximately 2.3 times compared to the case without induction (Fig. 7Β).
[0089] 図 8に CYP1A2の誘導能の検討の結果を示す。ヒト骨髄由来間葉系幹細胞を〔2— 2〕の方法で分化誘導した肝細胞様細胞 (分化誘導 25日目)につ ヽて、オメブラゾー
ル (OPZ)5, 15, 50 /z Mを含む培地により 2日間処理を行い、〔5— 2〕の方法にて CYP の活性を測定した。図 8から明らかなように、 CYP1A2の活性はオメブラゾール (OPZ) の濃度依存的に増大した。このことは、本発明の方法により得られたヒト肝細胞様細 胞が初代正常ヒト培養肝細胞と同様にヒト CYP450酵素の誘導を起したことを示してお り、本発明のヒト肝細胞様細胞力 薬剤のスクリーニングに利用できることを示唆して いる。 [0089] FIG. 8 shows the results of an examination of the CYP1A2 inducibility. Regarding 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]. As is clear from FIG. 8, the activity of CYP1A2 increased depending on the concentration of omebrazole (OPZ). This indicates that 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.
[0090] 〔実施例 6〕分ィ匕誘導したヒト臍帯血由来の間葉系幹細胞力 得た肝細胞様細胞のグ ルコース代謝機能分析 [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
〔6— 1〕実験方法 [6-1] Experimental method
〔2— 2〕で得た肝細胞様細胞の生化学的機能を分析する為に、まずインスリンによ つて処理した肝細胞様細胞の培養上清中のグルコース濃度について検討を行なつ た。 In order to analyze the biochemical function of hepatocyte-like cells obtained in [2-2], we first examined the glucose concentration in the culture supernatant of hepatocyte-like cells treated with insulin.
得られた肝細胞様細胞を 96wellプレートに 2.0X104/wellずつ播種し、およそ 24時間 後に PBSで洗浄し、その後それぞれの濃度のインスリンを添加し、 48時間後の培養上 清中のグルコース濃度を測定した。なお、グルコース濃度についてはグルコース測定 用 グルコース Cn-テストヮコー(ムタロターゼ 'GOD法)(和光純薬工業株式会社)を 使用し分光高度計を用いて得た結果力 グルコース濃度を求めた。 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. As for 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.
〔6— 2〕実験結果 [6-2] Experimental results
図 9に示すようにそれぞれの培養液中にインスリンを添カロすることにより、インスリン の濃度依存性にグルコース濃度の低下を認めた。 As shown in FIG. 9, a decrease in glucose concentration was observed depending on the concentration of insulin by adding insulin to each culture solution.
[0091] 〔実施例 7〕ヒト臍帯血由来間葉系幹細胞から分化誘導して得られた肝細胞様細胞の C型肝炎ウィルス感染実験 [Example 7] Hepatitis C virus infection experiment of hepatocyte-like cells obtained by inducing differentiation from human umbilical cord blood-derived mesenchymal stem cells
〔7— 1〕実験方法 [7-1] Experimental method
コラーゲンあるいはマトリゲルをコートした 12穴と 6穴の細胞培養プレートにヒト臍帯 血由来間葉幹細胞から分化誘導して得られた肝細胞様細胞を播種した。細胞が充 分に着床した後、 Williams E培地で 1回洗浄した。この細胞に感染性 HCVが存在し ていることが確認されている HCV感染者血清を接種した。接種 HCV量は、細胞あたり の HCV遺伝子量として、それぞれ 0. 5コピー及び 1. 0コピーとした。 37度の COイン
キュベータで 3時間ウィルスを細胞に吸着後、 Williams E培地で 3回洗浄し、未吸着 の HCVを除去した。 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.
これに肝細胞様細胞の培養液を添カ卩し、 37°Cの COインキュベータで培養した。ゥ This was supplemented with a culture solution of hepatocyte-like cells and cultured in a 37 ° C CO incubator. U
2 2
ィルス培養開始後、 1から 10日間にわたり毎日細胞を採取した。プレートから培養液 を除去し、 5M濃度の塩酸グァ-ジンを添加することにより細胞を採取した。塩酸グァ 二ジンで溶解した細胞液は、 HCV-RNAを抽出するまで—80°Cに保存した。塩酸グ ァ-ジンで溶解した細胞液から定法に従って RNAを抽出し、リアルタイム PCR法に より HCV-RNA量を定量した。 HCV-RNA量は、本発明者らが確立し、報告した方法 により疋量した (T. Takeuch. et al. Real-Time Detection System for wuantincation of Hepatitis C Virus Genome. Gastroenterology 1999, 116:636—642)。 Cells were harvested daily for 1 to 10 days after the start of virus culture. The culture solution was removed from the plate, and cells were harvested by adding 5 M guanidine hydrochloride. The cell solution dissolved with guanidine hydrochloride was stored at −80 ° C. until HCV-RNA was extracted. RNA was extracted from the cell solution dissolved in guanidine hydrochloride according to a conventional method, and the amount of HCV-RNA was quantified by real-time PCR. The amount of HCV-RNA was determined by the method established and reported by the present inventors (T. Takeuch. Et al. Real-Time Detection System for wuantincation of Hepatitis C Virus Genome. Gastroenterology 1999, 116: 636-642 ).
[0092] 〔7— 2〕実験結果 [0092] [7-2] Experimental results
図 10に C型肝炎ウィルス(以下 HCV)感染後の時間と HCV— RNA量の関係を示す 。ヒト臍帯血由来間葉系幹細胞から分化誘導した肝細胞様細胞を用いて HCVの感 染培養を行った結果、ウィルスの感染および増殖が確認された。この事実から、本発 明の方法で得られる成熟肝細胞様細胞は、 HCV感染阻害剤や HCV増殖阻害剤の スクリーニングに利用できることが確かめられた。 産業上の利用可能性 Figure 10 shows the relationship between the time after hepatitis C virus (HCV) infection and the amount of HCV-RNA. As a result of 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. Industrial applicability
[0093] 本発明は、 in vitroにおいて、間葉系幹細胞力 肝細胞様細胞への分ィ匕を誘導しう る方法を提供した。本発明は、幹細胞の肝細胞への分化機構を分子レベルで解明 するツールとして有用である。また本発明によって、成熟度の高い肝細胞様細胞が 提供された。本発明によって得ることができる成熟肝細胞様細胞は、肝臓に対する化 合物の影響の評価に利用することができる。従来は、生体に対する化合物の影響の 研究(実験)は、しばしば生体を利用して行われていた。たとえば、発がん実験、ある いは食品添加物ゃ抗がん剤などの安全評価試験には、実験動物が利用される。本 発明の成熟肝細胞様細胞は、これらの試験方法において、実験動物に代わる新しい アツセィモデルとして利用することができる。 [0093] 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. In the past, 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.
[0094] 例えば、毒性評価試験や食品安全性評価試験などは多くの場合、ラットを用いた 検査が主流である。動物を使った検査には、必然的に動物の飼育スペースなどが必
要である。そのため、一度に評価できる化合物の数はスペースに制約される。また動 物試験においては、通常、ラットで得られた結果に基づいて、ヒトに対する影響が予 測される。このとき行われる予測は、特に外揷 (extrapolation)と呼ばれる。しかしヒトと ラットでは動物種が大きく異なる。したがって、通常、外挿の精度を確認することは容 易ではない。また動物の犠牲を伴う試験方法には、社会的な批判もある。そこで近年 においては、ヒトの培養細胞を評価試験に用いる方法に移行しつつある。本発明に よって、安定的に、かつコスト的にも比較的安価に、試験に必要な肝細胞を得ること ができる。 [0094] For example, in many cases, tests using rats are the mainstream in toxicity evaluation tests and food safety evaluation tests. Animal inspection space is inevitably required for testing using animals. It is important. Therefore, the number of compounds that can be evaluated at one time is limited by space. In animal studies, effects on humans are usually predicted based on results obtained in rats. The prediction made at this time is particularly called extrapolation. However, animal species differ greatly between humans and rats. Therefore, it is usually not easy to check the accuracy of extrapolation. There are also social criticisms of test methods involving animal sacrifice. Therefore, in recent years, the method of using human cultured cells for evaluation tests is shifting. According to the present invention, hepatocytes necessary for the test can be obtained stably and relatively inexpensively.
[0095] 本発明で得られるヒト肝細胞様細胞は、肝炎ウィルスを感染させ、感染した状態を 維持することができる。 [0095] The human hepatocyte-like cells obtained in the present invention can be infected with hepatitis virus and maintained in the infected state.
「したがって、本発明の成熟肝細胞様細胞は、肝炎ウィルスによる疾病の予防あるい は治療の研究に利用することができる。特に、 C型肝炎ウィルスはヒトに対する種特異 性が高い。現在のところ、ヒト C型肝炎ウィルスを感染させることができる実験動物は、 チンパンジーの他に確認されていない。チンパンジーは、ワシントン条約で絶滅の恐 れが高い国際希少野生動植物種に指定された動物種である。学術研究を目的とす る移動は認められているものの、希少であることには変わりはない。また in vitroにお ける C型肝炎ウィルスの効率的な培養を可能にする細胞系も確立されて 、な 、。つ まり、 C型肝炎ウィルスの研究に必要な、感染モデルや培養方法すら十分でないの が現状である。 C型肝炎ウィルスの培養が難しいことは、その予防や治療の研究にお ける大きな障害となっている。 “Accordingly, the mature hepatocyte-like cells of the present invention can be used for research on prevention or treatment of diseases caused by hepatitis virus. In particular, hepatitis C virus is highly species-specific to humans. In addition to chimpanzees, 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. Although 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.
本発明によって得られた成熟肝細胞様細胞には、 C型肝炎ウィルスの感染が確認 された。したがって、本発明の成熟肝細胞様細胞によって、 in vitroの系で、すなわち 細胞レベルで、 C型肝炎ウィルスの増殖や感染実験を行うことができる。したがって本 発明の成熟肝細胞様細胞は、ヒト C型肝炎ウィルスの研究に貢献する。 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.
[0096] 更に、本発明の成熟肝細胞様細胞は、肝臓細胞の機能を高度に再現して 、る。し たがって、本発明の成熟肝細胞様細胞は、肝機能を代替する人工臓器に利用する ことができる。たとえば、本発明の成熟肝細胞様細胞を浸透膜に充填し、血液を循環 させることによって、人工肝臓とすることができる。人工透析や人工心肺においては、
ヒトに対する抗原性が低い浸透膜が既に実用化されている。更に、血液回路におい て患者血液を処理し、患者に戻す治療行為も日常的に行われている。このような血 液回路中で本発明の成熟肝細胞様細胞を患者血液に接触させることによって、肝臓 の代謝機能を代替させることができる。
[0096] Furthermore, 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. For example, 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. In addition, 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.
Claims
[1] オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩、および TGF- β の存在下で間葉系幹細胞を培養し、間葉系幹細胞を成熟肝細胞様細胞に分化させ る工程を含む、成熟肝細胞様細胞の製造方法。 [1] Maturation, including culturing mesenchymal stem cells in the presence of oncostatin M, dexamethasone or a derivative or salt thereof, and TGF-β, and differentiating mesenchymal stem cells into mature hepatocyte-like cells A method for producing hepatocyte-like cells.
[2] 間葉系幹細胞が、臍帯血、骨髄、脂肪組織、胎盤、および抹消血からなる群から選 択される 、ずれかの組織に由来する間葉系幹細胞である請求項 1に記載の成熟肝 細胞様細胞の製造方法。 [2] The mesenchymal stem cell according to claim 1, wherein the mesenchymal stem cell is a mesenchymal stem cell derived from any tissue selected from the group consisting of umbilical cord blood, bone marrow, adipose tissue, placenta, and peripheral blood. A method for producing mature hepatocyte-like cells.
[3] 間葉系幹細胞が、臍帯血に由来する間葉系幹細胞である請求項 2に記載の成熟 肝細胞様細胞の製造方法。 [3] The method for producing mature hepatocyte-like cells according to claim 2, wherein the mesenchymal stem cells are mesenchymal stem cells derived from umbilical cord blood.
[4] 間葉系幹細胞が、骨髄に由来する間葉系幹細胞である請求項 2に記載の成熟肝 細胞様細胞の製造方法。 4. The method for producing mature hepatocyte-like cells according to claim 2, wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow.
[5] 間葉系幹細胞が、少なくとも 6日間培養される請求項 1に記載の成熟肝細胞様細胞 の製造方法。 [5] The method for producing mature hepatocyte-like cells according to claim 1, wherein the mesenchymal stem cells are cultured for at least 6 days.
[6] オンコスタチン Μ、デキサメタゾン、および TGF- βの間葉系幹細胞を培養するため の培地における濃度力 それぞれ 1〜: LOOng/mL、 0. 1〜: LO /z M、そして 0. 2〜20n g/mLである請求項 1に記載の成熟肝細胞様細胞の製造方法。 [6] Oncostatin concentrations in media for culturing mesenchymal stem cells of sputum, dexamethasone, and TGF-β 1 to: LOOng / mL, 0.1 to: LO / z M, and 0.2 to 2. The method for producing a mature hepatocyte-like cell according to claim 1, which is 20 ng / mL.
[7] 請求項 1〜請求項 6に記載のいずれかの方法によって製造することができる成熟肝 細胞様細胞。 [7] A mature hepatocyte-like cell that can be produced by the method according to any one of claims 1 to 6.
[8] 請求項 1〜請求項 6に記載のいずれかの方法によって製造することができる成熟肝 細胞様細胞を含む、肝臓疾患の治療剤。 [8] A therapeutic agent for liver diseases, comprising mature hepatocyte-like cells that can be produced by the method according to any one of claims 1 to 6.
[9] 次の工程を含む、被験化合物の肝における代謝を検出する方法であって、成熟肝 細胞様細胞が、オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩、 および TGF- |8の存在下で間葉系幹細胞を培養することによって、間葉系幹細胞か ら分化した成熟肝細胞様細胞である方法; [9] A method for detecting metabolism of a test compound in the liver, comprising the steps of: mature hepatocyte-like cells in the presence of oncostatin M, dexamethasone or a derivative or salt thereof, and TGF- | 8 A mature hepatocyte-like cell differentiated from mesenchymal stem cells by culturing mesenchymal stem cells in
(1)成熟肝細胞様細胞に被験化合物を接触させる工程、および (1) contacting the test compound with mature hepatocyte-like cells, and
(2)成熟肝細胞様細胞における被験化合物の代謝を検出する工程。 (2) A step of detecting metabolism of a test compound in mature hepatocyte-like cells.
[10] 次の工程を含む、被験化合物の肝毒性を検出する方法であって、成熟肝細胞様 細胞が、オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩、および
TGF- |8の存在下で間葉系幹細胞を培養することによって、間葉系幹細胞から分ィ匕 した成熟肝細胞様細胞である方法; [10] A method for detecting hepatotoxicity of a test compound, comprising the following steps, wherein the mature hepatocyte-like cell comprises Oncostatin M, dexamethasone or a derivative or salt thereof, and A method of mature hepatocyte-like cells separated from mesenchymal stem cells by culturing mesenchymal stem cells in the presence of TGF- | 8;
(1)成熟肝細胞様細胞に被験化合物を接触させる工程、および (1) contacting the test compound with mature hepatocyte-like cells, and
(2)成熟肝細胞様細胞の障害が検出されたときに被験化合物の肝毒性が検出され る工程。 (2) A step in which hepatotoxicity of a test compound is detected when a disorder of mature hepatocyte-like cells is detected.
[11] 次の工程を含む、肝疾患の治療剤のスクリーニング方法であって、成熟肝細胞様 細胞が、オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩、および TGF- |8の存在下で間葉系幹細胞を培養することによって、間葉系幹細胞から分ィ匕 した成熟肝細胞様細胞である方法; [11] A method for screening a therapeutic agent for liver disease, comprising the steps of: A method wherein the cells are mature hepatocyte-like cells separated from mesenchymal stem cells by culturing mesenchymal stem cells;
(1)成熟肝細胞様細胞に被験化合物を接触させる工程、 (1) contacting the test compound with mature hepatocyte-like cells,
(2)被験化合物を接触させた成熟肝細胞様細胞の機能を測定する工程;および (2) measuring the function of mature hepatocyte-like cells contacted with the test compound; and
(3)対照と比較して、成熟肝細胞様細胞の機能を亢進させる作用を有する化合物 を選択する工程。 (3) A step of selecting a compound having an action of enhancing the function of mature hepatocyte-like cells as compared with the control.
[12] 次の工程を含む、肝炎ウィルスの感染阻害剤のスクリーニング方法であって、成熟 肝細胞様細胞が、オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩 、および TGF- |8の存在下で間葉系幹細胞を培養することによって、間葉系幹細胞 から分化した成熟肝細胞様細胞である方法; [12] A method for screening an inhibitor of hepatitis virus infection, comprising the steps of: mature hepatocyte-like cells in the presence of oncostatin M, dexamethasone or a derivative or salt thereof, and TGF- | 8 A method that is a mature hepatocyte-like cell differentiated from a mesenchymal stem cell by culturing the mesenchymal stem cell;
(1)成熟肝細胞様細胞に被験化合物の存在下で肝炎ウィルスを接触させるか、ま たは成熟肝細胞様細胞に肝炎ウィルスを接触させた後に被験化合物を接触させる 工程、 (1) contacting the hepatitis virus with mature hepatocyte-like cells in the presence of the test compound, or contacting the test compound after contacting hepatitis virus with mature hepatocyte-like cells;
(2)成熟肝細胞様細胞への肝炎ウィルスの感染のレベルを測定する工程;および (2) measuring the level of hepatitis virus infection of mature hepatocyte-like cells; and
(3)対照と比較して、成熟肝細胞様細胞への肝炎ウィルスの感染レベルが低い化 合物を選択する工程。 (3) A step of selecting a compound having a lower infection level of hepatitis virus to mature hepatocyte-like cells than the control.
[13] 次の工程を含む、ウィルス性肝炎の治療剤のスクリーニング方法であって、成熟肝 細胞様細胞が、オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩、 および TGF- |8の存在下で間葉系幹細胞を培養することによって、間葉系幹細胞か ら分化した成熟肝細胞様細胞である方法; [13] A method for screening a therapeutic agent for viral hepatitis, comprising the steps of: mature hepatocyte-like cells in the presence of oncostatin M, dexamethasone or a derivative or salt thereof, and TGF- | 8. A method that is a mature hepatocyte-like cell differentiated from a mesenchymal stem cell by culturing the mesenchymal stem cell;
(1)肝炎ウィルスを感染させた成熟肝細胞様細胞に被験化合物を接触させる工程
(2)成熟肝細胞様細胞における肝炎ウィルスの増殖を測定する工程;および(1) A step of bringing a test compound into contact with a mature hepatocyte-like cell infected with hepatitis virus (2) measuring the growth of hepatitis virus in mature hepatocyte-like cells; and
(3)対照と比較して、肝炎ウィルスの増殖の阻害作用が検出された化合物を選択す る工程。 (3) A step of selecting a compound in which an inhibitory effect on the growth of hepatitis virus is detected compared to the control.
[14] 成熟肝細胞様細胞に肝炎ウィルスを感染させる工程を含む肝炎ウィルスの培養方 法であって、成熟肝細胞様細胞がオンコスタチン M、デキサメタゾンまたはその誘導 体もしくはその塩、および TGF- |8の存在下で間葉系幹細胞を培養することによって 、間葉系幹細胞から分化した成熟肝細胞様細胞である肝炎ウィルスの培養方法。 [14] A method for culturing hepatitis virus comprising a step of infecting hepatitis virus into mature hepatocyte-like cells, wherein the mature hepatocyte-like cells are Oncostatin M, dexamethasone or a derivative thereof or a salt thereof, and TGF- | A method for culturing hepatitis virus which is a mature hepatocyte-like cell differentiated from mesenchymal stem cells by culturing mesenchymal stem cells in the presence of 8.
[15] オンコスタチン M、デキサメタゾンまたはその誘導体もしくはその塩、および TGF- β を含む、間葉系幹細胞から成熟肝細胞様細胞への分化誘導試薬。 [15] A reagent for inducing differentiation of mesenchymal stem cells into mature hepatocyte-like cells, comprising Oncostatin M, dexamethasone or a derivative or salt thereof, and TGF-β.
[16] オンコスタチン Μ、デキサメタゾンまたはその誘導体もしくはその塩、および TGF- β を含む、培地。 [16] A medium containing Oncostatin IV, dexamethasone or a derivative or salt thereof, and TGF-β.
[17] 間葉系幹細胞を成熟肝細胞様細胞に分化させるための、請求項 16に記載の培地 [17] The medium according to claim 16, for differentiating mesenchymal stem cells into mature hepatocyte-like cells.
[18] オンコスタチン Μ、デキサメタゾンまたはその誘導体もしくはその塩、および TGF- β の培地における濃度力 それぞれ 1〜: LOOng/mL、 0. 1〜: LO /z M、そして 0. 2〜20n g/mLである請求項 16又は 17に記載の培地。
[18] Oncostatin Μ, dexamethasone or its derivatives or salts thereof, and TGF-β concentration in the medium 1 to: LOOng / mL, 0.1 to: LO / z M, and 0.2 to 20 ng / The medium according to claim 16 or 17, which is mL.
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Cited By (5)
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WO2013068557A1 (en) * | 2011-11-09 | 2013-05-16 | Katholieke Universiteit Leuven | Virus infectable stem cells |
WO2016029267A1 (en) * | 2014-08-27 | 2016-03-03 | Prince Henry's Institute Of Medical Research Trading As The Hudson Institute Of Medical Research | A method for culturing mesenchymal stem cells |
CN108823148A (en) * | 2018-07-23 | 2018-11-16 | 广东唯泰生物科技有限公司 | A kind of method that fat mesenchymal stem cell is induced to differentiate into liver like cell |
CN111073843A (en) * | 2018-10-22 | 2020-04-28 | 立沃生物科技(深圳)有限公司 | Method for maturing and amplifying liver-like cells |
CN115025121A (en) * | 2022-06-23 | 2022-09-09 | 深圳凯兰赛尔健康管理有限公司 | Compound cell preparation and preparation method and application thereof |
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WO2016109947A1 (en) * | 2015-01-07 | 2016-07-14 | Frontier Bio-Drug Development Limited | Employing human adipose-derived stem cells to propagate serum-derived hepatitis c virus and use thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005095138A (en) * | 2003-08-19 | 2005-04-14 | Effector Cell Institute Inc | Method for inducing differentiation of cell having pluripotency |
-
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- 2006-04-03 JP JP2006102350A patent/JP2009153383A/en active Pending
-
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005095138A (en) * | 2003-08-19 | 2005-04-14 | Effector Cell Institute Inc | Method for inducing differentiation of cell having pluripotency |
Non-Patent Citations (4)
Title |
---|
HONG S.H. ET AL.: "In vitro differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 330, no. 4, 2005, pages 1153 - 1161, XP004844850 * |
LEE K.-D. ET AL.: "In vitro hepatic differentiation of human mesenchymal stem cells", HEPATOLOGY, vol. 40, 2004, pages 1275 - 1284, XP003018526 * |
TERATANI T. ET AL.: "Hito Kanyokei Kansaibo Yura Seijuku Kansaibo no Kanshikkan Kenkyu eno Oyo", ANNUAL MEETING OF THE JAPAN CANCER ASSOCIATION KIJI, vol. 64, 2005, pages 143 + ABSTR. NO. W-210, XP003018527 * |
TERATANI T. ET AL.: "Hito Kanyokei Kansaibo Yurai Kansaibo o Mochiita Kanshikkan Chiryo ni Taisuru Hyoka", ANNUAL MEETING OF THE JAPAN CANCER ASSOCIATION KIJI, vol. 65, 28 August 2006 (2006-08-28), pages 504 + ABSTR. NO. P-1221, XP003018528 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013068557A1 (en) * | 2011-11-09 | 2013-05-16 | Katholieke Universiteit Leuven | Virus infectable stem cells |
WO2016029267A1 (en) * | 2014-08-27 | 2016-03-03 | Prince Henry's Institute Of Medical Research Trading As The Hudson Institute Of Medical Research | A method for culturing mesenchymal stem cells |
CN108823148A (en) * | 2018-07-23 | 2018-11-16 | 广东唯泰生物科技有限公司 | A kind of method that fat mesenchymal stem cell is induced to differentiate into liver like cell |
CN111073843A (en) * | 2018-10-22 | 2020-04-28 | 立沃生物科技(深圳)有限公司 | Method for maturing and amplifying liver-like cells |
CN115025121A (en) * | 2022-06-23 | 2022-09-09 | 深圳凯兰赛尔健康管理有限公司 | Compound cell preparation and preparation method and application thereof |
CN115025121B (en) * | 2022-06-23 | 2023-10-03 | 深圳凯兰赛尔健康管理有限公司 | Composite cell preparation and preparation method and application thereof |
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TW200806793A (en) | 2008-02-01 |
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