TW200806793A - Method of preparing mature hepatocyte-like cell - Google Patents

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

200806793, IX. Description of the Invention: [Technical Field of the Invention] The present invention relates to mature hepatocyte~~ cel Is 〇 [Prior Art] Stem cells are cells having the ability to differentiate into various forms of cells. For example, cells having the ability to differentiate into liver, muscle, nerve, skin, or blood cells are known. In order to be applied to regenerative medicine, it is eagerly expected to efficiently differentiate stem cells into cells having specific organ functions. In particular, differentiation from stem cells to hepatoblasts (hepa1: 〇cyte-like cells) is expected to be applied to the development of biological livers (biol〇glcal art 1 flciai 1 iver), or in cell transplantation therapy (qu transplantation) Therapy). Cell transplantation therapy is useful as a treatment for liver disease and liver disease. And in the development of new drugs (called discovery), hepatocytes can also be used as a drug metabolism or toxicity analysis system or screening system. As a method of inducing hepatocytes from stem cell differentiation, a method of utilizing the regenerative ability of a wounded animal or a method of cocultivating by a cell is known. However, in order to produce a large amount of functional hepatocytes, it is desirable to have a culture line which induces differentiation into stem cells from stem cells in vitro. Various attempts have been made to report on differentiation into hepatocytes in vitro. Miyajima et al. revealed that by immature hepatocytes derived from the fetus, they were cultured with oncostatin M (OSM) and dexamethasone to induce 2125-8759-PF; Kai 5 200806793, differentiated into hepatocyte-like cells. The method. Alternatively, in the presence of an extracellular matrix, immature hepatocytes derived from the fetus are cultured by adding 0SM and glucocorticoid, and differentiation into hepatocyte-like cells is observed (Non-Patent Document 1 and Patent Document 1-2). . In addition, the Sugaoka scorpion uses a proliferation factor (gr〇wth fact〇r) or a differentiation inducing factor to confirm the nucleated cells isolated and modulated from human umbilical cord blood, and can express human white. Protein gene and cultured cells of human albumin (Patent Document 7). The performance of albumin is one of the characteristics of liver cells. In the report of Teraoka et al., especially the combination of FGF, HGF, LIF, and SCF, the best results were obtained. Human leukemia inhibitory factor (hLIF) > human stem cell factor (hSCF), fibroblast growth factor (FGF), human hepatocyte growth factor; hHGF), OSM, and dexamethasone or the like, revealing an embryonic stem cell (EScell), or bone marrow-derived mesenchyma stem cells (BM hMSC), differentiation-inducing class The method of liver cells. In this report, OSM, HGF, and FGF are essential differentiation growth factors (Patent Document 4). Embryonic stem (ES) cells were obtained from cells of 2125-8759-PF; Kai 6 200806793 - mice, rats and monkeys, and the mesenchymal stem cells between the bone marrow were used from human cells.

Lee et al. (Non-Patent Document 2, Patent Document 6) disclose a method for inducing differentiation into hepatocyte-like cells from phylloblastic stem cell MSCs derived from bone marrow by a two-stage differentiation induction method. In the first stage, a medium containing hgf as an essential factor and a medium for maturation in the second stage was used, and a medium using 0SM as an essential factor was used. Further, 'Hong et al. (Non-Patent Document 4) adopts the method of Lee et al. (Non-patent Document 6), and confirms that differentiation from cord blood stem cells is induced into hepatocyte-like cells. [Patent Document 1] Miyajima, et al: JP2000/287680 [Patent Document 2] Miyajima, et al: W002/074937 [Patent Document 3] Miki, et al: JP2005 - 523328 [Patent Document 4] Ochiya, et al·· JP2006 - 254896 [Patent Document 5] Tomisawa, JP 2005 - 2533 74 [Patent Document 6] Lee, et al: US 2005/0233449 Al [Patent Document 7] Teraoka, et al: JP2002 - 360243 [Non-Patent Document 1] Miyajima , et al: EMB〇J. V〇1.18, N〇. 8, pp. 21 27 - 21 36 (1 999) [Non-Patent Document 2] Lee, et al: HEPATOLOGY vo 1. 40 : 1 275 -1284 ( 2004) [Non-Patent Document 3] 8 - £¥3, 6:7:31:1乂1:〇1:1161^口7¥〇1·6,Ν〇·5,476 — 486 /486(2004) Non-Patent Document 4] SH Hong, et al: Biochemical and Biophysical Research Communications 330: 1153-1161 2125-8759-PF; Kai 7 200806793, (2005) [Non-Patent Document 5] Κ· Teramoto et al· : J Hepatobi 1 Iary Pancreat Surg ( 2005 ) 1 2:1 96 - 202 [Non-Patent Document 6] Lee, et al: Blood vol. 1 03:1 669 -1675 (2004) [Summary of the Invention [Problems to be solved by the invention] The object of the present invention is to provide a high degree of maturity (maturi 1; y) for producing hepatocyte cells of the method. Or the object of the present invention is to provide a liver cell which can be produced by the present invention and use thereof. [Methods for Solving the Problem] In the past, it has been indispensable for the differentiation of stem cells into hepatocyte-like cells and the hepatocyte growth factor (HGF). However, the inventors of the present invention found that even in the absence of HGF, differentiation into mature liver cells can be sufficiently induced by utilizing a specific proliferation differentiation factor. Based on this insight, 湓古^ established a method for producing hepatocytes such as 鬲 maturity, and completed the present invention. (4) The present invention relates to a manufacturer of the following liver-like fines and a liver cell obtainable by the production method, and various uses thereof. [1] A method for producing mature hepatocytes, which is described in the case of sputum sputum, dexamethasone or a derivative thereof or a salt thereof, and TGF~汐> Leaf stem cells, and the steps of dividing mesenchymal stem cells into _ scaffold mature liver cells. [2] The method for producing mature hepatocytes according to [1], wherein the mesenchymal stem cell line is selected from the group consisting of cord blood, bone marrow: fat makeup, fat tissue, placenta, and terminal 2125-8759-PF; Kai 8 200806793 One of the groups formed by a little blood - [3] such as the [2] manufacturing mature liver line from the cord blood. Weaving between leaf system stem cells. Cell method 'middle mesenchymal stem cells, middle mesenchymal stem cells, middle mesenchymal stem cells [4] such as [2] The method for producing mature hepatocytes is derived from bone marrow. [5] The method for producing mature hepatocytes as in [1] is cultured for at least 6曰. [6] such as [1] for the manufacture of mature liver fine tires, sputum, and method of cultivating the middle leaves

In the medium of stem cells, the tumor suppressor M is derived from the element M, the dexamethasone, and the TGF-泠

The concentration 'is 1 to l ng/mL, 〇ι 〜1f] M • and 〇·2 to 2 〇 ng/mL. [7] - A variety of mature hepatocytes can be produced by the method of manufacturing mature liver cells using U [6]. [6] The manufacture of [8] is a therapeutic agent for liver diseases, including mature hepatocytes that can be produced by the method of [i-cooked liver cells. (8) A method for detecting the metabolism of a test compound in the liver, comprising the steps of: (1) contacting the mature liver cell with the test compound, and (2) detecting the metabolism of the test compound in the mature liver cell; The hepatocyte cell line is cultured from the presence of M, dexamethasone or a derivative thereof or a salt thereof, and TGF-e in the presence of mature hepatocytes differentiated between leaf stem cells and mesenchymal stem cells. [10] - A method for hepatotoxicity of a test compound comprising the steps of: (1) contacting a mature liver cell with a test compound, and (2) detecting a damaged mature liver cell Hepatic toxicity of test compound; 2125-8759-PF; Kai 9 200806793 The mature liver cell line is cultured between the presence of oncostatin, dexamethasone or its derivatives or its salts 'and TGF — /5 It is a mature hepatocyte differentiated by stem cells and mesenchymal stem cells. [11] A method for pulverizing a therapeutic agent for liver diseases, comprising the steps of: (1) bringing mature human hepatocytes into contact with a test compound, and (2) 'the function of mature hepatocytes after contact with a test compound; And (3) comparing the effects of the group with a compound that has the function of promoting the function of mature hepatocytes; such mature liver cells are derived from oncostatin, dexamethasone or a derivative thereof or a salt thereof, and In the presence of TGF 10,000, mesenchymal stem cell culture is carried out, and differentiated from mesenchymal stem cells. [1 2 ] A method for infecting an infection inhibitor of serotonin hepatitis virus, comprising the steps of: contacting a test compound with a hepatitis hepatitis virus in the presence of a test compound; (1) causing the mature liver cell to be subjected to a virus, or a class Mature hepatocytes contact, (2) Hepatitis virus infection mature liver - ..., Qing xinxin 卞, 夂,, (8) compared with the control group, select hepatitis virus for low-level compounds of mature liver cells ~ ~ Such mature hepatic cell line is due to the presence of oncostatin M, dexamethasone or its salt, and the presence of TGF-^ in the presence of 八何办, 仃 仃 茱 干 干 干 干 干 干 干Stem cells differentiate. [13] A method for screening a therapeutic agent for viral hepatitis, comprising the steps of: 2125-8759-PF; Kai 1〇200806793 (1) contacting a mature hepatocyte such as a hepatitis virus with a test compound (2) Proliferation of hepatitis blast virus in mature hepatocytes; and (3) selection of the main compound to be detected compared with the control group; inhibition of proliferation of f serotonin t such mature liver cell line by inhibition of organism or its salt , the preservation of the hormone: dexamethasone or its derivatives, in the next 'to carry out mesenchymal stem cell culture, and differentiated from the mesenchymal stem cells. [14] A method for cultivating a hepatitis virus, g 3 is a step of infecting a mature liver cell with a hepatitis virus, such a mature liver is fine, and you are wrong because of the tumor suppressor, the dexamethasone or a derivative thereof or a salt thereof In the presence of TGF1, mesenchymal stem cell culture is carried out, and differentiated from mesenchymal stem cells. [15] - a drug for inducing differentiation of mesenchymal stem cells into mature hepatocytes, comprising oncostatin M, dexamethasone or a derivative thereof or a salt thereof, and tgf ten [16] a medium containing tumor inhibition M, dexamethasone or a derivative thereof or a salt thereof, and TGF - /3. [17] The medium of [16] is used to differentiate mesenchymal stem cells into mature hepatocytes. [1 8] The medium according to [1 6 ] or [17], wherein, in the medium, the inhibitory concentration of estramin, dexamethasone or a derivative thereof or a salt thereof, and tgf and a cold are each 1~ lOOng/mL, 〇·1 ~ίο" μ, and 0·2~20ng/mL. [Effect of the Invention] The present invention provides a method for producing a mature liver cell. The method of the present invention can induce differentiation from mesenchymal stem cells to a mature liver-like fine 2125-8759-PF; Kai 11 200806793 cells. For example, the expression of a liver-specific gene as a hepatocyte maturity ‘I If$ Μ彳± is required to be cultured for at least several weeks in order to differentiate stem cells into mature liver cells. However, in the method of the present invention, it is possible to confirm the expression of the mature hepatocyte-specific gene within a short period of 6 days after the initiation of the culture. Hepatocytes, which are obtained by the ugly and memorable period, have the same characteristics as the hepatocytes. Further, the liver squama pack which can be obtained according to the present invention also has characteristics of hepatocytes at the point of cell function. Specifically, in a preferred aspect of hepatocytes such as the present invention, it is confirmed that the following liver cells have characteristic functions. High hepatocyte specific gene expression, glucose metabolism, albumin production, and CYP activity

In addition, in a preferred embodiment, hepatocytes such as the present invention can be infected with hepatitis C virus. This fact can support the fact that hepatocytes which can be obtained according to the present invention are cells whose maturity south is highly similar to human mature liver cells. [Embodiment] The best mode for carrying out the invention relates to a method for producing mature hepatocytes, which comprises culturing mesenchymal stem cells in the presence of oncostatin, dexamethasone, and TGF-5, and The step of differentiating mesenchymal stem cells into maturation-like hepatocytes. 2125~8759-PF; Kai 12 200806793 In the present invention, the mesenchymal stem cells use leaf-derived stem cells derived from mammals including humans. Preferably, the leaf system stem cells are human mesenchymal stem cells. Mesenchymal stem cells (MSC) have been reported to have the ability to differentiate into diverse cells such as adipocytes, chondrocytes, bone cells, cardiomyocytes, and neuron. Unlike mesenchymal stem cells that are only present in the fetus, mesenchymal stem cells, which are one type of tissue stem cells, can be isolated from the tissues of patients. Therefore, it is valued as a material for regenerative medicine. In the present invention, the source organization of the mesenchymal stem cells is not limited. A mesenchymal stem cell, which has been found to be a bone marrow stem cell that differentiates into cells other than blood cells. Thereafter, in addition to bone marrow, mesenchymal stem cells have also been found in adipose tissue, placenta, cord blood, terminal blood, and odontogenic tissue. Such mesenchymal stem cells derived from tissues other than bone may also be utilized in the present invention. For example, leaf cell stem cells derived from human blood and human bone marrow are preferred interfamily stem cells in the present invention. Bone marrow can be obtained from individuals of the fetus to adult at any stage of growth. Therefore, based on the present invention, mature hepatocytes such as autotransplant can be obtained. Methods for isolating mesenchymal stem cells are well known. For example, mesenchymal stem cells can be recovered using the cell surface marker (CD271) as an indicator. A kit for isolating human mesenchymal stem cells, which is an antibody against CD271, is also available for sale in the market of "Foliar Cell Stem Cell Separation and Culture Box - CD271 (LNGFR)" (manufactured by Miltenyi Biotec Co., Ltd., trade name). Or commercially available pre-separated leaf line stem cells can be utilized in the present invention. Furthermore, not only the mesenchymal stem cells are differentiated, but also the undifferentiated state can be proliferated. Therefore, the mesenchymal stem cell proliferation can also be 2125-8759-PF after separation; Kai 13 200806793. Used in the present invention. In the present invention, it is preferred to increase the mesenchymal stem cells before differentiation. The mesenchymal stem cells can be cultured using a medium commercially available as a medium for mesenchymal stem cell proliferation. Specifically, MF medium (Τ0Υ0Β0 company, Japan), MSCG medium (Cambrex, USA), and the like can be used for proliferation of mesenchymal stem cells. Alternatively, alpha MEM medium containing bovine serum can be used for proliferation of mesenchymal stem cells. The mesenchymal stem cells can be propagated by culturing in a cell culture under normal conditions. The general culture conditions are, for example, cultured at 37 ° C in a humidified atmosphere containing about 5% of C 02 . For the purpose of amplifying the amount of the stem cell, the collagen-free culture container is generally used. In the method for producing mature hepatocytes of the present invention, the mesenchymal stem cells are cultured in the presence of oncostatin, dexamethasone, and TGF - /3. In the present invention, the medium for culturing the mesenchymal stem cells is usually 1 ng/ml to 1 ng/mi, preferably 5 ng/ml to 50 ng/ml. Similarly, the concentration of dexamethasone is usually 〜10//M, preferably 0.5/M to 5//M. Further, the concentration of TGF - /3 is usually from 0.2 ng / ml to 20 ng / ml, preferably from 1 ng / ml to 1 ng / ml. Oncostatin M (〇ncostatin M), a cytokine of the IL-6 family. Oncostatin is identified as a factor that inhibits the proliferation of human melanoma ((5) "(4) (10) (eight) cell lines (Pr〇c. Natl, Acad. Sci. USA V〇l 83, pi 9739-9743, December 1 986). There are many reports on the differentiation of blood cells or stem cells. Human oncostatin M, a protein composed of 252 amino acids and having a molecular weight, contains a secretion signal, and the N-terminal 25 is removed by treatment (Pr〇CeSSlng). The amino acid residue is a mature protein composed of 2125-8759-PF; Kai 200806793 2 2 7 amino acid residues. Further, it is known that 31 amino acid residues on the c-terminal side are The mature protein with a molecular weight of about 22 kDa consisting of 169 amino acid residues is removed. The physiological activity of these mature proteins is 5 to 6 times higher than that of the parent uranium-driven protein ([ins 1 ey, eta 1 ·, 1 990 ' Mol_ Cel 1 · Biol · l〇: 1882 — 1 890 ). SEQ ID NO: 13 shows the amino acid sequence of human oncostatin (Gengank number (Accession No.) AAA36388). Serial number: 13 Of the 252 residues of the amino acid sequence 'from the N-terminal side, 26-25 227 residues in position 2 or 196 residues in positions 26 to 221 are comparable to the mature protein of human oncostatin μ. Both the prostaglandin and the mature protein have the same effect on cells. In the present invention, the prostaglandin pro-protein and the mature protein or both may be used. However, the preferred anti-tumor Μ in the present invention is a physiologically active mature oncostatin 序列 (SEQ ID NO: 13 In the middle of 26-221 of 196 amino acids. In addition, the previously expressed concentration of tumor suppressor f μ in the medium is used for the form of the precursor protein f. Therefore, it is assumed that the fatty protein is replaced by the mature protein. %, the concentration of the antitumor in the medium may be 〇·8 ng/mL~(10), preferably about 4·(tetra)/乩~43 ng/mL.

In the present invention, the anti-tumor sputum is not euthanized as long as it is cultured in the form of a precursor cell, and the cell is induced to differentiate into a mature hepatocyte I. A clear amino acid sequence of oncostatin. Human Oncostatin M: GenBank No. AAA36388, A AAD31435, ΝΡ-〇〇399〇 Mice of Oncostatin: GenBank No. ΝΡ—035707, ΑΑΗ1 3738 2125-8759-PF; Kai 15 200806793 Dexamethasone (Dex Am et has one; 9 -Fluor-11/?,17,21-trihydroxy-16 α-methyl-pregnant-1,4-diene-3, 2 0-dione; CAS accession number 50 — 02 — 2) 'A synthetic steroid having a physiological action similar to glucocorticoidation (3) (3) "丨(3)丨^). Like the steroid hormone, moving to the nucleus simultaneously with the receptor is considered to be involved in transcription (transcripti〇n) regulation For clinical use, it is a compound such as astringent, analgesic, anti-inflammatory, ophthalmic, antipruritic, digestive organ, etc. In the present invention, dexamethasone can be used instead, and the same effect can be utilized. Derivatives of dexamethasone, such as dexamethasone and an acid ester, are known to have the same physiological activity as dexamethasone. For example, a derivative thereof or a salt thereof can be utilized as dexamethasone in the present invention. Dexamethasone 21 - acetate (CAS registration number 177_87 one; one Dexamethasone propionate (CAS Registry No. 55541-3〇-5); Dexamethasone Sodium Phosphate (CAS Registry Number 2392 — 39 — 4); Transforming Growth Factor — beta; TGF — 10,000), which is a protein with a dimeric structure, it is known that there are three kinds of isomers with similar structures in mammals. This isomer is called /5 1 , /5 2 , and 3 In the following, unless otherwise specified, "TGF 10,000" is used to include the entire term of the same isomer. This isomer can be used in the present invention. Hereinafter, the TGF of the present invention will be exemplified. 9 amino group I sequence Human TGF - /5 1 amino acid sequence (GenBank accession number AAQ1 8641) as shown in SEQ ID NO: 14. Sequence number: 14 amino acid sequence lacks 6 residues on the N-terminal side The 112 residues (7 - 11 8 positions) are the amino acid sequence of the mature protein of TGF-like cold 1. Contains the sequence of 2125-8759-PF/Kai 16 200806793: 14 of the amino acid sequence of the n-terminal a protein having a side amino acid sequence of 112 residues is preferred as TGF-5 in the present invention. Human TGF — /5 1 : GenBank No. AAQ18 64 NP—00 065 1, AAA51458, AAL27646, AAQ18642, AAV71148, AAN86616, AAT77144, ΑΑΠ7143, AAX32228, P01137, CAA29283, AAH01180, AAH00125, and AAP35909 Human TGF — /5 2 : GenBank No. (Accession No.) Y〇〇〇83, Μ19154 Human TGF-A 3 : GenBank No. J03241, Χ144149 Mouse TGF - /5 1 : GenBank No. NP 00 1 01 3383, AAH99866, and CAI25749 Mouse TGF — /9 2 : GenBank No. X5 7413 Mouse TGF— /5 3 : GenBank No. M32745 In the present invention, the tumor suppressor f Μ & TGF ten is as long as it is to induce differentiation of the leaf cells in the pre-drive cell form between the cells. Mature liver cells are not limited in their origin. In particular, the TGF-million amino acid sequence is known to be highly conserved among mammals. For example, in the case of tgf-stone, the amino acid sequence identity of human and mouse is 99%. Therefore, in general, the difference between TGF and 被 is considered to be almost ridiculous. However, it is preferable that mature hepatocytes can be used in human medical treatment in accordance with the present invention to utilize human T G F - /5 . In the present invention, the antitumor oxime may also be used in a person having the same domain as the natural molecule, and based on the aforementioned amine group and TGF - /3, a natural, active gene recombinant may be used. This technology acid sequence information, or encoded as 2125-8759-PF/Kai 200806793 salt-based alignment information, can produce anti-tumor secret or TGF 10,000 gene recombinant. Alternatively, a recombinant gene of oncostatin or TGF-dot is also commercially available. For example, the companies shown below sell genetic recombinants of these proteins. Therefore, such commercially available genetic recombinants can also be utilized in the present invention. Human or small prostaglandin (pre-proprotein)

United States Biological, ProSpec-Tany TechnoGene Ltd, etc. Human or mouse anti-tumor M (mature protein) R&D Systems Inc., etc. Human or mouse TGF-stone: CHEMICON Internati onal, Inc,

Fitzgerald Industries Inti·'s RDI division can be added to the mesenchymal stem. The base culture of the present invention and the vitamin 鲎, generally, the above-mentioned proliferation and differentiation factor, the medium medium for the cell differentiation of the animal cells, and the representative composition of the inorganic salt, the saccharide, and the amino acid-based medium are as follows Show. Inorganic salts:

Chlorinated sodium 4000 — 8000mg/L Calcium carbonate 200 — 330mg/L Potassium nitrate G— 0. lmg/L Sodium selenite 0— 〇. 〇2mg/L anhydrous calcium chloride 20 — 1 65mg/L anhydrous magnesium sulfate 0 — l〇〇mg/L 2125-8759-PF; Kai 18 200806793 Disodium hydrogen sulphate 100 — 250mg/L Sugar: Dextr〇se 1200 — 4500mg/L Amino acid:

L - alanine 5 - 25mg / L

L-arginine HC1 84 — 450mg/L

L one day aspartic acid · H20 15 — 30mg/L

L - aspartic acid 10 - 30mg / L

L - cystine 2 Na salt 0-90mg/L

L - cysteine H20 0-40mg/L

L-glutamic acid 10-80mg/L

Glycine 5 - 30mg / L

L-histamine HC1 · H20 20 — 50mg/L

L -isoleucine 2 - llOmg/L

L-leucine 10 – 110 mg/L

L-isoamine acid HC1 30 — 1 50mg/L

L — 曱 thiamic acid 3 — 30mg/L

L - phenylalanine 4 - 80mg/L

L-proline 50 - 70mg/L

L-serine 1 0 — 5 0 m g / L

L-threonine 10 - 1 00mg/L

L-tryptophan 2-20mg/L

L-tyrosine 2 Na · 2H20 5 - 1 1 0mg/L

L - leucine 1 0 - 1 0 0 mg / L 2125-8759-PF; Kai 19 200806793

• L - sulphate 140 - 600mg / L vitamins, etc.:

Folic Acid l - 5 mg / L Inositol 7 - 20 mg / L in Nitrotinic Acid Amide 0. 3 - 5mg / L Riboflavin O. 02 - 0 · 4mg /L Thiamine HCl 0.3-5mg/L Biotin (d-Biot in) 0.007-0. 0 8mg/L Pantothenic Acid #5 Salt 0.2-5mg/L Pyruvic Acid Sodium Salt 1 00 — 250 mg/L Vitamin B-12 0.01 — 2 mg/L Pyridoxine HCl 0.05 — 5 mg/L Choline Chloride 4 — 15 mg/L Buffer:

Phenol red, sodium salt 〇 - 15 mg / L HEPES, acid type 〇 - 6000 mg / L HEPES, 1 steel salt 〇 - 68Omg / L sodium bicarbonate 1 0 00 - 3500mg / L In general, in the basal medium of the present invention, A medium composition of a commercially available basal medium can be utilized. For example, a culture medium such as the following may be used as a base medium in the present invention. IMDM medium (iscove, 3 Modified DMEM) (Sigma, USA) F12K medium (F-12 Nutrient Mixture (Ham, s F12) 2125-8759-PF; Kai 20 200806793

Kaighn's Modification) (Invitr〇gen HCM medium (Cambrex), etc., may also contain serum to improve the cell basal medium 'in addition to the above-mentioned representative composition plus ingredients. For example, by adding albumin or animal development Supporting ability. In the present invention, the mesenchymal stem cells are differentiated into mature liver cells by culturing the mesenchymal stem cells in the presence of oncostatin M 'dexamethasone and (10)-oxime. It is cultured by a general animal cell culture method. Specific culture conditions are, for example, about 5% of a c〇2 atmosphere, and a temperature of about 3 。. Further, in the present invention, it is used to induce differentiation into mature hepatocytes. It is advantageous to use a collagen-coated culture container. A commercially available collagen-coated plate (made of AsahiTechno glass) or the like can be used in the method of the present invention. In the present invention, the number of cells at the start of differentiation induction supports cell survival. And the monthly sputum can be appropriately adjusted in the range of inducible differentiation. Specifically, the case can be inoculated with 5·0x1 〇3~5· 0x1 〇6 cells/culture cells. The conditions for culturing for 3 days or more, usually 5 days or longer, preferably 6 days or more, or more than 10 days, are various conventional techniques for inducing differentiation of mesenchymal stem cells into mature hepatocytes as characteristics of mature hepatocytes. By observing these markers, it is possible to confirm that the cells have been differentiated into mature hepatocytes, "field cells. The present invention, the mature liver cells, for example, have the following characteristics (1) one (4) complex φ $ " One of the cells of Ningxia Xi, especially with these indicators

The cells of both (2) and (3) are preferably used for compound screening or regenerative medicine. Or go to H cells with 4 inch sign (4), as hepatitis virus infection 2125-8759-PF; Kai 21 200806793 test host cells are useful. (1) Morphological characteristics: The whole has a circular shape; the cytoplasm contains particles; has a distinct nucleus and a clear nucleolus. The above-mentioned 4-inch 彳 开, can be observed by microscopy. Confirmation (2) characteristic gene expression of hepatocytes (at least one, preferably plural, desirably all kinds of albumin (ALB); 酉-aminoglycine aminotransferase (TAT); 3-oxidase (TD02) ) tryptophan cytochrome P450 (CYP1A2, CYP3A4, CYp2D6); thyroxine protein (TTR); Dole-resistant protein (MRP, MRP2, MRP3) multidrug-related protein (MDR1, MDR3) The primers for each gene are used to amplify the primers. Further, it is possible to combine the probes for the hybridization of the genes for each gene, and specifically combine the amplification products, 7 w or /. ATAC - PCR 'is a representative method for detecting the amplification of PCR products using 'needle detection'. 0 (3) Functional characteristics: (at least 1 kind is preferred for most species, hopefully for all types of glucose production Ability; gas metabolism capacity; 2125-8759-PF; Kai 22 200806793 曰 protein production capacity; urea synthesis ability to confirm the above-mentioned functions of the square 匕 *, π #, such as the production of glucose, b analysis of glucose in the culture supernatant by glucose oxidase method Horizontal and exact. Gas ice i L Yue Yue I Portuguese word sugar, "H ° shot moon b force" can be changed by indophenol (indnPhenol) method (^nDB & SquireCR' Chim·八咖.i4: i85 1 966), confirming the ammonia level in the culture medium and confirming that the albumin production energy h can be confirmed by measuring the albumin concentration in the culture solution by measuring the concentration of serum albumin I. For example, it can be confirmed by a colorimetric assay (Colorimetric assay, Sigma). (4) Viral sensitivity: Human hepatitis C virus-sensitive hepatocyte-like infection of human hepatitis C virus can be exemplified by, for example, "also It is not known to be confirmed. That is, by the RT-PCR using the template recovered from the cells as a template, the proliferation of the hepatitis G virus can be achieved. The primer for amplifying the RNA of the human hepatitis C virus is known (T. Takeuch). M al.

Real-Time Detection System f〇r Quantification 〇f

Hepatitis C Virus Genome. Gastroenterology 1999, 116:636 — 642). In general, a cell having both a morphological characteristic and a gene expression profiling similar to that of a normal human cultured liver cell is called a liver-like, a cell-cell, and a specific, for example, a cytochrome. (cyt〇chr〇me) P450 (CYP), multidrug resistance-associated protein (MRP), and multi-dose tolerance (MDR) protein cells, including hepatocytes 23 2125-8759-PF; Kai 200806793 (hepatocyte — 1 ike t , e cells). Among the hepatocytes, there are cells with similar characteristics to those of the first generation of human cultured hepatocytes, especially hepatocytes with high degree of sputum. ° ‘For example, as recorded in the aforementioned feature (3), a cell having liver functional characteristics can be a cell of high maturity. Therefore, hepatocytes having the aforementioned hepatocyte characteristics (2) to (4) are included in the cell of the present invention (fflatUrehepatocyte-llkeceUs). In the present invention, it is preferable to provide at least two kinds of characteristics of the mature hepatocyte functional characteristics (3), preferably more than two types, more preferably all types of cells, which are included in the mature liver cells of the present invention. In the present invention, the proliferation-differentiating factor, dexamethasone M, dexamethasone, and TGF-Wan, which are induced to differentiate from mature hepatocytes of Lizhu and cytoplasm, induce differentiation from mesenchymal stem cells to mature hepatocytes. The test is useful. That is, the present invention relates to a drug comprising a tumor suppressor, a dexamethasone Ha-/5, which induces differentiation from a mesenchymal stem cell to a mature liver cell. In the present invention, by adding the tumor suppressor M to the milk, and cooling the tgf to the basal medium, it is possible to prepare a medium for inducing the use of the stem cells from the sputum to the mature hepatocytes. Induction of the present invention in the test of differentiation of mature hepatocytes, in the above-mentioned induction and cultivating, together with the amount required to provide the concentration of each proliferation and differentiation factor, Dexamethasone, and TGF — /3. For example, in the case of the proliferation-differentiation factor of the above-mentioned amount per liter of the culture medium, the present invention can be made into a test for the differentiation of mature liver cells. Inhibin Μ : 1 // g~1 0 0 // g, compared to the death of 1 soil is 5"g~50/zg 2125-8759-PF; Kai 24 200806793 dexamethasone: 〇 · lmM ~ 1〇 mM, preferably 〇 5 mM to 5 mM TGF - /3 : 〇 · 2 " g 〜 2 〇 / / g, preferably i " g 〜 1 〇 vg The invention induces differentiation into mature hepatocytes In the medicine, the ingredients to be added are added to the differentiation-inducing reagent of the present invention, for example, L-guanamine which is often added to the basal medium. Further, various antibiotics or additional nutrients may be blended. " The present invention provides the function of a mature hepatocyte produced according to the method of the present invention, and the function or gait of a mature hepatocyte produced by the method of the present invention, and the function of a hepatocyte manufactured according to a conventional method. Or morphology: more similar to the characteristics of human mature hepatocytes. It is apparent that mature hepatocytes such as the present invention are useful, for example, in the medical field. More specifically, ancient liver cells according to the present invention, It is useful as a regenerative medical work 1. A mature liver can be obtained according to the present invention. The cells are, for example, inoculated in a culture dish for various tests in vitro. Or, by injecting the mature liver cells of the present invention into the body, you can reconstruct the liver tissue. By reconstituting the liver tissue, it can be treated. Liver disease. 士' : The cells after the knife is treated with a solution containing the enzyme and placed in a test tube to recover the cells under mild conditions, and the hepatocytes of the present invention are concentrated. The enzyme treatment of the cells + You can use Shengyuan egg white enzyme (C〇Uage job e) or neutral protease (...ah), etc. As a mild condition for recovering cells, you can use the physical properties like centrifugation at a lower speed. Biochemical operation. The shape of the musk cell is cultivated for various purposes, and it can be recovered 2125-8759-PF; Kai 25 200806793

The mature liver sputum of the present invention, M ..., ,, ' I, is released in a suitable culture container. Culture vessel: Use a well dish or a 24-well plate. Alternatively, the cells may be administered to a living body. The mature hepatocytes of the present invention may be suspended in a suitable culture solution or buffer and then injected into a living body. The cell suspension can be administered intravenously or via the portal. Alternatively, hot hepatocytes such as the present invention can be administered to a living body by subcutaneous administration or intraperitoneal administration. Furthermore, it is also possible to treat the disease by transplanting the mature liver cells embedded in the living affinity material boat Xiweig; As the living affinity material, a known material can be used for collagen or polyurethane vinegar. Alternatively, mature hepatocytes provided in accordance with the present invention may be utilized as an artificial liver. In the present invention, the person who compensates for liver function, w. transplanted to the living body "hepatic function. The body = ΓΓ contact with the patient's body fluid." The liver has a mature cell structure to maintain the blood of the patient's blood and mature stem cells For example, the machine t is used for transplanting an artificial liver in a living body, and for example, a serum-permeable human, a cell-holding material retains a mature-like liver cell. The liver has been transplanted into an adult liver to contact with serum to carry out blood components. The metabolism of 〆 力 企 企 ' ' 将 将 将 将 ' ' ' ' ' ' ' ' ' ' ' 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 = = = = = = = = = = = = = After being taken out to the living body, the human mature liver cells are contacted, and the blood is returned to the patient's cattle: membrane, in the treatment method, the mature liver cells can be passed through, for example, a blood-permeable blood, too _/month through the 14 membrane. The patient's blood contact has been transmitted through the dialysis membrane 'Mengjing' from the mature hepatocytes, so that the components in the serum are metabolized by the action of the mature liver cells 26 2l25-8759-PF; Kai 200806793. The serum after contact is returned to the bloodstream through the dialysis membrane. 'This invention S provides the use of mature hepatocytes such as those manufactured by the method described in the present invention. That is, the present invention provides a composition according to the present invention. The method provides a therapeutic agent for liver diseases such as mature hepatocytes. Further, a method for treating liver diseases, comprising the step of administering such mature liver cells to a patient having liver diseases. The liver disease of the present invention comprises the energy Various diseases such as hepatic diseases, such as liver cirrhosis, fuiminanthepatitis, biliary atresia, liver cancer, hepatitis, etc. Hepatitis includes, for example, viral hepatitis or Alcoholic Hepatitis In addition, the human mature hepatocytes of the present invention are also useful for the research field for the purpose of treating liver diseases. For example, in the research and development of artificial liver, τ uses mature hepatocytes such as the present invention. The silk hepatocytes of the present invention are also useful in the development field of pharmaceuticals or foods, etc. Specifically, they can be used for test compounds. Evaluation of metabolism or hepatotoxicity, screening of therapeutic agents for diseases, inhibitors of hepatitis virus infection, or therapeutic agents for viral hepatitis. $ Human mature liver cells, which are manufactured by the method of the present invention, and heart-shaped sputum Metabolic or hepatotoxicity. That is, the present invention provides a method for "metabolism of a guanidine compound in the liver, comprising the steps of: (1) bringing mature liver cells into contact with a test compound, and (2) measuring mature hepatocyte tissue. The test compound is metabolized; the mature hepatocyte cell line is differentiated from mesenchymal stem cells by culturing mesenchymal stem cells in the presence of oncostatin M, dexamethasone, and (10) 2l25-8759~PF; Kai 27 200806793 Come. Or the present invention provides a method for detecting hepatotoxicity of a test compound, comprising the steps of: (1) contacting a mature liver cell with a test compound, and (2) detecting when the mature liver cell is damaged. Hepatotoxicity of test compounds; Mature liver cell lines differentiated from mesenchymal stem cells by culturing mesenchymal stem cells in the presence of oncostatin M, dexamethasone, and /5. In the present invention, the test compound is not particularly limited. For example, living foreign bodies, natural compounds, organic compounds, inorganic compounds, white matter, peptides, and the like, a compound library of a substance, a product of a gene bank, a cell extract, a cell culture supernatant, a fermented microorganism product, a marine organism extract Living foreign matter such as objects and plant extracts contains various living foreign bodies that are foreign bodies to the living body. For example, a candidate compound of an agent or a food, an existing drug or food σα °, the test compound can usually be contacted with a mature liver cell by adding a test compound to a medium or a culture solution. In addition, the test compound and the mature liver can be made by causing the two to be exposed to H by co-culturing with the cell producing the test compound by expressing the gene encoded as the test compound in the mature liver cell. Cell contact. Further, the metabolism of the oral compound can be measured by a method known to those skilled in the art. For example, the condition that the metabolite of the test compound is detected by Uber 2125-8759-PF; and the shape of Kai 28 200806793 determines that the test compound has been metabolized. Further, when the test compound is contacted, the expression of an enzyme gene such as cytochrome (cytochr(10)e) p45〇, a surface, or MRp is induced, or the activity of the enzyme is increased, and it can be judged that The test compound has been metabolized. Methods for predicting metabolites are well known. For example, a test compound or a metabolite thereof can be extracted from a culture of hepatocyte-like cells, and analyzed by liquid chromatography or mass analysis. ^In the case of predicting a metabolite in advance, the presence of the metabolite can be confirmed by such an analysis method, in the case of predicting metabolism to carbon dioxide or water, etc., if a radiolabeled compound is used as a test It is confirmed that the metabolism is carbon dioxide or water by combining the vertical radioactivity of mu W. Alternatively, in the case of the expression of genes related to drug metabolism such as CYP, _, and _, the knife J detects the mRNA of the gene.

It can be tested by +-, #, ai mKNA by RT-PCR and other methods. For example, the method described in the examples below can be used. Or ' can also be used as the enzyme activity of CYP. A reagent for measuring the enzyme activity of (10) is commercially available. ,,which performed. The second evaluation of hepatotoxicity is followed by measuring the degree of damage to the mature hepatocytes in contact with the test compound. The degree of damage can be measured, for example, by using a human-like mature liver fine tire & six-t using field survival rate or G()T "GpT and other liver indicators. Read '^ yourself as, for example, 'hunting because of human culture of mature liver cells, the human body turned over #1 ^^ plus double test, and the survival rate of the hepatocytes was reduced, and the test compound was judged. If there is ^, then there is - liver f sex. On the other hand, in the case where the survival rate is not significantly changed from 2125 to 8759, as in the case of 29 200806793, it is judged that the aroma and the compound of the formula 5 have no hepatotoxicity. Further, for example, when a test compound is added to a human heart-like mature hepatocyte, the test compound is determined by the case where GOT戎GPT i & τ is raised in the culture solution.

Has liver toxicity. Similarly, in the case where there was a significant change in G0T 忒/, it was judged that the test compound did not have hepatotoxicity. Further, the hunting was performed by quantitatively evaluating the hepatotoxicity of the test compound by using a compound which has been judged to have hepatotoxicity as a control. Evaluation of metabolism or hepatotoxicity of the conjugate: In the past, animals and the like were used, but the number of test compounds that can be evaluated at one time is limited. Moreover, there will be $ _ > in the animal model, etc. The main 伃 之 sweat can not be applied to human problems in the original state. Therefore, the recent evaluation of the use of human hepatoma cell lines or the first generation of human cultured hepatocytes in the first place, such as 1 & ^, I. I T is not lingering, because human liver cancer cell lines are cancer cells Therefore, the evaluation of human liver cancer cells and human liver cancer cell lines is still not applicable to the human right η - the possibility that humans are hanging liver cells. Hey, the first generation of normal humans = hepatocytes have problems with stable supply or cost. In addition, the first generation of human hepatocytes was cultured as an undead cell line, and compared with the case of not dead, it showed that CYP3A4 activity was degraded. 低η+ + . + low (international J0urnai of (10) 咖^❿111614··663 — 668 2_', such as -^., can be used to solve such problems as mature hepatocytes produced by the method of the present invention. Mature liver cells such as ',,, and Uf can be used in sieves. K-hepatic disease therapeutic agent, which is a method for screening a therapeutic agent for liver diseases, comprising the following steps: (1) bringing mature liver cells into contact with a test compound, 2125~8759 -PF;Kai 30 200806793 • (2) Measure the function of mature hepatocytes such as fish sputum/μ human ^, and sputum compound contact; and (3) 舁 舁 舁 比较 , , 选择a compound that functions as a function of cells; several 'hot hepatic cell lines differentiate from mesenchymal stem cells by culturing mesenchymal stem cells in the presence of oncostatin M, dexamethasone, and (10) 10,000 In this turtle, after being found in contact with the test compound In the case of mature hepatocytes, in the case of hyperactivity, the therapeutic effect of the test compound on the liver can be measured. In the screening method of the present invention, the test compound can be evaluated by the above-described metabolism or hepatotoxicity. The test compound is treated in the same manner to be in contact with mature liver cells. & The function of the mature hepatocytes in the present invention can utilize, for example, glucose production energy, force, ammonia metabolism ability, albumin production capacity, urea synthesis The activity of enzymes such as CYP and other factors are evaluated as indicators. The glucose production capacity can be confirmed by glucose oxidase analysis of the glucose level in the culture supernatant. The ammonia metabolism ability can be changed by the phenol method (Horn DB & Squire CR, Chim· Acta· 14: 185 - 194 1 966 ), which is determined by analyzing the ammonia level in the medium. The albumin production capacity can be determined by measuring the serum albumin concentration by analyzing the albumin concentration in the culture solution. Further, the urea synthesis ability can be confirmed using, for example, a colorimetric assay (Colorimetric assay, Sigma). The cYp of the present invention is not For the limitation, for example, CYP1A:1, CYP2C8, CYP2C9, CYP3A4, etc. can be used. The method for measuring the activity of CYP can be carried out by a method known in the art of 31 2l25-8759-PF; Kai 200806793. Any test compound to be evaluated for the improvement of liver function is used. Specifically, a library of natural substances or synthetic compounds can be used as the test compound. The natural substance is extracted from plants, animals, insects, or microorganisms. ingredient. Alternatively, commercially available libraries of compounds can also be screened by the methods of the invention. The function or morphology of mature hepatocytes produced by the method of the present invention is similar to that of mature hepatocytes, and thus can be infected by hepatitis virus. Therefore, mature hepatocytes such as the present invention can be utilized as a method for screening hepatitis virus infection inhibitors. That is, the present invention provides a method for screening an infection inhibitor of hepatitis virus, comprising the steps of: (1) contacting a mature liver cell with a hepatitis virus in the presence of a test compound, or contacting the mature liver cell with a hepatitis virus Determination of the level of mature hepatocytes infected with hepatitis virus (2) compared with the control group; and (3) selection of compounds with low levels of hepatitis virus infection of mature liver cells compared with the control group; The cell line is differentiated from mesenchymal stem cells by culturing mesenchymal stem cells in the presence of oncostatin, dexamethasone, and butyl gf-10,000. By the screening method of the present invention, it is possible to screen for infection inhibitors of various viruses which infect heat-forming hepatocytes such as the present invention. Specifically, C human liver disease type f hepatitis A virus and hepatitis B virus can be used as targets. These hepatitis viruses may be either strained or infected with hepatitis virus: 2125-8759-PF/Kai 32 200806793. Further, it may be in a state of being purified, or may be in a crude state (for example, a state of serum obtained from an infected person). The buccal mature hepatocytes are examined by the amount of hepatitis virus in the cells of π π 1 Μ. Cell Long-term virus I can be determined, for example, by using the amount of RNA of a hepatitis virus in a cell as an index. The RNA* of the liver inflammation virus can be measured according to the method. In addition, the method established by the inventor and the like is measured (τ·心(四)eHtai.

Real — Time Detection System f〇r fh ,.,. ior Quantification of

Hepatitis C Virus Genome. Gastm〇 + Ί , gastroenterology 1999, 116:636 — 642). Alternatively, a mature liver cell package obtained in accordance with the present invention is also useful for screening for therapeutic agents for viral hepatitis. #即, The present invention provides a method for screening a therapeutic agent for cancerous hepatitis, comprising the steps of: (a contacting a mature liver cell such as a hepatitis virus with a test compound, and (2) measuring a mature liver cell. Proliferation of hepatitis virus; and (3) selecting a compound that detects inhibition of hepatitis virus proliferation compared to a control group;

The mature hepatocyte cell line is differentiated from mesenchymal stem cells by culturing mesenchymal stem cells in the presence of oncostatin, dexamethasone, and TGF/5. The compound for inhibiting the proliferation of hepatitis virus of the present invention comprises a compound such as the following. 1) Compounds that inhibit hepatitis virus 2125-8759-PF/Kai 33 200806793, 2) Compounds that completely inhibit hepatitis virus proliferation, and 3) Disappear the hepatitis virus compared with the case where the test compound is not contacted Proliferation or elimination of the compound hepatitis virus ^ (3) The error can be checked by measuring the liver inflammation in the cells. The method for screening a hepatitis virus inhibitor of the present invention or the method for treating a therapeutic agent for a sputum hepatitis can be used as a virulence virus infection inhibitor or a virus. Arbitrarily character. Specifically, a natural substance " can be used as a test compound. Natural substances, no rhinoceros g s攸 plants, animals 'Kun microorganisms and other components extracted. Alternatively, the mites or ._n J will be commercially available and screened according to the method of the present invention. As mentioned above, the hepatitis virus can be cultured by using mature liver cells such as the stalker of the scorpion. The method of hepatitis virus has included the step of raising the nutrient; the mature liver cell line is derived from the parental sense of R, such as M, dexamethasone, and

- The presence of mesenchymal stem cells in the presence of 10,000, and TGF. According to the present invention, the cell is differentiated, the cell is differentiated, and the side of the silkworm hepatitis virus is

Hepatitis virus. L 仏 毋 is C. The method of cultivating hepatitis virus of the present invention is useful for subculture or amplification. Alternatively, the hepatitis virus is isolated using the invention, the isolated disease or a sample of the patient. For example, the hepatitis virus isolate of the blood culture test of the virus of the present invention is carried out by taking the loop sample as a sample. / , can be used from the 2125-8759-PF/Kai 34 200806793 Leuk Hepatitis virus strain to use the amplification of viral genes to measure the debt. However, due to the amplification method, Lees will still detect the virus even if the virus is lost. In addition, in the other way, the virus that actually maintains the infectivity can be separated by the method of separating the disease and the virus. The mother-in-law T is used in the infection model of the C-type hepatic fire virus reported in vitro and in vitro, and is used for infection experiments without the alpha, alpha, and knives. Therefore, it is not practical to use hepatocytes in vitro to develop infection inhibition of hepatitis C virus. And because the ideal model of viral infection is still going to be the main, there is no progress in the study of the life history of hepatitis C virus. In contrast, the inventors of the present invention found that mature hepatocytes such as the present invention are highly efficiently infected by a d-type hepatitis B virus. This result shows that the mature hepatocytes such as Shishi and Yishen can be screened for the infection of c-type liver disease, which inhibits the sword-type liver fire d A liver fire treatment agent, which can solve the life history of hepatitis C virus.

In addition, all the prior technical documents cited by the field A τ in the book of the brothers are added to the specification as a spring. ν ^ ', ,, [Examples] [Example 1] Culture of human mesenchymal stem cells will be taken from MediP〇st Bi (10) edical Research Institute (庠早国) from human umbilical cord blood phylogenetic stem cells In the swelling medium (Τ0Υ0Β0 company, 曰太,, 卢丄^J Japan), it was cultured and propagated on a tissue culture plate in a humidified atmosphere containing 5% c〇2 and 3rc. The cells were subcultured at a ratio of 2 to 3 days. On the other hand, phylloblastic stem cells derived from human bone marrow 2125-8759-PF; Kai 35 200806793 • (Cambrex Corporation, USA) were cultured and propagated in the same manner as described above using MSCG medium (Cambrex, USA). [Example 2] Differentiation induction of human mesenchymal stem cells [2-1] Modulation of medium In the IMDM medium (Sigma, USA), the following components were added to prepare a medium. Inhibin M used for the preparation of the medium is a recombinant (a thermogenic protein composed of 227 amino acid residues) which is obtained by coliformation of human oncostatin. On the other hand, TGF is a cold protein, which is a mature protein composed of human recombinant TGF-amino acid residues obtained by c-shave cells. 1 # dexamethasone (Dexamethas〇ne) { Sigma, USA}, l〇ng/ml Oncostatin m (〇SM) {Peprotech, USA}, 2ng/ral TGF-A 1 { r&d , USA}, ITS-A (insulin, transpiercing, selenium mixture, §igma, USA), L-faced amine (Sigma, USA), PSA (penicillin, streptomycin, nodular streptococcus B) Mixture, Invitrogen, USA) For the basal medium, F12K (InVitr〇gen) or HCM medium (Cambrex) was used to obtain the same result in differentiation induction in addition to the above-mentioned ΜΜ medium. Hereinafter, those which are based on such a medium and added with the above components are described below. Using IMDM as the basic medium·· medium; F12K as the basic medium··FKD〇T medium; 2125-8759-PF; Kai 36 200806793 • Using HCM as the basic medium: HD0T medium [2 2] differentiation induction The leaf-derived stem cells derived from human umbilical cord blood cultured in the first embodiment were separated by a 0.05% trypsin® solution (Sigma, USA) k plastic disk. After the cells were washed, they were suspended in the above I(10)T medium, and inoculated on a J-type collagen protein-coated plate (Asahi Techno Glass Co., Ltd.) at a ratio of 30,000 to 60,000 per 1 cm 2 . The cells inoculated on the plate were cultured in 3 rc in a humidified atmosphere under 5% c〇2 to induce differentiation of the cells. Further, the same results were obtained by using the ID0T medium instead of the HD〇T medium. For the stem cells derived from human bone marrow cultured in Example 1, the HD0T medium was also used to induce differentiation in the same manner as described above. Further, the same results were obtained by using HD0T medium instead of using F〇〇T medium or id〇t medium. [Example 3] Morphology of hepatocytes derived from human mesenchymal stem cells The cells derived from the phylogenetic stem cells derived from human cord blood and the stem cells derived from human bone marrow were observed under a microscope. The cells cultured in the undifferentiated human mesenchymal stem cells or the medium to which the differentiation inducing factor is not added exhibit an elongated form of fibroblasts. On the other hand, the cells cultured in the medium supplemented with dexamethasone (Dexamethassne), Oncostatin M (〇SM), and TG{? 1/5 1 have been presented on the 2nd day after the start of the culture. Round shape. Up to the week after the start of culture, the cytoplasmic king has granules with a distinct nucleus and clear nucleoli such as hepatocyte shape 2125-8759-PF; Kai 37 200806793, after which the particles increase with time. This phenomenon represents a cell cultured in a medium to which a differentiation-inducing factor is added, and is morphologically differentiated into hepatocyte-like cells with high efficiency. In Fig. 1, the morphological changes from stem cells derived from human umbilical cord blood and from bone marrow to hematopoietic cells are shown. Figure A of Figure 1 shows the morphology of leaf cell stem cells between undifferentiated human cord blood, showing the morphology of fibroblasts. B shows the morphology of the leaf axillary stem cells between the human J γ blood on the 5th day after the initiation of differentiation induction, and it was found that the whole was in the form of a circle. C is the stem cell shape of the human bone marrow on the 2nd day after the initiation of differentiation induction, and the stem cell between the bone marrow is also rounded in the second cell. D shows the morphology of the leaf cell stem cells between human bone marrow on the 9th day after the initiation of differentiation induction, and it is known that the whole is in a circular shape as in the case of sputum. Also, the nuclear is white, and the nucleolus is clearly visible. Fig. 2 shows the form of the fifth day when hepatic cells are induced by differentiation of phylogenetic stem cells between human bone marrow. The ID0T, HD0T, and FKD0T in Fig. 2 show the difference in the basal medium to which the differentiation factor is added. On the left is a low magnification shirt image (xlOO) ‘on the right is a high magnification image (χ2〇〇). It can be known from the case of culture in any of the culture bases that the whole body is rounded on the 5th day after the start of differentiation, and in the double-magnified image, the nucleus is white and the nucleolus is clearly visible. [Example 4] Analysis of gene expression [4 1 ] Analysis of gene expression analysis Human mesenchymal stem cells were supplemented with dexamethasone (DeXamethaS〇ne), oncostatin M (OSM), and TGF - /3 1 Medium 2125-8759-PF; Kai 38 200806793. The gene expression of the cultured cells was analyzed by PCR. The genes that were expressed were as follows. Albumin (ALB); tyrosine aminotransferase (TAT); tryptophan 2,3-dioxygenase (TD02); cytochrome P450 (CYP1A2, CYP3A4, CYP2D6); scorpion protein (TTR) And /3 - Actin [4-2] analytical method First, the undifferentiated stem cells derived from human cord blood and the stem cells derived from human cord blood are differentiated and induced. The cells were treated with DNase I (amplification grade reagent; Takara, Kyoto, Japan) using total RNA isolated from IS0GEN solution (Nippongene, Japan). The RT-PCR reaction was carried out using Superscript II reverse transcriptase (Invitrogen, USA). Enlarged into cMA sheets of a single band ^ and, in addition, the identification of the § § has been confirmed using the pcR method using a specific primer. The PCR primers used in RT-PCR are as follows. Further, the cytochrome P450 (CYP1A2, CYP3A4, CYP2D6) gene is used as a primer for the real-time (real-time) pCR of Takara~bio. Albumin (ALB) 5' A GTC ACC AAA TGC TGC ACA GA-3, / Serial Number:! 5' An ACG AGC TCA ACA AGT GCA GT-3' /Sequence Number·· 2 Tryptophan 2, 3 —Dioxide (TD02) 5 — CTG AAG AAA AAG AGG AAC AG—3'/Serial Number: 3 39 2l25-8759-PF; Kai 200806793 5'-TCT GTG CAC CAT GCA CAC AT-3' / SEQ ID NO: 4 tyrosine-aminotransferase (TAT) 5, a TGA GCA GTC TGT CCA CTG CCT-3, / SEQ ID NO: 5 5' - ATG TGA ATG AGG AGG ATC TGA G-3' / SEQ ID NO: 6 Thyroxin (TTR) (pre-albumin, starch-like deposition type 1) 5' A TAC TGG AAG GCA CTT GGC AT-3' /Sequence Number:7 5, One TTC CTT GGG ATT GGT GAC GA-3' /Sequence Number:8 P450 7A1 5' One AGG ACG GTT CCT ACA ACA TC-3' /Serial Number:9 5 , A CGA TCC AAA GGG CAT GTA GT-3' /Sequence Number·· 10 /3 Actin 5' A CAA GAG ATG GCC ACG GCT GCT-3' /Serial Number: 11 5' One TCC TTC TGC ATC CTG TCG GCA-3' / SEQ ID NO: 12 [4 - 3 ] Analytical results by phylogenetic stem cells from human cord blood to dexamethasone (Dexamethasone), oncostatin M(OSM) and TGF — /? 1 were differentiated and cultured, and the expression of hepatocyte-specific genes (albumin, TD02, TAT) was observed 6 days after the start of culture. On the other hand, TD02 and TAT gene expression was not observed in cells treated with dexamethasone (Dexamethasone), Oncostatin M (OSM), and TGF-1 negative medium. This indicates that cells cultured in the presence of dexamethasone (Dexamethasone), oncostatin M (0SM), TGF /5 1 2125-8759-PF; and Kai 40 200806793 also efficiently differentiate into gene expression levels. Hepatocyte-like cells. The 6th day after the start of the culture - the analysis result of PCR is shown in Fig. 3. In Fig. 3, line 1 represents the leaf cell stem cells from human cord blood, and line 2 represents the analysis results of cells after 6 days of differentiation, a is in case actin, B is albumin, C is TAT, and D is TD02RT. - Analytical results of PCR. The cells of the sixth day of differentiation induction have confirmed the gene expression of hepatocyte-specific molecular albumin, TAT, and TD02. Undifferentiated stem cells derived from human umbilical cord blood, and these gene expressions were not confirmed. The results of the decantation are as shown in Figure 4, RT-PCR, 12 days after the start of the culture. In Fig. 4, line 1 is an undifferentiated RT-PCR analysis result from the phylogenetic stem cells derived from human bone marrow, and line 2 is the cell from the 12th day of phylogenetic stem cell differentiation between human bone marrow, line 3 is RT-PCR analysis of the 12th day of phylogenetic stem cell differentiation from human bone marrow with line 2 as different donors, 'Line 4 is RT-PCR from day 12 of stem cell differentiation between human cord blood The results of the analysis, line 5 and line 4 are RT-PCR results of different donors from the human umbilical cord blood stem cell differentiation 帛 12 days, line 6 is undifferentiated from the human umbilical cord blood between the leaf system RT ~ PCR analysis of stem cells. Hepatocyte-specific molecular albumin and TD〇2 gene expression were also confirmed in the differentiation of stem cells derived from human bone marrow. Among the hepatocytes of any type of donor stem cells derived from different human cord blood, the performance of the stone is enhanced and the gene expression of hepatocyte-specific molecule TM is enhanced. In addition, in the differentiation-inducing cells, 2125-8759-PF was observed; Kai 200806793, CYP3A4 showed induction and enhanced expression of CYP2D6. [Scheme 5] Determination of cYp activity of stem cells induced by differentiation [5-1] Determination of CYP activity Human mesenchymal stem cells were supplemented with dexamethasone (Dexamethasone), oncostatin M (〇SM), KF - The cyp activity of the cultured cells was measured by the GL method (pr (10) ega). The active CYP was determined to be CYP3A4 and CYP1A2. [5-2] Measurement method Cells obtained by differentiation induction were separated from the disk by using collagenase/neutral protease, and HCM medium containing 50//L of a luminescent substrate was added to 1 χ1〇5 cells in a suspended state. Further, cells which were not subjected to differentiation induction were cultured on a disk, and the medium in the disk was extracted, washed once with HCM medium, and then HCM medium containing a light-emitting substrate was added. HCM medium containing luminescent substrate, in the case of 24 well plates, add 200 # L, and in the case of 96 well plates, add 50 // L. The luminescent substrate used for the measurement was as follows. P450 - Glo CYP1A2 test (Promega Cat·# V8772), or P450-GloM 〇γρ3Α4 test (promega V88〇1) The plate was incubated at 37 ° C for 4 hours, and after the reaction was completed, the cells were cultured on a month/month. (5 〇 / / L) Moved to a white multi-well plate for Luminescence measurement. Furthermore, add an equal amount of luciferin detection reagent and stir for 1 second. After reacting at room temperature for 20 minutes, the relative luminescence amount (RLU) was measured by a reader. Further, when performing the activity measurement, the culture was carried out at 37 ° C for 4 hours, and the sample was measured in the same manner as the sample, and as the background value of the non-existing fine 2125-8759-PF; Ka i 42 200806793, Correction of the measured value is performed.疋 ^ ^ ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' _ After 3 days of culture, the cYp activity was measured by the above method. [5 - 3 ] The results were obtained by differentiation of human umbilical cord blood stem cells by the method of [2-2], and the W3A4 was measured with time. Activity. Figure 5 shows the results. In Figure 5, it was confirmed that CYP3A4 activity and differentiation-induced sputum increased Dependently. Figure 6 shows a comparison of CYP3A4, 壬 也, „ ^ ' human primary cultured hepatocytes. Line A represents the CYP 3 A 4 activity from cells of the 34th sputum of stem cell differentiation between human bones, and the line B represents human primary cultured hepatocytes (second! Music: #77) (10) A4 activity, line. Represents CYP3A4 activity from human:: between the phylogenetic stem cell differentiation, line 23 represents CYP3A4 activity from the human umbilical cord blood 荦rvpQA/1 ^ threshold stem cell differentiation on the 20th day, line e represents and Line c, n also Ding human umbilical cord blood between the eight children can be I wood π

Shiyan 1茱糸 Stem Cell Knife Degraded 2nd Day CYP3A4 Activity. The surface of CYP3A4 derived from the phylogenetic cell of the leaf line of the human bone marrow is derived from the differentiated cell of the phylogenetic stem cells between human cord blood. / Tongue, which has about the same life as human hepatocytes in the first generation. Secondly, for the stem cells from the human umbilical cord blood, the 21-way I stem cells are induced by [2 - 10,000]. Eli 2125-8759-PF; Kai 43 200806793 rifampcin (RIF) as an inducer of CYP3A4 (Μ·Yueh, Kawahara, J. Raucy, Drug Metab. Dispos. 33, 38-48 (2005) ). Figure 7 shows the ability of CYP3A4 to induce phylogenetic stem cells in human cord blood to differentiate into hepatocytes after induction [2-2] to contain rifampicin (rif ampc i η) 1, 3, 1 0 // The medium of sputum was treated for 2 to 3 days, and the activity of CYP was measured by the method of [5-2]. In Fig. 7, A is the cell activity on the 62nd day of differentiation induction and B is the 17th day. As can be seen from Fig. 7, the concentration of CYP3A4 and the concentration of rifampcin depended on each other. In the case of exposure to rifampicin (r丨f 1 〇 “33 in Μ, compared with uninduced, it increased by about 2.3 times (Fig. 7β). Figure 8 shows the results of CYP1A2 inducing ability. The facial hepatocytes derived from the human bone minerals are differentiated and induced by the method of [2-2] (differentiation induction on the 25th day) to contain Ogilre / (10) 咐az〇le) (0PZ) 5 The medium of 15 and 5 MM was measured for 2 days, and the CYP activity was measured by the method of [Bu 2]. As can be seen from Fig. 8, the concentration of the substance and Ogilvy (10) PZ increased Dependently. The obtained human hepatocytes are cultured with the first-generation normal human liver: in the same way, the human CYP45 fermentation can be induced to be induced by the human CYP45 fermentation, and the human turf can be used for drug screening. Example 6] Analysis of glucose metabolism function of hepatocytes obtained from differentiation-induced human meridian blood. "Stem cells [6-1] experimental method for the survival of human liver cells obtained from [2-2] Chemical function into 2125-8759-PF; Kai 44 200806793 First analysis of hepatocytes like insulin treatment Keep the glucose concentration in production were discussed. The obtained hepatocytes were inoculated into the 96 well plate in wells of 2·0Χ10ν, and washed with PBS after about 24 hours, then added with insulin at each concentration, and then determined to culture the supernatant after 48 j, %. Glucose concentration. Further, the glucose wave degree was determined by using a glucose CII-TESTWAK0 (variable chymase/G0D method) for glucose measurement (Wako Pure Chemical Industries, Ltd.) and using a spectrophotometer. [6-2] Experimental results As shown in Fig. 9, it was found that by adding insulin to each culture solution, the glucose concentration and the concentration of insulin depended on each other. [Recognition 7] Hepatitis C virus infection experiment from hepatocytes derived from the differentiation of phylloblastic stem cells derived from human umbilical cord blood [7-1] The experimental method was applied to wells coated with collagen or matrigel 12 The cell culture plate of the 6 well was inoculated with hepatocytes derived from the differentiation of stem cells derived from human umbilical cord blood. After the cells were fully implanted, they were washed once with a wu Hams E medium. For this cell, the serum of HCV-infected persons in which the presence of infectious HCV has been confirmed is inoculated. The amount of HCV inoculated was 0.5 copies (copy) and 丨· 副本 copies for each cell. At 37. The (3) 2 incubator was allowed to adsorb the cells to the cells for 3 hours, and then washed three times with Wi丨丨iams E medium to remove unadsorbed HCV. The culture medium containing hepatocytes was added thereto, and cultured in a C 2 incubator at 37 °C. After the virus culture begins, from 1 to 1 Q, daily sampling is fine 2125-8759-PF/Kai 45 200806793. The culture solution was removed from the disk, and 5 M concentration of guanidine hydrochloride was added to perform cell sampling. The cell solution dissolved in guanidine hydrochloride was stored at 80 ° C before extraction of HCV-RNA. RNA was extracted from the cell solution dissolved in guanidine hydrochloride according to the method, and the amount of HCV-RNA was quantified by the real-time PCR method. The amount of HCV-RNA was quantified by the method established and reported by the inventors of the present invention (T. Takeuch. et al. Real - Time Detection System for Quantification of Hepatitis C Virus Genome. Gastroenterology 1999, 1 1 6: 636 - 642 ). [7-2] Experimental results Figure 10 shows the relationship between the time after infection with hepatitis C virus (hereinafter HCV) and the amount of HCV-RNA. The infection and culture of HCV were carried out using hepatocytes derived from the differentiation of phylloblastic stem cells derived from human umbilical cord blood, and the infection and proliferation of the virus were confirmed. From this fact, it was confirmed that mature hepatocytes obtained in the method of the present invention can be utilized for screening of HCV infection inhibitors or HCV proliferation inhibitors. [Industrial Applicability] The present invention provides a method capable of inducing differentiation into hepatocyte-like cells from the mesenchymal stem cells, in vitro, in vitro. The present invention is useful as a tool for molecular-level analysis of differentiation of stem cells into differentiation mechanisms of hepatocytes. Further, the present invention provides a liver cell of high maturity. Mature hepatocytes, which can be obtained in accordance with the present invention, can be used to evaluate the effects of compounds on the liver. In the past, studies on the effects of compounds on living organisms (experiments) often used living organisms in 2125-8759~PF; Kai 46 200806793:. For example, a carcinogenic test or a safety evaluation test such as a food additive or an anticancer agent uses an experimental animal. The mature hepatocytes of the present invention, such as the test method, can be used as a new experimental model instead of experimental animals. For example, in many cases, such as a toxicity evaluation test or a food safety evaluation test, examination using a rat is mainstream. In the inspection of animals, it is necessary to have an animal breeding space and the like. Therefore, the number of compounds that can be evaluated at one time is subject to the work. Moreover, in animal experiments, the effects on humans are generally predicted based on the results obtained in rats. The prediction made at this time is called external (eXtraPQlatiQn). However, humans and rats are not different in animal species. Therefore, in general, it is not easy to confirm the accuracy of extrapolation. Moreover, there have been criticisms on the test methods accompanying the sacrifice of animals. Therefore, in recent years, it has gradually shifted to a method of evaluating cells using cultured cells. According to the present invention, it is possible to obtain a liver run that is required for the test. The cost of d is also relatively inexpensive, and the human liver obtained in the present invention maintains an infected state. It can be infected with hepatitis virus. Therefore, mature hepatocytes such as the present invention can be utilized for research on prevention or treatment of diseases prescribed by hepatitis virus. In particular, the species of human beings are highly specific. Now the liver dog disease I for the brother in the month b to infect the human c to create a fat fire test animal, in addition to the chimpanzee, the liver disease, the mother of the disease has not been confirmed 0 black 猸煺 helmet poor β on the treaty was designated as Animal species of high altitude, '", +-page plant species. The international rareness of the academic field has not changed the scarcity. Moreover, although the movement for this is recognized, it can be cultivated efficiently in vitro. Hepatitis 4 7 2125-8759-PF; Kai 200806793: The cell line of the virus has not yet been established. That is to say, the research on hepatitis virus: the nucleus type or culture method is not sufficient. The cf hepatitis virus is difficult to raise. It has caused significant obstacles to the research of its prevention or treatment. In the mature liver cells such as the main and the known, it is confirmed that the hepatitis C disease can be in vitro, that is, the cell level, according to the mature liver cells of the present invention. U-hepatitis virus proliferation or infection test. The mature liver cells of the present invention are beneficial to human hepatitis C disease research. Furthermore, the mature liver cells of the present invention make the function of liver cells highly: also: now .therefore' The mature hepatocytes of the present invention are being used as a dead organ for replacing liver function. For example, the mature liver cells of the present invention are filled in the permeation ' and can be used to circulate blood by circulating blood. Human dialysis or artificial In the heart and lungs, the osmotic membrane with low antigenicity for humans has been put into practical use. Furthermore, the treatment of treating the patient's blood in the blood flow path and returning to the patient has also been carried out daily. By being in this blood flow path The invention can be used to replace the hepatocytes of the patient with the blood of the patient, and can replace the metabolic function of the liver. [Simple description of the figure] Fig. 1 shows the change of the morphological changes of the hepatocytes from the stem cells derived from the human squeezed blood. Photo: a is an undifferentiated stem cell from the human shame ▼ blood. B is the stem cell from the human cord blood on the 5th day after differentiation induction. c is the second inducing differentiation induction. Leaf cell stem cells between human bone marrow. D is the phylogenetic stem cell derived from human bone marrow at the 9th point of differentiation induction. 2l25*-8759-PF; Kai 48 200806793 Figure 2 shows human beings from bone Photographs of the morphology of the leaves on the 5th day after the hepatocytes were isolated. Figure 3 of the knife showed the undifferentiated stem cells from the human stem cells and the RT cells of the liver cells after 6 days of differentiation induction. (10) A photograph showing the fact that the social deduction is derived from phylloblastic stem cells between human cord blood and 2 is a hepatocyte after differentiation. Fig. 4 is a photograph showing the results of the analysis of hepatocytes after 12 days of differentiation induction. Fig. 5 is a graph showing the increase in Cyp3A4 activity accompanying the number of days of differentiation culture. Fig. 6 is a graph showing the activity of CYP3A4 in hepatocytes and differentiation-induced hepatocytes in human primary culture. Figure 7 shows hepatocytes after differentiation induction. A graph showing the increase in CYP3A4 activity and rifampcin concentration. Figure 8 shows CYP1A2 activity and omeprazole in hepatocytes after differentiation induction. Sitting (0ΡΖ) the wave of dependence and rising. Figure 9 shows the results of analysis of glucose metabolism function of hepatocyte-like cells. Fig. 10 shows an experiment of hepatitis C virus infection of hepatocytes induced by differentiation of human umbilical cord blood mesenchymal stem cells. Shows the relationship with HCV-mRNA levels over time after HCV inoculation into hepatocytes. [Main component symbol description] No 0 2125-8759-PF; Kai 49

Claims (1)

200806793 X. Patent application scope: 1 · A method for manufacturing mature hepatocytes, comprising the steps of: cultivating a tumor in the presence of oncostatin, dexamethasone or a derivative thereof or a salt thereof, and TGF-stone The leaf cells are stem cells and the mesenchymal stem cells are differentiated into a type of mature hepatocytes. 2. The method for producing a mature hepatocyte according to claim 1, wherein the mesenchymal stem cell is derived from a leaf cell stem cell between any of the following groups: cord blood, bone marrow, adipose tissue, Placenta, and terminal blood stasis • A method for producing mature hepatocytes as described in Section 2 of the sputum, wherein the mesenchymal stem cells are derived from cord blood stem cells between cord blood. 4. The method of producing mature hepatocytes according to the scope of claim 2, wherein the mesenchymal stem cell line is derived from mesenchymal stem cells between the bone marrow. 5. The method of producing mature hepatocytes according to the invention of claim i, wherein the mesenchymal stem cells are cultured for at least 6 曰. 6. In the medium for the cultivation of mesenchymal stem cells among the mature hepatocytes of the manufacturing type described in the first paragraph of the patent application, the tumor suppressor M, n-base, and the degree of each are 1 to ~β and 〇 · 2~2〇ng/mL. The middle L. hepatocytes are manufactured by the method of claim 1 to 6 of the patent application. 8·-Therapeutic agent for liver diseases, including the first to sixth items # s Mature liver cells made by the method of 9 kinds/one of the patents. - A method for detecting the metabolism of a test compound in the liver, including the following 2125-8759-PF; Kai 50 200806793 Procedure: (1) contacting the mature liver cells with the test compound, and = 贞 ^ such mature liver cells The metabolic condition of the test compound; , : (4) mature hepatocytes, in the presence of antitumor M, dexamethasone or a derivative thereof or a salt thereof, and TGF — /3 in the presence of Cultured mesenchymal stem cells 'differentiated from mesenchymal stem cells. Ίο. A method of detecting hepatotoxicity of a test compound, comprising the steps of: (1) contacting a type of mature hepatocyte with a test compound, and (2) detecting damage to the mature hepatocyte The hepatotoxicity of the test compound is measured; wherein the mature hepatocytes are cultured by mesangial stem cells in the presence of oncostatin M, dexamethasone or a derivative thereof or a salt thereof, and TGF-/3 Differentiated from mesenchymal stem cells. 11 . A screening method for a therapeutic agent for liver diseases, comprising the steps of: (1) contacting a type of mature hepatocytes with a test compound; (2) determining the function of mature hepatocytes after contact with the test compound; and (3) Comparing with a control group, a compound having a function of hyperactivity of the mature liver cells is selected; wherein the mature liver cells are cooled by a tumor suppressor, dexamethasone or a derivative thereof or a salt thereof, and TGF The mesenchymal stem cells are cultured in the presence of the mesenchymal stem cells. 12 · Screening method for hepatitis virus infection inhibitors, including the following steps 2125-8759-PF; Kai 51 200806793 (1) contacting the mature liver cell with a hepatitis virus in the presence of a test compound, or contacting the test compound with the mature liver cell after contacting the hepatitis virus; (2) determining the hepatitis virus infection The degree of mature hepatocytes; and (3) compared with the control group, the hepatitis C virus has a low degree of infection with the mature liver cells; wherein the mature liver cells are obtained by the tumor suppressor M and the ground. Dexamethasone or a derivative thereof or a salt thereof, in which the mesenchymal stem cells are cultured in the presence of A TGFj, is differentiated from mesenchymal stem cells. A screening method for a therapeutic agent for viral hepatitis, comprising the steps of: compound; ° (2) measuring the proliferation of hepatitis virus in such mature hepatocytes; and (3) detecting hepatic liver compared with the control group * Compounds that inhibit the proliferation of hepatitis virus; such mature hepatocytes, by right as, say I μ, Ignita in the tumor, dexamethasone or its derivatives or its salts, and TGF - In the presence of sputum, the mesenchymal stem cells are cultured and differentiated from mesenchymal stem cells. U.-A method for cultivating a hepatitis virus, comprising the step of infecting a type of mature hepatocyte-hepatitis virus. The mature liver cell line is obtained by using a tumor suppressor, dexamethasone or a derivative thereof or a salt thereof, and τ And the presence of TGF-/3 in the presence of sputum stem cells differentiated from mesenchymal stem cells to 2125-8759-PF; Kai 200806793 • 1 5 · A differentiation inducement of mesenchymal stem cells into mature hepatocytes Medicated' includes oncostatin, dexamethasone or a derivative thereof or a salt thereof, and TGF - /5 ° 1 6 · a medium comprising oncostatin, dexamethasone or a derivative thereof or a salt thereof, and TGF ~ call. 1 7. The medium of claim 16, wherein a leaf line stem cell is differentiated into a type of mature liver cell. The medium according to claim 16 or 17, wherein the concentration of 'inhibin guanidine, dexamethasone or a derivative thereof or a salt thereof, and TGF - /3 is 1 ~l〇〇ng/mL, 0·1 ～10//Μ, and 0.2 〜20 ng/mL 0 2125-8759-PF; Kai 53 200806793 SEQUENCE LISTING &lt;110&gt; EFFECTOR CELL INSTITUTE, INC. &lt;120&gt; Method for producing mature hepatocyte-like cells &lt;130〉 ΚΑΝ-A0601P &lt;150> JP 2006-102350 &lt;151> 2006-04-03 &lt;160> 14 &lt;170&gt; Patent In version 3.3 &lt;210〉 1 &lt;211> 20 &lt;212&gt; DNA <213>Artificial &lt;220> &lt;223&gt; An artificially synthesized primer sequence &lt;400> 1 gtcaccaaat gctgcacaga 20 &lt;210> 2 &lt;211> 20 &lt;212> DNA <213> Artificial &lt;220> 20 &lt;223&gt; An artificially synthesized primer sequence &lt;400&gt; 2 acgagctcaa caagtgcagt &lt;210>3 &lt;211> 20 &lt;212> DNA &lt;213>Artificial &lt;220&gt;;223&gt; An artificially synthesized primer sequence &lt;400&g t; 3 ctgaagaaaa agaggaacag 20 &lt;210> 4 &lt;211> 20 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&lt;223&gt; An artificially synthesized primer sequence &lt;400&gt; 4 tctgtgcacc atgcacacat 20 &lt;210 〉 5 &lt;211> 21 &lt;212> DNA &lt;213&gt; Artificial &lt;220&gt;&lt;223&gt; An artificially synthesized primer sequence &lt;400&gt; 5 tgagcagtct gtccactgcc t &lt;1 v(1) 21 200806793 &lt;210>6 &lt;;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;&lt;223&gt; An artificially synthesized primer sequence &lt;400&gt; 6 atgtgaatga ggaggatctg ag 22 &lt;210&gt; 7 &lt;211> 20 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;&lt;223&gt; An artificially synthesized primer sequence <400> 7 tactggaagg cacttggcat 20 &lt;210>8 &lt;211> 20 &lt;212> DNA &lt;213&gt; Artificial <220> &lt;;223&gt; An artificially synthesized primer sequence &lt;400〉 8 ttccttggga ttggtgacga 20 &lt;210>9 &lt;211> 20 &lt;212>DNA &lt;213>Artificial &lt;2 20> &lt;223&gt; Artificially synthesized primer sequence &lt;400&gt; 9 aggacggttc ctacaacatc 20 &lt;210&gt; 10 &lt;211> 20 &lt;212> DNA &lt;213&gt; Artificial &lt;220&gt;&lt;223&gt; Artificially synthesized primer sequence &lt;400&gt; 10 cgatccaaag ggcatgtagt 20 &lt;210>11 &lt;211> 21 &lt;212> DNA &lt;213&gt; Artificial <220> &lt;223&gt; Artificially synthesized primer sequence &lt;400> 11 caagagatgg ccacggctgc t 21 &lt; 210>12 &lt;211> 21 &lt;212&gt; DNA v(2) 200806793 &lt;213> Artificial &lt;220&gt;&lt;223&gt; Artificially synthesized primer sequence &lt;400&gt; 12 tccttctgca tcctgtcggc a &lt;210> 13 &lt;211> 252 &lt;212> PRT &lt;213> Homo sapiens <220> &lt;221&gt; SIGNAL &lt;222> (1)·. (25) &lt;220> &lt;221&gt; mat_peptide &lt;222&gt;(26)&quot;. (221) &lt;400> 13 Met Gly Val Leu Leu Thr Gin Arg Thr Leu Leu Ser Leu Val Leu Ala -25 -20 -15 -10 Leu Leu Phe Pro Ser Met Ala Ser Met Ala Ala lie Gly Ser Cys Ser - 5 one 1 1 5 Lys Glu Tyr Arg Val Leu Leu Gly Gin Leu Gin Lys Gin Thr Asp Leu 10 15 20 Met Gin Asp Thr Ser Arg Leu Leu Asp Pro Tyr lie Arg Me Gin Gly 25 30 35 Leu Asp Val Pro Lys Leu Arg Glu His Cys Arg Glu Arg Pro Gly Ala 40 45 50 55 Phe Pro Ser Glu Glu Thr Leu Arg Gly Leu Gly Arg Arg Gly Phe Leu 60 65 70 Gin Thr Leu Asn Ala Thr Leu Gly Cys Val Leu His Arg Leu Ala Asp 75 80 85 Leu Glu Gin Arg Leu Pro Lys Ala Gin Asp Leu Glu Arg Ser Gly Leu 90 95 100 Asn Ile Glu Asp Leu Glu Lys Leu Gin Met Ala Arg Pro Asn I le Leu 105 110 115 Gly Leu Arg Asn Asn lie Tyr Cys Met Ala Gin Leu Leu Asp Asn Ser 120 125 130 135 Asp Thr Ala Glu Pro Thr Lys Ala Gly Arg Gly Ala Ser Gin Pro Pro 140 145 150 Thr Pro Thr Pro Ala Ser Asp Ala Phe Gin Arg Lys Leu Glu Gly Cys 155 160 165 Arg Phe Leu His Gly Tyr His Arg Phe Met His Ser Val Gly Arg Val 170 175 180 200806793 Phe Ser Lys Trp Gly Glu Ser Pro Asn Arg Ser Arg Arg His Ser Pro 185 190 195 His Gin Ala Leu Arg Lys Gly Val Arg Arg Thr Arg Pro Ser Arg Lys 200 205 210 215 Gly Lys Arg Leu Met Thr Arg Gly Gin Leu Pro Arg 220 225 &lt;210> 14 &lt;211&gt; 118 &lt;212> PRT &lt;213&gt; Homo sapiens &lt;220> &lt;221&gt; SIGNAL &lt;222&gt; (1)..(6) &lt;220&gt;&lt;221>mat_pprint tide &lt;222>(7) 7. (118) &lt;400&gt; 14 Met Pro Pro Ser Gly Leu Arg Leu Leu Pro Leu Leu Leu Pro Leu Leu A 5 -11 5 10 Trp Leu Leu Val Leu Thr Pro Gly Arg Pro Ala Ala Gly Leu Ser Thr 15 20 25 Cys Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile Glu Ala 30 35 40 I le Arg Gly Gin I le Leu Ser Lys Leu Arg Leu Ala Ser Pro Pro Ser 45 50 55 Gin Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala Val Leu Ala Leu 60 65 70 Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala Glu Pro Glu 75 80 85 90 Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu Val Thr Arg Val Leu 95 100 105 Met Val Glu Thr His Asn 110