HK40008841A - Method for preparing human hepatocyte progenitor cells - Google Patents

Description

Preparation method of human liver precursor cell

Technical Field

The present invention relates to a preparation method of human liver precursor cell.

Background

An effective treatment for severe liver disease is now only liver transplantation, but absolute donor deficiency becomes a problem. In order to replace liver transplantation, attempts to induce hepatocytes by differentiation of iPS cells and use them for transplantation therapy have been continued so far.

However, since iPS cells have totipotency of differentiation and high proliferation ability, formation of teratomas following transplantation into immunodeficient mice has also been reported, and when hepatocytes are prepared from iPS cells, even if undifferentiated iPS cells are slightly mixed, there is a risk of tumor formation after transplantation. Further, since an induction method for efficiently differentiating into endoderm cells such as liver using iPS cells has not been established, it is not possible to produce liver cells that can replace liver functions from iPS cells at present.

In response to this situation, the presence of few hepatic precursor cells in adult livers has also begun to be considered as a source of available cells. In addition, recent studies have shown that: in chronic hepatitis, mature hepatocytes are reprogrammed to be precursor cells having a double differentiation ability to differentiate into hepatocytes and biliary epithelial cells (non-patent documents 1 to 4).

Documents of the prior art

Non-patent document

Non-patent document 1 Yanger K.et.al, Genes & Development, pp.719-724,27,2013

Nonpatent document 2, Tanimizu N.et. al, Journal of Biological Chemistry, pp.7589-7598,289(11),2014

Non-patent document 3 Yimlamai D.et.al., Cell, pp.1324-1338,157,2014

Non-patent document 4 Tarlow B.D.et.al., Cell Stem Cell, pp.605-618,15,2014

Disclosure of Invention

The present invention aims to provide a method for reprogramming human mature hepatocytes into human liver precursor cells capable of self-replication in vitro.

The present inventors have conducted extensive studies to solve the above problems, and as a result, have found that human mature hepatocytes can be reprogrammed to self-replicating human hepatocyte precursor cells by culturing in vitro human mature hepatocytes in a medium containing serum, a-83-01(TGF- β signal inhibitor) and CHIR99021(GSK3 inhibitor), thereby completing the present invention.

That is, the present invention provides a method for producing human hepatic precursor cells as described below.

Item 1. a method for preparing human hepatocyte precursor cell, comprising culturing human mature hepatocyte in a medium containing serum, a-83-01 and CHIR 99021.

Item 2. the method of preparing human liver precursor cells according to item 1, wherein the human mature hepatocytes are from an infant.

Item 3. the method of preparing human hepatoprecursor cells according to item 1 or 2, wherein said serum is fetal bovine serum.

Item 4. a human hepatocyte precursor cell, prepared by culturing human mature hepatocytes in a medium containing serum, a-83-01 and CHIR 99021.

An adult hepatocyte according to item 5, induced by the human hepatocyte precursor according to item 4.

Item 6. A method for screening a test substance, which comprises using the mature hepatocyte described in item 5.

An item 7. a method for culturing a hepatitis virus, comprising using the mature hepatocyte described in item 5.

An animal model of human liver obtained by transplanting the mature hepatocytes of item 5 into a non-human mammal.

Item 9. A kit for evaluating the metabolism and/or hepatotoxicity of a test substance using human liver precursor cells or mature hepatocytes,

comprising human hepatoblasts prepared by culturing human mature hepatocytes induced by the human hepatoblasts in a medium containing serum, A-83-01 and CHIR99021, or mature hepatocytes.

According to the present invention, a method for reprogramming human mature hepatocytes into self-replicating hepatocyte precursor cells in vitro can be provided.

Drawings

FIG. 1 is a phase contrast micrograph showing the results of culturing human mature hepatocytes in AC-F medium (A), YAC-F medium (B), or YAC medium (C).

FIG. 2 is a phase contrast micrograph showing the results of culturing human mature hepatocytes in AC-F medium.

FIG. 3 is a phase contrast micrograph showing the results of culturing human mature hepatocytes in AC-F medium or FBS medium.

FIG. 4 is a phase contrast micrograph showing the results of culturing human mature hepatocytes in AC-F medium or FBS medium.

FIG. 5 is a graph showing the change with time of human specific albumin in blood after administration of hepatic precursor cells to cDNA-uPA/SCID mice.

FIG. 6 is a photograph showing immunostaining for expression of human CYP2C9 in the medial right leaf 70 days after administration of hepatic precursor cells to cDNA-uPA/SCID mice.

FIG. 7 is a photograph showing immunostaining for expression of human CYP2C9 in the medial left lobe 70 days after administration of hepatic precursor cells to cDNA-uPA/SCID mice.

FIG. 8 is a graph showing the change with time of human specific albumin in serum after administration of hepatic precursor cells to TK-NOG mice.

FIG. 9 is a photograph of immunostaining showing the expression of human CYP2C9 in the liver 60 days after administration of hepatoprogenitor cells to TK-NOG mice.

FIG. 10 is a photograph of immunostaining showing the expression of human CYP2C9 in the liver 60 days after administration of hepatoprogenitor cells to TK-NOG mice.

FIG. 11 is a graph showing the activity of the metabolic enzyme CYP1A2 in the liver precursor cells of the present invention or in mature hepatocytes differentiated from liver precursor cells (differentiated mature hepatocytes).

Fig. 12 is a graph showing the activity ratio of the metabolic enzyme CYP1a2 under the induction of presence or absence of omeprazole in the mature hepatocytes (differentiated mature hepatocytes) differentiated from the hepatocyte or the hepatocyte of the present invention.

FIG. 13 is a graph showing the activity of the metabolic enzyme CYP3A4 in the liver precursor cells of the present invention or in mature hepatocytes differentiated from liver precursor cells (differentiated mature hepatocytes).

FIG. 14 is a graph showing the activity ratio of the metabolic enzyme CYP3A4 in the presence or absence of phenobarbital induction in the liver precursor cells of the present invention or mature hepatocytes (differentiated mature hepatocytes) differentiated from the liver precursor cells.

Fig. 15 is a graph showing the gene expression level of ALB in the liver precursor cells of the present invention or in mature hepatocytes differentiated from liver precursor cells (differentiated mature hepatocytes).

FIG. 16 is a graph showing the gene expression level of TAT in the liver precursor cells of the present invention or in mature hepatocytes differentiated from liver precursor cells (differentiated mature hepatocytes).

Fig. 17 is a graph showing the gene expression level of TDO2 in the liver precursor cells of the present invention or in mature hepatocytes differentiated from liver precursor cells (differentiated mature hepatocytes).

FIG. 18 is a graph showing the gene expression level of TTR in the liver precursor cells of the present invention or mature hepatocytes differentiated from liver precursor cells (differentiated mature hepatocytes).

FIG. 19 is a graph showing the gene expression level of G6PC in the liver precursor cells of the present invention or in mature hepatocytes differentiated from liver precursor cells (differentiated mature hepatocytes).

Fig. 20 is a graph showing gene expression levels of NTCP in the hepatocyte or a mature hepatocyte differentiated from the hepatocyte (differentiated mature hepatocyte) of the present invention.

FIG. 21 is a graph showing the gene expression levels of CYP1A2 in the liver precursor cells of the present invention or in mature hepatocytes differentiated from liver precursor cells (differentiated mature hepatocytes).

FIG. 22 is a graph showing the gene expression levels of CYP2B6 in the liver precursor cells of the invention or in mature hepatocytes differentiated from liver precursor cells (differentiated mature hepatocytes).

FIG. 23 is a graph showing the gene expression levels of CYP2C9 in the liver precursor cells of the invention or in mature hepatocytes differentiated from liver precursor cells (differentiated mature hepatocytes).

FIG. 24 is a graph showing the gene expression levels of CYP2C19 in the liver precursor cells of the invention or in mature hepatocytes differentiated from liver precursor cells (differentiated mature hepatocytes).

FIG. 25 is a graph showing the gene expression levels of CYP2D6 in the liver precursor cells of the present invention or in mature hepatocytes differentiated from liver precursor cells (differentiated mature hepatocytes).

FIG. 26 is a graph showing the gene expression levels of CYP3A4 in the liver precursor cells of the invention or in mature hepatocytes differentiated from liver precursor cells (differentiated mature hepatocytes).

Fig. 27 is a graph showing the gene expression levels of CYP7a1 in the hepatocyte of the present invention or a mature hepatocyte differentiated from a hepatocyte (differentiated mature hepatocyte).

FIG. 28 is a photograph of the liver precursor cells of the present invention before transplantation of cDNA-uPA/SCID mice.

FIG. 29 is a photograph showing cells obtained by transplanting the liver precursor cells of the present invention to a cDNA-uPA/SCID mouse, then removing the cells and culturing the cells for 4 days.

FIG. 30 is a graph showing the activity of CYP1A2 in hepatocytes obtained from cDNA-uPA/SCID mice.

FIG. 31 is a graph showing the activity of CYP3A4 in hepatocytes taken from cDNA-uPA/SCID mice.

FIG. 32 is a photograph of the liver precursor cells of the present invention before transplantation of cDNA-uPA/SCID mice.

FIG. 33 is a photograph showing cells obtained by transplanting the liver precursor cells of the present invention to a cDNA-uPA/SCID mouse, then removing the cells and culturing the cells for 4 days.

FIG. 34 is a graph showing CYP1A2 activity in hepatocytes taken from cDNA-uPA/SCID mice.

FIG. 35 is a graph showing the activity of CYP3A4 in hepatocytes taken from cDNA-uPA/SCID mice.

FIG. 36 is a photograph of the liver precursor cells of the present invention before transplantation of cDNA-uPA/SCID mice.

FIG. 37 is a photograph showing cells obtained by transplanting the liver precursor cells of the present invention to a cDNA-uPA/SCID mouse, then removing the cells and culturing the cells for 4 days.

FIG. 38 is a graph showing the activity of CYP1A2 in hepatocytes taken from cDNA-uPA/SCID mice.

FIG. 39 is a graph showing the activity of CYP3A4 in hepatocytes taken from cDNA-uPA/SCID mice.

Detailed Description

The invention relates to a preparation method of human liver precursor cells, which comprises the step of culturing human mature liver cells in a culture medium containing serum, A-83-01 and CHIR 99021.

[ human mature hepatocytes ]

The human mature hepatocytes used in the present invention can be obtained from living liver tissues by any known method. In the present specification, the living liver tissue refers to a liver tissue obtained from a human liver after birth. The donor of live liver tissue may survive and die. The age of the individual to whom the living liver tissue is supplied is not limited, but from the viewpoint of cell proliferation, the age is preferably 20 years or less, more preferably 10 years or less, even more preferably an infant (0 to 7 years), and most preferably an infant (0 to 2 years).

The human mature hepatocytes used in the present invention may be cells obtained from a living liver tissue and then cryopreserved, or may be cells obtained from a living liver tissue and then cryopreserved and thawed repeatedly as appropriate, as long as they have the characteristics of mature hepatocytes. The human mature hepatocytes used in the present invention may be commercially available human mature hepatocytes.

In addition, the human mature hepatocytes used in the method of the present invention comprise fully differentiated hepatocytes that have no proliferative capacity in vitro.

The human mature hepatocytes used in the method of the present invention may be hepatocytes obtained by differentiation induction of iPS cells (induced pluripotent stem cells), ES cells (embryonic stem cells), and the like.

[ serum ]

The serum used in the present invention includes, for example, human serum, Fetal Bovine Serum (FBS), bovine serum, calf serum, goat serum, horse serum, pig serum, sheep serum, rabbit serum, rat serum, and the like, preferably FBS, calf serum, and bovine serum, and more preferably FBS. The serum used in the present invention may be derived from serum components such as albumin (bovine, porcine, human, dog, rabbit, rat, mouse, chicken, etc.) and human platelet lysate. The serum used in the present invention may be a commercially available product.

The amount of the serum used in the present invention is 0.1 to 30 v/v%, preferably 1 to 20 v/v%, more preferably 5 to 15 v/v%, even more preferably 8 to 12 v/v%, and most preferably 10 v/v% based on the total amount of the culture medium.

[A-83-01]

A-83-01(CAS No.909910-43-6) is one of TGF- β signal inhibitors, and is capable of selectively inhibiting TGF- β type I/activating receptor-like kinase (ALK5), type I activating/nodal receptor kinase (ALK4), and type Inodal receptor kinase (ALK 7). A-83-01 is also known as 3- (6-Methyl-2-pyridyl) -N-phenyl-4- (4-quinolyl) -1H-pyrazole-1-carbothioamide (3- (6-Methyl-2-pyridyl) -N-phenyl-4- (4-quinolyl) -1H-pyrazole-1-carbothioamide.) although not limited thereto, A-83-01 is available from Wako pure chemical industries, Ltd.

The content of A-83-01 used in the present invention is 0.0001. mu.M to 5. mu.M, preferably 0.001. mu.M to 2. mu.M, more preferably 0.01. mu.M to 1. mu.M, still more preferably 0.05. mu.M to 0.7. mu.M, and most preferably 0.5. mu.M, based on the total amount of the medium.

[CHIR99021]

CHIR99021(CAS No.252917-06-9) is one of GSK-3 β (Glycogen Synthase Kinase 3 β ) inhibitors, and is currently known as the most selective inhibitor, CHIR99021 is also known as 6- [ [2- [ [4- (2,4-dichlorophenyl) -5- (5-methyl-1H-imidazol-2-yl) -2-pyrimidinyl ] amino ] ethyl ] amino ] -3-pyridinecarbonitrile (6- [ [2- [ [4- (2,4-dichlorophenyl) -5- (5-methyl-1H-imidazol-2-yl) -2 pyrilinyl ] amino ] ethyl ] amino ] -3-pyridininebutyril).

The content of CHIR99021 used in the present invention is 0.001. mu.M to 20. mu.M, preferably 0.01. mu.M to 10. mu.M, more preferably 0.1. mu.M to 5. mu.M, still more preferably 0.3. mu.M to 4. mu.M, and most preferably 3. mu.M, based on the total amount of the medium.

[ ROCK inhibitor ]

In the present invention, the medium for culturing human mature hepatocytes may further contain a ROCK inhibitor. The ROCK inhibitor is not limited to, but examples thereof include Y-27632(CAS No.146986-50-7), GSK269962(CASNO.850664-21-0), Fasudil (Fasudil hydrochloride) (CAS No.105628-07-7), and H-1152(CAS No.871543-07-6), preferably Y-27632. Y-27632 is a selective and potent inhibitor of ROCK (Rho-associated coiled coil forming protein kinase)/Rho binding kinase). Y-27632 is also substituted as trans-4- [ (1R) -1-aminoethyl]-N-4-pyridinyl-cyclohexanecarboxamide (trans-4- [ (1R) -1-aminoethyl)]-N-4-pyridinyl-cycloheximide) are known. Y-27632 may be in the form of an episome, a salt such as a hydrochloride or sulfate, or a hydrate. GSK269962A is also referred to as N- [3- [ [2- (4-amino-1,2, 5-)Oxadiazol-3-yl) -1-ethyl-1H-imidazo [4,5-c]Pyridin-6-yl]Oxy radical]Phenyl radical]-4- [2- (4-morpholinyl) ethoxy]Benzamide (N- [3- [ [2- (4-Amino-1,2,5-oxadiazol-3-yl) -1-ethyl-1H-imidazo [4, 5-c)]pyridin-6-yl]oxy]phenyl]-4-[2-(4-morpholinyl)ethoxy]benzamide) are well known. Fasudil (Fasudil hydrochloride) is also known as Fasudil hydrochloride (1- (5-thioisoquinoline) piperazine dihydrochloride (1- (5-isoquinonesulfonyl) homopiperazine hydrochloride)). Fasudil (fasuil hydrochloride) can be in the form of free body, hydrochloride, sulfate and other salts, and can also be in the form of hydrate. H-1152 is also used as (S) - (+) -2-methyl-1- [ (4-methyl-5-isoquinolinyl) sulfonyl]-hexahydro-1H-1, 4-dinitrogenDihydrochloride salt (S) - (+) -2-methyll-1-[(4-methyl-5-isoquinolinyl)sulfonyl]hexahydro-1H-1,4-diazepine dihydrochloride) are known. Although not limited thereto, Y-27632 is available from Wako pure chemical industries, Ltd., SK269962A is available from Axon medchem, Inc., Fasudil (Faudel hydrochloride) is available from Tocrisis Bioscience, Inc., and H-1152 is available from Wako pure chemical industries, Inc., etc.

The ROCK inhibitor used in the present invention is contained in an amount of 0.001. mu.M to 50. mu.M, preferably 0.01. mu.M to 30. mu.M, more preferably 0.1. mu.M to 20. mu.M, still more preferably 1. mu.M to 15. mu.M, and most preferably 10. mu.M, based on the whole culture medium.

In the present invention, 1 to a plurality of genes among various genes known as an induction technique, such as ES cells and iPS cells, or gene products (such as protein and mRNA) thereof, or drugs, etc. may be added to a medium for culturing human mature hepatocytes. In addition, 1 to a plurality of genes among various genes known as an induction technique, such as ES cells and iPS cells, or gene products (such as proteins and mrnas) thereof may be expressed or introduced into mammalian cells.

In the present invention, for example, inhibitors against TGF receptor tyrosine kinase, MEK (mitogen-activated protein kinase)/ERK (extracellular signal-controlled kinases 1 and 2) pathway, low molecular inhibitors [ SU5402 and PD184352 ] of three types of FGF receptor tyrosine kinase 3, MEK/ERK pathway and GSK3, low molecular inhibitors [ PD0325901 ] of MEK/ERK pathway and GSK3, inhibitors against histone methylase G9a, i.e., low molecular compounds [ BIX-01294(BIX) ], azacytidine, trichostatin A (TSA), 7-hydroxyflavone (7-hydroxyflavanone), lysergic acid diethylamide (lysergic acid amide), inhibitors against TGF receptor kinase [ TGF-5942 ], TGF receptor kinase, TGF receptor antagonist III-9622, TGF-598, TGF-597-actinomycin/activin-1 ], TGF-5938, TGF-kinase inhibitor against TGF receptor kinase [ TGF-599, TGF-5938, TGF-599, TGF-59352 ], TGF-kinase inhibitor against TGF-5938, TGF-and TGF-5938, and so on.

In a medium for culturing human mature hepatocytes, small rna (micro rna) used for producing ES cells, iPS cells, and the like may be further used to increase the efficiency of induction into hepatic precursor cells.

In the present invention, various inhibitors or antibodies that inhibit or neutralize the activity of TGF- β and the like may be used in the medium for culturing human mature hepatocytes, and examples of the inhibitor of TGF- β include a TGF- β RI inhibitor, a TGF- β RI kinase inhibitor, and the like.

In the culture of the present invention, it is also preferable to culture on a coated culture dish. As the coating, matrigel coating, collagen coating, gelatin coating, laminin coating, fibronectin coating, or the like can be used. Preferably, a matrigel coating is used as the coating.

Examples of the basic Medium usable in the present invention include ES Medium [ 40% Dulbecco's Modified Eagle Medium (DMEM), 40% F12 Medium (Sigma), 2mM L-glutamine or GlutaMAX (Sigma), 1% of an unnecessary amino acid (Sigma), 0.1mM β -mercaptoethanol (Sigma), 15-20% Knockout serum substitute (Invitrogen), 10. mu.g/ml gentamycin (Invitrogen), 4-10 ng/ml bFGF (FGF2) ] (hereinafter referred to as "CHIES Medium"), a Medium obtained by culturing a mouse embryo in an ES Medium with 0.1mM of β -mercaptoethanol removed therefrom for 24 hours, a Medium prepared by adding 0.1mM of a acclimation Medium such as fibroblast-64-mercaptoethanol (CHIVERES Medium) and 10mM of a Medium such as a Medium (CHIVERAGE), a Medium prepared by adding thereto an optimum Medium such as a culture Medium for culturing mouse embryos for 24 hours (STEELOPTIME), and a Medium for which is not suitable for the culture of human cells under the conditions such as the REPELECSTRESF (STEELS, STEELS-A culture Medium, STEELS Medium (STEELS-10 mM) and the optimum Medium for maintaining the culture (STEELAGENTS culture, STEELS-5, STEELS, and the methods for the non-S culture Medium for the non-S culture, such as the method for the REPELS culture Medium (REPELS culture Medium under the methods for the Invitrogen culture, the methods for the Invitrogen culture Medium, the methods for the use of the methods of the Invitrogen (STEELS, the Seedes Medium, the methods of the Invitrogen (STEELS, the methods of the Invitrogen (STEELS, the methods of the Invitrogen incorporated herein, the CORPORS, the methods of the.

In the present invention, the optimum culture conditions for preparing adult human liver precursor cells from human mature hepatocytes may be appropriately changed according to a conventional method. Although not limited thereto, the optimum culture conditions for preparing adult human liver precursor cells from human mature hepatocytes are as follows, for example.

The culture temperature is as follows: preferably 15 to 45 ℃, more preferably 25 to 40 ℃, and most preferably 37 ℃.

CO₂Concentration: preferably 1% to 10%, more preferably 3% to 8%, and most preferably 5%.

During the culture period: preferably 1 day (24 hours) to 30 days, and more preferably 3 days to 20 days.

In the present invention, any method commonly used by those skilled in the art for culturing ES cells, iPS cells, and the like can be used as a method for propagation culture or subculture of hepatic precursor cells. For example, the following methods can be exemplified: the medium was removed from the cells, washed with PBS (-), added with a cell-detaching solution and left to stand, then centrifuged with D-MEM (high glucose) medium containing 1 × antibiotic-antifungal agent and 10% FBS, and after further removing the supernatant, 1 × antibiotic-antifungal agent, mTeSR1 and 10 μ M Y-27632 were added, and a cell suspension was inoculated into a matrigel-coated, gelatin-coated or collagen-coated culture dish inoculated with MEF, thereby subculturing.

The liver precursor cells of the present invention may have the ability to differentiate into mature hepatocytes or biliary epithelial cells. The hepatic precursor cells may be cryopreserved cells, or may be cells that are suitably repeatedly cryopreserved and thawed. In the present invention, cryopreservation can be performed by suspending the hepatic precursor cells in a cryopreservation solution known to those skilled in the art and cooling the suspension. The suspension can be performed by peeling cells with a peeling agent such as trypsin, transferring the cells to a cryopreservation vessel, appropriately treating the cells, and adding a cryopreservation solution to the vessel.

The cryopreservation solution may contain DMSO (Dimethyl sulfoxide) as a freezing injury protective agent, but since DMSO is cytotoxic, it is preferable to reduce the DMSO content. Examples of the DMSO substitute include glycerin, propylene glycol, and polysaccharides. In the case of DMSO, the DMSO concentration is 5% to 20%, preferably 5% to 10%, and more preferably 10%. In addition, the additives described in WO2007/058308 may be contained. Examples of the cryopreservation solution include those provided by BioVerde, Japan Genetics, Inc., REPROCELL, ZENOAQ, Cosmo Bio, Kohjin-Bio, Thermo Fisher Scientific, and the like.

When the suspended cells are stored in a frozen state, they may be stored at a temperature of from-80 ℃ to-100 ℃ (for example, -80 ℃), and any freezer capable of achieving this temperature may be used. Although not particularly limited, the cooling rate may be appropriately controlled by using a program freezer in order to avoid a sudden temperature change. The cooling rate may be appropriately selected depending on the components of the cryopreservation liquid, and may be performed according to the instructions of the manufacturer of the cryopreservation liquid.

The upper limit of the storage period is not particularly limited as long as the cells cryopreserved under the above conditions retain the same properties as before freezing after thawing, and examples thereof include 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 1 year or more. Since the storage at a lower temperature can suppress the cell damage, the cells can be transferred to a gas phase (about-150 ℃ or lower to-180 ℃ or lower) on liquid nitrogen for storage. In the case of preservation in the gas phase on liquid nitrogen, preservation containers known to those skilled in the art can be used. Although not particularly limited, for example, in the case of storage for 2 weeks or more, it is preferable to store the cells in a gas phase on liquid nitrogen.

Thawing of cryopreserved hepatic precursor cells can be performed by methods well known to those skilled in the art. For example, a method of standing or shaking in a thermostatic bath or a hot water bath at 37 ℃ can be exemplified.

By further culturing the hepatic precursor cells obtained by the present invention, differentiation into mature hepatocytes, biliary epithelial cells, or the like can be achieved.

The mature hepatocytes thus obtained (hereinafter referred to as "differentiated mature hepatocytes") are selected from ALB (Albumin)), AFP (Alpha Fetoprotein), TAT (Tyrosine Aminotransferase), TDO2 (Tryptophan 2,3-dioxygenase), TTR (Transthyretin), G6PC (Glucose-6-phosphatase), NTCP (sodium taurocholate cotransporter), and Cnx32, as compared to the hepatic precursor cells, the expression level of at least 1 gene among CYP1a1 (Cytochrome) P1a1), CYP1a2 (Cytochrome) P1a2), CYP2B6 (Cytochrome) P2B6), CYP2C9 (Cytochrome) P2C9), CYP2C19 (Cytochrome) P2C19), CYP2D6 (Cytochrome) P2D6), CYP3a4 (Cytochrome) P3a4), and CYP7a1 (Cytochrome) P7a1) is significantly increased. Although not limited thereto, the differentiated mature hepatocytes are characterized in that the expression level of the above-mentioned genes is increased 10 to 50000 times as compared with that of the hepatic precursor cells.

The induction of differentiation from human hepatic precursor cells into differentiated mature hepatocytes (hereinafter also referred to as "differentiation" or "induction") can be carried out, for example, by adding oncostatin M and dexamethasone to a basal medium (such as the aforementioned basal medium). The amount of the oncostatin M required to give the concentration of each proliferative differentiation factor in the differentiation induction medium is, for example, 1. mu.g to 100. mu.g, preferably 5. mu.g to 50. mu.g, per 1L of the medium. Dexamethasone is used in an amount required to give a concentration of each proliferative differentiation factor to the differentiation induction medium, for example, 0.1mM to 10mM, preferably 0.5mM to 5mM, per 1L of the medium.

The induction of differentiation from human hepatic precursor cells into differentiated mature hepatocytes can also be performed by transplanting human hepatic precursor cells into the liver or spleen of an animal. The animal to be transplanted is not particularly limited as long as it is an animal capable of inducing differentiation of human hepatocyte precursor cells into differentiated mature hepatocytes, and is preferably a mammal, and examples thereof include a rabbit, a dog, a cat, a guinea pig, a hamster, a mouse, a rat, a sheep, a goat, a pig, a miniature pig, a horse, a cow, a monkey, and the like, and more preferably a mouse, a rat, a miniature pig, and a monkey.

Further, the mature hepatocytes differentiated from the hepatic precursor cells (differentiated mature hepatocytes) have, for example, glucose-producing ability, ammonia-metabolizing ability, albumin-producing ability, urea-synthesizing ability, and the like as functions. The glucose-producing ability can be confirmed by analyzing the glucose level in the culture supernatant by, for example, a glucose oxidase method. The ammonia metabolizing ability can be confirmed, for example, by analyzing the ammonia level in the medium by the modified indophenol method (Horn DB & Squire CR, Chim. acta.14: 185-194.1966). The albumin-producing ability can be confirmed by analyzing the albumin concentration in the culture medium by a method of measuring the serum albumin concentration, for example. The ability to synthesize urea can be confirmed by, for example, a Colorimetric assay (Sigma).

In addition, the present invention can provide the hepatic precursor cells produced by the method of the present invention and mature hepatocytes differentiated from the hepatic precursor cells (differentiated mature hepatocytes). The function and morphology of the hepatocyte produced by the method of the present invention and the mature hepatocyte differentiated from the hepatocyte (differentiated mature hepatocyte) have characteristics closer to those of human mature hepatocyte than those of the hepatocyte produced by the conventional method. The hepatocyte produced by the method of the present invention and a mature hepatocyte differentiated from the hepatocyte (differentiated mature hepatocyte) have a feature of functioning even in vitro. Therefore, the liver precursor cells of the present invention and mature hepatocytes differentiated from the liver precursor cells (differentiated mature hepatocytes) are useful, for example, in the medical field (e.g., the regenerative medical field).

For example, liver diseases can be treated by using the liver precursor cells of the present invention. For example, liver diseases can be treated by a method of directly transplanting liver precursor cells or mature liver cells (differentiated mature liver cells) differentiated from liver precursor cells via the portal vein of the liver, or a method of transplanting the liver precursor cells or the mature liver cells (differentiated mature liver cells) in the form of being embedded in collagen, polyurethane, or other known biocompatible materials. As described above, the present invention also provides use of the hepatic precursor cells produced by the above-described steps or mature hepatocytes differentiated from hepatic precursor cells (differentiated mature hepatocytes). More specifically, a therapeutic agent for liver diseases comprising hepatic precursor cells or mature hepatocytes differentiated from hepatic precursor cells (differentiated mature hepatocytes) is provided. In addition, a method for treating liver diseases using the cell is provided. Specific examples of the diseases include, but are not limited to, liver cirrhosis, fulminant hepatitis, chronic hepatitis, viral hepatitis, alcoholic hepatitis, hepatic fibrosis, autoimmune hepatitis, fatty liver, drug-induced liver injury, hemochromatosis, hemosiderosis, wilson's disease, primary biliary cirrhosis, primary sclerosing cholangitis, liver abscess, chronic active hepatitis, chronic persistent hepatitis, biliary atresia, and liver cancer.

Furthermore, cholangiocytes differentiated from hepatic progenitors can be identified from their morphological characteristics, markers for epithelial cells, and the like.

The present invention can also provide cholangiocytes differentiated from the hepatic progenitors produced by the method of the present invention (hereinafter referred to as "differentiated cholangiocytes"). The function and form of the differentiated biliary epithelial cells produced by the method of the present invention are more similar to those of human biliary epithelial cells than those produced by conventional methods. The differentiated cholangioepithelial cells produced by the method of the present invention are characterized by functioning even in vitro. Therefore, the liver precursor cells and bile duct epithelial cells (differentiated bile duct epithelial cells) differentiated from the liver precursor cells of the present invention are useful, for example, in the medical field (e.g., regenerative medical field).

For example, the inventive hepatoprogenitor cells or cholangioepithelial cells can be used for treatment of bile duct diseases, such as cholelithiasis, gallbladder polyp, gallbladder cancer, cholangiocarcinoma, cholangitis, cholecystitis, Alagille syndrome (AGS), cholecystolithiasis, cholecystadenomyosis, cystic hyperplasia, non-calculous biliary tract pain, Primary Sclerosing Cholangitis (PSC), and the like, by using the inventive hepatoprogenitor cells.

The hepatic precursor cells of the present invention are also useful in the field of research for the purpose of, for example, treating hepatic diseases. For example, the present invention can be used for research and development of artificial organs (artificial liver, etc.). The human hepatocytes of the present invention are also useful in the field of development of pharmaceuticals, foods, and the like. Specifically, the present invention can be used for the metabolism of a test substance, the evaluation of hepatotoxicity, and the screening of a therapeutic agent for liver diseases, an inhibitor of hepatitis virus infection, or a therapeutic agent for viral hepatitis.

By using the hepatic precursor cells produced by the method of the present invention, mature hepatocytes differentiated from the hepatic precursor cells (differentiated mature hepatocytes) or biliary epithelial cells (differentiated biliary epithelial cells), the metabolism and hepatotoxicity of the test substance can be evaluated.

In the evaluation of metabolism and hepatotoxicity of test substances, animal models and the like have been used in the past, but there are problems as follows: the number of test substances that can be evaluated at one time is limited, and evaluations obtained by animal models and the like cannot be directly applied to humans. Therefore, evaluation methods using human liver cancer cell lines and cultured hepatocytes of primary normal persons have been used. However, since the human hepatoma cell line is a cancer cell, there is a possibility that the evaluation by the human hepatoma cell line cannot be applied to a normal human hepatocyte. In addition, cultured hepatocytes of primary normal persons have problems in terms of stable supply and cost. Further, the cell line immortalized by primary normal human cultured hepatocytes showed a decrease in CYP3A4 activity as compared with the non-immortalized cell line (International Journal of Molecular Medicine 14: 663-668,2004, Akiyama I.et.). Such a problem can be solved by using the hepatic precursor cells produced by the method of the present invention, mature hepatocytes differentiated from the hepatic precursor cells (differentiated mature hepatocytes), or biliary epithelial cells (differentiated biliary epithelial cells).

The differentiated mature hepatocytes of the present invention can be used for screening test substances. The screening of the test substance of the present invention can be performed by using, as an index, analysis of metabolic enzymes, analysis of metabolic pathways, analysis of metabolites, analysis of metabolic activity, analysis of cytotoxicity, analysis of genetic toxicity, analysis of oncogenic expression, analysis of mutagenicity, analysis of hepatotoxic expression, analysis of action on the liver, and the like.

The present invention provides a method for evaluating the metabolism of a test substance. In this method, the test substance is brought into contact with the hepatic precursor cells produced by the method of the present invention or with mature hepatocytes differentiated from the hepatic precursor cells (differentiated mature hepatocytes). Next, the metabolism of the test substance brought into contact with the cell is measured.

The test substance used in the present invention is not particularly limited. Examples thereof include, but are not limited to, foreign substances, natural compounds, organic compounds, inorganic compounds, proteins, peptides, single compounds, libraries of compounds, gene library expression products, cell extracts, cell culture supernatants, fermentation microorganism products, marine organism extracts, plant extracts, pharmaceutical materials, cosmetic materials, food materials, pharmaceutical additives, cosmetic additives, food additives, and supplementary ingredients.

More specifically, Rifampin (Rifampin), Dexamethasone (Dexamethasone), Phenobarbital (Phenobital), Ciglitazone, Phenytoin (Phenytoin), Efavirenz (Efavirenz), Simvastatin (Simvastatin), β -Naphthoflavone (β -Napthoflavone), Maillarazole (Omepazole), Clotrimazole (Clotrimazole), 3-Methylcholanthrene (3-Methyhaloxanthene), and the like can be exemplified.

The "contact" in the present invention is usually carried out by adding a test substance to a culture medium or a culture solution, but is not limited to this method. When the test substance is a protein or the like, the test substance can be "contacted" by introducing a DNA vector expressing the protein into the cell.

The metabolism of the test substance can be measured by methods known to those skilled in the art. For example, when a metabolite of the test substance is detected, it is determined that the test substance is metabolized. When the test substance is brought into contact with the test substance, the test substance is determined to be metabolized when the expression of an enzyme gene such as CYP (cytochrome p450), MDR (MultiDrug Resistance, ABCB1), MPR (MultiDrug Resistance-associated Protein), ABCC2, or the like is induced or when the activity of these enzymes is increased.

Analysis of metabolic enzymes analysis of enzymes involved in the metabolism of a test substance can be performed by, for example, contacting the test substance with the differentiated mature hepatocytes of the present invention and then analyzing the change in the structure of the test substance. Specifically, there are exemplified identification of enzymes involved in metabolism of a test substance by analyzing a change in the structure of the test substance by an inhibitory/antagonistic substance for each enzyme or a neutralizing antibody for each enzyme after the test substance is brought into contact with the differentiated mature hepatocytes of the present invention, analysis of an enzyme reaction mechanism by analyzing a change in the structure of the test substance based on the contact of the test substance with the cells, analysis of substrate specificity, and the like.

Analysis of metabolic pathways and analysis of metabolic products the analysis of metabolic pathways and the analysis of metabolic products of a test substance can be performed by, for example, analyzing a change in the structure of the test substance after contacting the test substance with the differentiated mature hepatocytes of the present invention. As a method for detecting a metabolite of a test substance, a known method can be used. For example, the metabolite can be detected by analyzing a medium or the like for differentiating mature hepatocytes, which is brought into contact with the test substance, by liquid chromatography, mass spectrometry, or the like.

The analysis of metabolic activity can be carried out by, for example, bringing a test substance into contact with the differentiated mature hepatocytes of the present invention, and detecting an increase in the metabolic activity of the test substance, an increase in the amount of an enzyme, an increase in the transcription amount of a gene encoding the enzyme, or the like, thereby promoting the activity of the test substance-metabolizing enzyme. Specifically, the analysis can be carried out by detecting an increase in cytochrome P450 enzyme activity, an increase in protein amount, and an increase in mRNA. As the detection method, known methods such as measurement of enzymatic activities corresponding to various P450 proteins, Western blotting corresponding to various P450 proteins, Northern hybridization corresponding to various P450 mRNAs, and RT-PCR can be used.

In addition, the present invention provides a method for evaluating hepatotoxicity of a test substance. In this method, the test substance is brought into contact with the hepatic precursor cells produced by the method of the present invention and the mature hepatocytes differentiated from the hepatic precursor cells (differentiated mature hepatocytes). Then, the degree of the disorder of the cell brought into contact with the test substance is measured. The degree of the disorder can be measured, for example, using as an index a liver injury marker such as survival rate of the cell, GOT (glutamate oxaloacetate transferase), GPT (glutamate pyruvate transaminase)).

The analysis of hepatotoxicity expression can be performed by, for example, contacting a test substance with the differentiated mature hepatocytes of the present invention to observe the expression of the cytotoxicity, or contacting the test substance with the cells, administering the test substance changed by the cells to other hepatocytes, liver slices, or test animals, removing the liver, or observing changes in cells, tissues, or organisms caused by the administration of the test substance, and analyzing the hepatotoxicity based on the metabolism of the test substance.

For example, when a test substance is added to a culture solution of hepatic precursor cells or mature hepatocytes differentiated from hepatic precursor cells (differentiated mature hepatocytes), if the survival rate of the cells decreases, the test substance is determined to have hepatotoxicity, and if the survival rate does not change significantly, the test substance is determined to have no hepatotoxicity. For example, when a test substance is added to a culture solution of the cell and GOT and GPT in the culture solution rise, the test substance is determined to have hepatotoxicity, and when there is no significant change in GOT and GPT, the test substance is determined not to have hepatotoxicity.

By using a substance for which the presence or absence of hepatotoxicity has been found as a control, it is possible to more accurately evaluate whether or not a test substance has hepatotoxicity.

For example, the test substance may be contacted with the differentiated mature hepatocytes of the present invention to analyze cytotoxicity based on a metabolite of the test substance. Specifically, the analysis is performed by observing changes in the morphology of the cells, changes in the number of living cells, leakage of intracellular enzymes, changes in the cell surface layer structure, changes in intracellular enzymes, and the like, which are caused by contact with the test substance.

For example, the genetic toxicity analysis may be performed by contacting a test substance with the differentiated mature hepatocytes of the present invention, and subjecting the cells to a chromosome abnormality test, a micronucleus test, or the like. The test substance is contacted with the differentiated mature hepatocytes of the present invention, and then the test substance that changes depending on the cells is evaluated by an appropriate evaluation system, and analyzed by a chromosomal abnormality test, a micronucleus test, a back-mutation test, or the like.

Analysis of carcinogenic expression the analysis of carcinogenicity based on the metabolism of a test substance can be carried out, for example, by bringing the test substance into contact with the differentiated mature hepatocytes of the present invention and subjecting the cells to a chromosome abnormality test, modification of DNA, or the like. In addition, the test substance can be analyzed by contacting the test substance with the differentiated mature hepatocytes of the present invention, and then evaluating the test substance that changes with the cells using a carcinogenic evaluation system based on an appropriate chemical substance.

Analysis of mutagenicity analysis based on the metabolism of a test substance can be performed, for example, by contacting the test substance with the differentiated mature hepatocytes of the present invention and subjecting the cells to a chromosomal abnormality test, a micronucleus test, or the like. In addition, the test substance can be analyzed by contacting the test substance with the differentiated mature hepatocytes of the present invention, then evaluating the test substance that changes depending on the cells using an appropriate evaluation system, and performing a chromosomal abnormality test, a micronucleus test, a back-mutation test, and the like.

In addition, the present invention can provide a method for screening a therapeutic agent for a liver disease or a bile duct disease. In this method, the test substance is brought into contact with the hepatic precursor cells produced by the method of the present invention or with mature hepatocytes differentiated from the hepatic precursor cells (differentiated mature hepatocytes). Subsequently, the function of the cell to which the test substance is brought into contact is measured. Then, a substance that enhances the function of the cell contacted with the test substance is selected.

The analysis of the action on the liver can be performed, for example, by contacting the test substance with the differentiated mature hepatocytes of the present invention, and then observing the expression of the change in the cells, or by contacting the test substance with the cells, then administering the test substance changed by the cells to other liver cells, liver slices, and then removing the liver, or an experimental animal, and then observing the change in the cells, tissues, or organisms caused by the administration. When the differentiated mature hepatocytes to which the test substance is brought into contact are found to have an increased cell function, the test substance is expected to have a therapeutic effect on the liver.

The function of the hepatocyte or a mature hepatocyte differentiated from the hepatocyte (differentiated mature hepatocyte) of the present invention can be measured by using, for example, glucose-producing ability, ammonia-metabolizing ability, albumin-producing ability, urea-synthesizing ability, and the activity of enzymes such as CYP as indices.

The glucose-producing ability can be confirmed by analyzing the glucose level in the culture supernatant by, for example, a glucose oxidase method. The ammonia metabolizing ability can be confirmed, for example, by analyzing the ammonia level in the medium by the modified indophenol method (Horn DB & Squire CR, Chim. acta.14: 185-194.1966). The albumin-producing ability can be confirmed by analyzing the albumin concentration in the culture medium by a method of measuring the serum albumin concentration, for example. The urea synthesis ability can be confirmed, for example, by using Colorimetric assay (Sigma). The CYP of the present invention is not particularly limited, and examples thereof include CYP1a1, CYP2C8, CYP2C9, CYP3a4 and the like. Methods for measuring CYP activity may be those known to those skilled in the art.

The hepatocyte produced by the method of the present invention and the mature hepatocyte differentiated from the hepatocyte (differentiated mature hepatocyte) have functions and forms closer to those of human mature hepatocyte, and therefore, can be infected with hepatitis virus.

The present invention provides a method for screening an inhibitor against hepatitis virus infection. In this method, the hepatic precursor cells produced by the method of the present invention or mature hepatocytes differentiated from the hepatic precursor cells (differentiated mature hepatocytes) are contacted with the hepatitis virus in the presence of the test substance. Next, the cells contacted with the hepatitis virus were examined for the presence or absence of the hepatitis virus infection. Next, a substance inhibiting hepatitis virus infection is selected. The contacting with the hepatitis virus of the cell can be carried out by a conventional method.

The hepatitis virus is not particularly limited, and includes hepatitis C virus, hepatitis A virus, and hepatitis B virus. These hepatitis viruses may be established strains of hepatitis viruses or hepatitis viruses isolated directly from hepatitis virus infected persons. The product may be in a purified state or in a crude state (for example, in the state of serum obtained from an infected person).

The presence or absence of hepatitis virus infection can be examined by using the amount of hepatitis virus in cells as an index. The amount of hepatitis virus in the cell can be determined, for example, by using the amount of hepatitis virus RNA in the cell as an index. The amount of hepatitis virus RNA can be measured by a conventional method. The measurement can be carried out by a method established by the present inventors (T. Takeuchi et al. real-Time Detection System for Quantification of Hepatitis C Virus genome. gastroenterology 1999,116: 636-642).

In addition, the present invention can provide a method for screening a therapeutic agent for viral hepatitis. In this method, the hepatitis virus is brought into contact with the hepatic precursor cells produced by the method of the present invention or with mature hepatocytes differentiated from the hepatic precursor cells (differentiated mature hepatocytes). Next, the test substance is brought into contact with the cells infected with hepatitis virus. Next, the proliferation of hepatitis virus in the cells contacted with the test substance is measured. Next, a substance that inhibits the growth of hepatitis virus is selected.

The substance that inhibits the growth of hepatitis virus includes 1) a substance that inhibits the growth of hepatitis virus compared to a case where the test substance is not brought into contact, 2) a substance that completely inhibits the growth of hepatitis virus, and 3) a substance that causes the hepatitis virus to disappear. The growth and disappearance of hepatitis virus can be examined by measuring the amount of hepatitis virus in cells.

In the present invention, the test substance can be brought into contact with the cells by adding the test substance to a medium or a culture solution for differentiating mature hepatocytes. Alternatively, a gene encoding a test substance may be expressed in a differentiated mature hepatocyte and brought into contact with the differentiated mature hepatocyte. Alternatively, the test substance may be brought into contact with cells producing the test substance by co-culturing the cells with differentiated mature hepatocytes.

The differentiated mature liver cells of the invention have functions and forms closer to those of the mature liver cells, so the differentiated mature liver cells can be infected by hepatitis viruses. Accordingly, the present invention can provide a method for culturing hepatitis virus. In the method for culturing hepatitis virus of the present invention, the type of virus is not particularly limited, and examples thereof include hepatitis a virus, hepatitis b virus, hepatitis c virus, hepatitis d virus and hepatitis e virus. The method for culturing hepatitis virus of the present invention is useful for passaging and amplifying the isolated virus. In addition, the method for culturing hepatitis virus of the present invention can isolate hepatitis virus from environmental or patient-derived samples.

Human liver model animal of the invention，Can be obtained by transplanting the differentiated mature hepatocytes of the present invention into a non-human mammal. The route of administration of the differentiated mature hepatocytes to be transplanted into the non-human mammal includes direct administration to the surface of the liver, intrahepatic administration, intraportal administration, intrasplenic administration, oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, sublingual administration, rectal administration, vaginal administration, transdermal administration, etc., and from the viewpoint of the survival rate of the differentiated mature hepatocytes of the present invention, direct administration to the surface of the liver, intrahepatic administration, intrasplenic administration, intraarterial administration, and intravenous administration are preferable, and intrahepatic administration, intrasplenic administration, and intrahepatic administration are more preferable.

The dose of differentiated mature hepatocytes in the human liver model animal of the present invention may vary depending on the non-human mammal and the administration route, and is usually 1 × 10 to 1 × 10¹⁰One/individual, preferably 1X 10²～1×10⁹More preferably 1X 10/individual³～1×10⁸Individual/individual. The dose may be administered in1 dose or in several doses.

The non-human mammal of the present invention is not particularly limited as long as it is a mammal, and examples thereof include a rabbit, a dog, a cat, a guinea pig, a hamster, a mouse, a rat, a sheep, a goat, a pig, a miniature pig, a horse, a cow, a monkey, and the like, and more preferably a mouse, a rat, a miniature pig, and a monkey.

In addition, the present invention can provide a kit comprising human hepatoblasts prepared by culturing human mature hepatocytes, which are induced by the human hepatoblasts, or mature hepatocytes (differentiated mature hepatocytes) prepared by culturing the human hepatoblasts in a medium containing serum, A-83-01, and CHIR 99021. Such a kit is used for evaluating the metabolism and/or hepatotoxicity of a test substance using human hepatic precursor cells or differentiated mature hepatocytes. In another embodiment, such a kit can also be used for screening test substances, specifically, screening of therapeutic agents for liver diseases or bile duct diseases, screening of hepatitis virus infection inhibitors, screening of viral hepatitis therapeutic agents, and the like. The specific evaluation methods and screening methods are as described above.

Examples

The present invention will be described in detail below with reference to examples and test examples, but the present invention is not limited to these examples and the like. Hereinafter, age and Month age information of the cells to be used are abbreviated as "M" (Month) and "Y" (Year), respectively.

(example 1)

Human frozen hepatocytes (Lot. ID: HC3-14, 45Y, Male, Caucasian, manufactured by Xenotech) were suspended in a thawing medium (William's E medium (manufactured by Life Technologies, 32551-020), 10% FBS (manufactured by Life Technologies), 10%^-4M insulin (manufactured by Sigma Co.) and 1 Xantibiotic/antifungal solution (manufactured by Life technologies Co.), were centrifuged at 500rpm (about 40 Xg) at 4 ℃ for 2min to recover human hepatocytes. The recovered cells were resuspended in an inoculation medium (L-15 medium (manufactured by Life Technologies, 11415-. To be 1 × 10⁴cells/cm²The method (1) comprises inoculating to collagen-coated plate (IWAKI), and adding CO₂Incubator (37 ℃, 5% CO)₂) In addition, the inoculation medium was replaced with a basal medium (hereinafter, "SHM medium" (DMEM) 3 to 5 hours after cell adhesion[ solution ] F12 medium (11320033, Life Technologies), 5mM HEPES (St. Louis, Mo., Sigma), 30mg/L L-proline (Sigma), 0.05% BSA (Sigma), 10ng/mL epidermal growth factor (Sigma), insulin-transferrin-serine (ITS) -X (Life Technologies), 10^-7M dexamethasone (Dex) (manufactured by Sigma Co., Ltd.), 10mM nicotinamide (manufactured by Sigma Co., Ltd.), 100mM ascorbate-2-phosphate (manufactured by Wako Co., Ltd.), and 1 Xantibiotic/antifungal solution (manufactured by Life Technologies Co., Ltd.) were added to an AC-F medium obtained by adding 10% FBS (manufactured by Life Technologies Co., Ltd.), 0.5. mu. M A-83-01 (manufactured by WAKO Co., Ltd.) and 3. mu.M CHIR99021 (manufactured by Axon Medchem Co., Ltd.), YAC-F medium obtained by adding 10% FBS (Life Technologies), 0.5. mu. M A-83-01 (WAKO), 3. mu.M CHIR99021(Axon Medchem) and 10. mu. M Y-27632 (WAKO), and YAC medium obtained by adding 0.5. mu. M A-83-01 (WAKO), 3. mu.M CHIR99021(Axon Medchem) and 10. mu.M M Y-27632 (WAKO) to the basal medium. Then, the medium was changed at a frequency of once every 2 to 3 days using each test medium in CO₂Incubator (37 ℃, 5% CO)₂) The culture is carried out.

As a result of observing the morphology of the cultured cells with a phase-contrast microscope after 17 days, many small cells were found in the cells cultured in the AC-F medium and the YAC-F medium, and since the nucleus/cytoplasm (N/C) ratio was high and white nuclei were observed, the cells were confirmed to be liver precursor cells (fig. 1A and 1B). On the other hand, since the cells cultured in YAC medium had a low N/C ratio after 12 days, black nuclei and also binuclear cells were observed (fig. 1C), it could be confirmed that these cells were not liver precursor cells. In addition, cells cultured in AC-F medium and YAC-F medium proliferated faster than cells cultured in YAC medium.

From the above, it was found that serum, A-83-01 and CHIR99021 must be contained in the medium in order to obtain the hepatic precursor cells from mature hepatocytes.

(example 2)

In the same manner as in example 1, human frozen hepatocytes (10M, Female, Hispinic, Celsis) were cultured in AC-F medium, and liver precursor cells were observed after 6 days (D6), 9 days (D9) and 12 days (D12) (FIG. 2). In FIG. 2, arrows indicate cells that are partially spontaneously differentiated and matured from hepatic precursor cells.

(example 3)

In the same manner as in example 1, human frozen hepatocytes (2Y, Male, Caucasian, Biopredic) were cultured in AC-F medium, and as a result, liver precursor cells were observed after 7 days and 14 days. On the other hand, when the test medium was cultured in FBS medium containing only 10% FBS (manufactured by Life Technologies), no hepatic precursor cells were observed (fig. 3).

Similarly, when human frozen hepatocytes (8M, Male, Caucasian, BiorecamationIVT) were cultured in AC-F medium, then, after 7 days and 14 days, precursor hepatocytes were observed, and when the test medium was FBS medium, no precursor hepatocytes were observed (FIG. 4).

(example 4)

The transplantation of the hepatic precursor cells of the present invention was performed on a cDNA-uPA/SCID mouse in which the uPA gene is specifically expressed in hepatocytes and chronic hepatic injury occurs due to congenital sustained damage to the liver, and it was confirmed that the hepatocytes derived from the hepatic precursor cells of the present invention were implanted into the liver of the cDNA-uPA/SCID mouse.

Cells cultured for 4 days in AC-F medium using human frozen hepatocytes (10M, manufactured by Female, Hispanic, Celsis Co., Ltd.) in the same manner as in example 1 were washed 2 times with PBS (-) and then peeled and collected with TrypLE Express (manufactured by Thermo Co., Ltd., SKU: 12604013) to measure the number of cells. After centrifugation (200 Xg, 5 min) of the cell suspension, the cell suspension was suspended in DMEM10 (10% FBS-DMEM) to 5X 10⁷cells。

cDNA-uPA/SCID mice (Tateno et al, 2015, 2-4 weeks old) were laparotomized under isoflurane anesthesia to expose the spleenDirty, 0.5X 10⁵～2×10⁶After cell/mouse implantation, the laparotomy was closed. After 1 week 1, 20 to 40uL of blood was collected from the eye sockets, serum was separated, and Human-specific albumin in the serum was measured using ALB Human ALB ELISA kit (product code: E88-129, manufactured by Bethyl corporation). The cells were dissected 8 weeks after transplantation to prepare whole blood and liver/spleen tissue samples (paraffin and frozen blocks).

(example 5)

Since the hepatocytes specifically express thymidine kinase, the hepatocytes can specifically induce cell death by ganciclovir administration to cause liver damage, and the transplantation of the hepatoprecursor cells of the present invention into these TK-NOG mice confirmed that the hepatocytes derived from the hepatoprecursor cells of the present invention were grown in the liver of the TK-NOG mice.

A cell suspension was prepared using human frozen hepatocytes (10M, manufactured by Female, Hispinic, Celsis Co., Ltd.) in the same manner as in example 4. For TK-NOG mice (Hasegawa et al, 2011, 7-8 weeks old, In-Vivo Science), 10MG of ganciclovir (GCV, Sigma, G2536-100MG) was weighed and dissolved In 16.7mL of PBS (-) 7 days and 5 days before cell transplantation, and was subjected to filtration sterilization using a 0.22um filter and intraperitoneal administration at 10uL/G body weight (6 MG/kg). TK-NOG mice were laparotomized under isoflurane anesthesia, with spleens exposed at 0.5X 10⁵～2×10⁶After cell/mouse administration, the laparotomy was closed. 20-40 uL of blood was collected from the tail vein 1 week after 1 week, serum was separated, and Human-specific albumin in serum was measured using ALB Human ALBELISA kit (product code: E88-129, manufactured by Bethyl corporation). The cells were dissected 8 weeks after administration, and whole blood and liver/spleen tissue samples (paraffin and frozen blocks) were prepared.

(example 6)

Test for confirming Gene expression for differentiation into hepatocytes

In the same manner as in example 1, human frozen hepatocytes were used, cultured in an AC-F medium to prepare liver precursor cells, cultured in a medium containing oncostatin M (OSM) and dexamethasone (Dex) for 6 days, and then cultured in matrigel to differentiate into hepatocytes. The differentiated hepatocytes were subjected to one-color micro-array-based gene expression analysis system (Agilent Technologies) using a SurePrint G3RatGE 8X60K Kit (G4853A) and SurePrint G3Mouse GE 8X60K Ver 2.0Kit (G4852B) according to the protocol. The intensity values were log-transformed to base 2 and the data were read into a Partek Genomics Suite6.6 (produced by Partek Inc, Chesterfield, Mo., USA). Analysis of Gene expression Using one-way ANOVA, genes with differences in expression were determined. In each analysis, the ratio of the P value to the change amount was calculated. Using Partek Genomics suitex 6.6, the expression of liver-specific genes was confirmed by unsupervised clustering (unsupervised clustering) and heatmap generation using Euclidean of average linking clustering of selected probe sets for all or sorted data sets.

Example 7 test for confirming differentiation into biliary epithelial cells

Secretin assay

In the same manner as in example 1, human frozen hepatocytes were used and cultured in an AC-F medium to prepare precursor hepatocytes, which were then cultured on MEF for 6 days in a medium containing mTeSR1 and YAC, and then cultured with 2% matrigel added for 2 days to differentiate into biliary epithelial cells. For the bile duct epithelial cells obtained by differentiation, 2X 10^-7After culturing for 30 minutes with rat secretin added to M (manufactured by Wako Co., Ltd.), the enlargement of the luminal area in the bile duct-like structure was observed with a phase-contrast microscope to confirm the differentiation into bile duct epithelial cells.

(example 8)

Fluorescein diacetate assay

The bile duct epithelial cells were differentiated from the hepatic precursor cells in the same manner as in example 7, and fluorescein diacetate was added to the bile duct epithelial cells obtained, followed by 15 minutes of culture and then replacement with a new medium. The incubation was continued for a further 30 minutes, thereby facilitating the transport of the decomposed fluorescein to the luminal area. Then, the medium was replaced with HBSS (+), and the distribution of fluorescein was observed with a fluorescence microscope to confirm the differentiation into bile duct epithelial cells.

(example 9)

Confirmation test of liver-specific Gene expression upon differentiation into hepatocytes

In The same manner as in example 6, hepatic precursor cells were differentiated into hepatocytes, and total RNA was extracted from The obtained hepatocytes using mirneasyMini Kit (QIAGEN, Venlo, The Netherlands). Reverse Transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Inc.) according to the protocol. Using the prepared cDNA as a template, PCR was carried out using Platinum SYBR Green qPCR Supermix UDG (manufactured by Invitrogen) to confirm the expression of the liver-specific gene.

(example 10)

In the same manner as in example 1, using human frozen hepatocytes (10M, Female, Hispinic, Celsis corporation), cells cultured for 11 days in AC-F medium were washed 2 times with PBS (-) and then detached and collected with TrypLE Express (SKU: 12604013, Thermo corporation) to measure the number of cells. After centrifugation (200 Xg, 5 min) of the cell suspension, the cell suspension was suspended in DMEM10 (10% FBS-DMEM) to 5X 10⁷cells。

cDNA-uPA/SCID mice (Tateno et. al, 2015, 2-4 weeks old, n-3) were laparotomized under isoflurane anesthesia to expose the spleen at 1X 10⁶After cell/mouse implantation, the laparotomy was closed. 20-40 uL of blood was collected from the eye socket 1 week and 1 time, and Human-specific albumin in the blood was measured using a Human ALB ELISA kit (product code: E88-129, manufactured by Bethyl). As shown in fig. 5, it was confirmed that human ALB was present in the blood of mice and that hepatocytes derived from hepatic precursor cells were grown on the livers of mice. Note that human ALB was 17.2mg/mL, 12.1mg/mL and 12.0m in blood 70 days after cell administrationg/mL。

In addition, the expression of human CYP2C9 in the liver of the mouse 70 days after cell administration was confirmed to be 94.6 to 96.1% (right inner leaf), 91.3 to 97.2% (left inner leaf), and 93.1 to 96.0% (all) respectively (fig. 6 and 7, respectively).

(example 11)

In the same manner as in example 10, using human frozen hepatocytes (10M, Female, Hispinic, Celsis corporation), cells cultured for 11 days in AC-F medium were washed 2 times with PBS (-) and then detached and collected with TrypLE Express (SKU: 12604013, Thermo corporation) to measure the number of cells. After centrifugation (200 Xg, 5 min) of the cell suspension, the cell suspension was suspended in DMEM10 (10% FBS-DMEM) to 5X 10⁷cells. Ganciclovir (GCV) was administered to TK-NOG mice (Hasegawa et al, 2011, 7-8 weeks old, In-Vivo Science, n.2), ALT was measured 1 week after administration, and individuals showing a value of 400-. In preparation of GCV, 500mg (DENOSINE for intravenous drip) was dissolved (50mg/mL) in 10mL of distilled water for injection (tsukamur) at the time of preparation of GCV, 0.2mL of each of the solutions was dispensed as a starting material, and a solution diluted 5 times with PBS (-) was prepared in advance at the time of transplantation, and the mouse was intraperitoneally administered 0.1mL per 10g of body weight, and the hepatocyte specifically induced cell death. TK-NOG mice were laparotomized under isoflurane anesthesia, with spleens exposed at 1X 10⁶After cell/mouse administration, the laparotomy was closed. 20-40 uL of blood was collected from the tail vein 1 week after 1 week, serum was separated, and Human-specific albumin in serum was measured using ALB Human ALB ELISAkit (product code: E88-129, manufactured by Bethyl corporation). As shown in fig. 8, the presence of ALB in the mouse serum was confirmed, and implantation of hepatocytes derived from hepatic precursor cells was confirmed. Note that the serum concentration of human ALB 60 days after cell administration was 8.1mg/mL and 2.2mg/mL in each individual.

In addition, confirmation of the expression of human CYP2C9 in the liver of the mouse 60 days after cell administration was performed, and as a result, it was confirmed that 57.5% and 30.6% were expressed in each individual (fig. 9 and 10).

(example 12)

In the same manner as in example 1, human frozen hepatocytes 1(10M, Femal, Hispanic, Celsis corporation) and human frozen hepatocytes 2(8M, Male, Caucasian, BioReclationIVT corporation) were cultured in AC-F medium to prepare precursor hepatocytes (referred to as "FCL", "DUX", respectively), and then they were cultured in the presence of oncostatin M (OSM, 5ng/ml) and dexamethasone (Dex, 10 ng/ml)^-6M) was cultured in a medium for 6 days, and then cultured in matrigel for 2 days to differentiate into hepatocytes. For the hepatic precursor cells and the differentiated hepatocytes, the activity of the metabolic enzyme CYP1a2 was measured using methanol (MeOH, 1% concentration) and omeprazole (OMP, 50 μ M). In addition, the activity of the metabolic enzyme CYP3A4 was measured using distilled water and phenobarbital (1 mM). The activities of the metabolic enzymes were carried out using the Luciferin1A2 kit and the Luciferin-IPA kit from Promega, respectively. The results showed that CYP1A2 and CYP3A4 were induced by differentiation of hepatic precursor cells into hepatocytes (FIGS. 11-14).

(example 13)

In the same manner as in example 1, human frozen hepatocytes (10M, Femal, Hispanic, Celsis) were cultured in AC-F medium to prepare precursor hepatocytes, which were then cultured in the presence of oncostatin M (OSM, 5ng/ml) and dexamethasone (Dex, 10 ng/ml)^-6M) was cultured in a medium for 6 days, and then cultured in matrigel for 2 days to differentiate into hepatocytes. The expression level of each metabolic enzyme and the like was measured by PCR on the hepatic precursor cells and the differentiated hepatic cells. The results showed that ALB, TAT, TDO2, TTR, G6PC, NTCP, CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP7A1 were induced by differentiating the hepatic precursor cells into hepatocytes (FIGS. 15 to 27).

(example 14)

In the same manner as in example 1, human frozen hepatocytes (10M, Femal, Hispinic, Celsis) were used to prepare precursor hepatocytes, and then cDNA-uPA/SCID mice (2-4M, Male, PhoenixBi) were anesthetized with isofluraneo Co.) was dissected to expose the spleen at a rate of 0.5X 10⁵～2×10⁶After cell/mouse implantation, the laparotomy was closed. On day 73 post-transplantation, the liver was removed and hepatocytes were isolated by reflux at 4X 10⁵cells/well were cultured on a 24-well collagen plate (IWAKI) for 4 days using 2% FBS-SHM medium. FIGS. 28 and 29 are photographs of the pre-transplantation hepatocyte cells and the cells that were taken out and cultured for 4 days after transplantation, respectively, and morphological observation revealed that the transplanted cells completely matured into hepatocytes in the liver of mice.

For hepatocytes removed from mice, the activity of the metabolic enzyme CYP1a2 was measured using methanol (MeOH, 1% concentration) and omeprazole (OMP, 50 μ M). In addition, the activity of the metabolic enzyme CYP3A4 was measured using rifampicin (RF, 10. mu.M), methanol (MeOH, 1% concentration), phenobarbital (1mM), and distilled water. The activities of the metabolic enzymes were carried out using the Luciferin1A2 kit and the Luciferin-IPA kit from Promega, respectively. The results showed that CYP1A2 was also induced by omeprazole, CYP3A4 was induced by rifampicin and phenobarbital for the liver precursor cells transplanted into the liver of animals and cultured (FIGS. 30 and 31)

(example 15)

In the same manner as in example 1, human frozen hepatocytes (8M, Male, Caucasian, manufactured by biorelevationivt) were used to prepare liver precursor cells, which were then transplanted and cultured in the same manner as in example 14. FIGS. 32 and 33 are photographs of the pre-transplant hepatocyte cells and the cells that were removed and cultured for 4 days after transplantation, respectively.

The metabolic enzyme activity of the cells taken out of the mice was measured in the same manner as in example 14, and the results showed that CYP1A2 was induced by omeprazole and CYP3A4 was induced by rifampicin and phenobarbital in the same manner as in example 14 (FIGS. 34 and 35)

(example 16)

In the same manner as in example 1, human frozen hepatocytes (1Y, Male) were used to prepare liver precursor cells, which were then transplanted and cultured in the same manner as in example 14. FIGS. 36 and 37 are photographs of the pre-transplant hepatocyte cells and the cells that were removed and cultured for 4 days after transplantation, respectively.

The metabolic enzyme activity of the cells taken out of the mice was measured in the same manner as in example 14, and the results showed that CYP1a2 was induced by omeprazole and CYP3a4 was induced by rifampicin and phenobarbital in the same manner as in example 14 (fig. 38 and 39).

Claims (9)

1. A method for preparing human liver precursor cell comprises culturing human mature liver cell in culture medium containing serum, A-83-01 and CHIR 99021.

2. The method for producing human hepatic precursor cells according to claim 1, wherein the mature hepatic cells are derived from infants.

3. The method for preparing human hepatocyte according to claim 1 or 2, wherein the serum is fetal bovine serum.

4. A human hepatocyte precursor cell is prepared by culturing human mature hepatocyte in a culture medium containing serum, A-83-01 and CHIR 99021.

5. A mature hepatocyte induced by the human hepatic precursor cell of claim 4.

6. A method for screening a test substance, comprising using the mature hepatocyte according to claim 5.

7. A method for culturing hepatitis virus, comprising using the mature hepatocyte of claim 5.

8. A human liver model animal obtained by transplanting the mature hepatocytes of claim 5 into a non-human mammal.

9. A kit for evaluating the metabolism and/or hepatotoxicity of a test substance using human hepatic precursor cells or mature hepatic cells,

comprising human hepatoblasts prepared by culturing human mature hepatocytes induced by the human hepatoblasts in a medium containing serum, A-83-01 and CHIR99021, or mature hepatocytes.