Method for producing hepatocytes

Abstract

The present invention provides a method for producing hepatocytes, said method comprising a step for carrying out suspension culturing of hepatoblasts in a medium that includes an oncostatin M receptor agonist, an ALK inhibitor, a cAMP agonist, and an adrenaline receptor agonist.

Description

Method for producing hepatocytes

[0001] The present invention relates to a method for producing hepatocytes, hepatocytes produced by the method, uses of the hepatocytes, and the like.

[0002] Many patients die each year from liver failure, including chronic liver failure such as cirrhosis and acute liver failure. The only curative treatment for cirrhosis is orthotopic liver transplantation. However, there remains a significant global shortage of donors, the primary source of liver grafts. Furthermore, due to transplant-associated rejection and the requirement that the donor and recipient have at least a partial HLA match, many patients are unable to receive a transplant at the appropriate time because they must wait for an HLA-matched donor from the limited donor pool.

[0003] One promising solution to the donor shortage is the use of pluripotent stem cells, such as human induced pluripotent stem cells (iPS cells). During mammalian development, the liver develops from the definitive endoderm, a type of early embryonic tissue, and is formed via hepatoblasts, which are embryonic liver progenitor cells that give rise to both hepatocyte and bile duct cell lineages. Several methods have been reported that mimic this developmental process to induce differentiation of human pluripotent stem cells into hepatocytes (e.g., Non-Patent Documents 1 and 2). The present inventors have also reported a method for inducing differentiation of pluripotent stem cells into hepatocytes (Patent Document 1).

[0004] International Publication No. 2016 / 104717

[0005] Lay Teng Ang et al., Cell Rep. 2018 Feb 20;22(8):2190-2205Maki Kotaka et al., Sci Rep. 2017 Dec 1;7(1):16734

[0006] However, it is difficult to say that hepatocytes induced by the above-mentioned conventional differentiation induction methods can fully exert therapeutic effects in terms of albumin production ability. Therefore, an objective of the present invention is to provide a method for inducing hepatocytes with improved albumin production ability by modifying the conventional hepatocyte induction method, and to provide hepatocytes obtained by said method.

[0007] The present inventors conceived the idea that various combinations of compounds used in conventional methods for inducing hepatocyte differentiation from pluripotent stem cells might be used to efficiently induce hepatocytes with improved albumin production. Therefore, we investigated the combination of hepatocyte growth factor (HGF), oncostatin M (OSM) (an oncostatin M receptor agonist), A83-01 (an ALK inhibitor), DAPT (a NOTCH inhibitor), phenylephrine (an adrenergic receptor agonist), and forskolin (a cAMP agonist). Surprisingly, HGF did not affect albumin production in the OSM-mediated hepatoblast differentiation induction method using suspension culture. Furthermore, the combination of OSM with A83-01, DAPT, and phenylephrine (ADPF) resulted in hepatocyte clusters with improved albumin production, and the morphology of these hepatocyte clusters was clearly different from that of other compound combinations (OSM, OSM + ADP). It was concluded that this morphological difference was due to forskolin. Further investigations focusing on OSM and forskolin demonstrated that DAPT did not affect albumin production. Based on these findings, the inventors concluded that the combination of an oncostatin M receptor agonist, an ALK inhibitor, a cAMP agonist, and an adrenergic receptor agonist is important for inducing the differentiation of hepatoblasts into hepatocytes with high albumin production. Based on these findings, the inventors conducted further research and completed the present invention.

[0008] That is, the present invention provides the following: [1] A method for producing hepatocytes, comprising a step of suspension culturing hepatoblasts in a medium containing an oncostatin M receptor agonist, an ALK inhibitor, a cAMP agonist, and an adrenergic receptor agonist. [2] The method according to [1], wherein the hepatocytes are in the form of cell aggregates. [3] The method according to [1] or [2], comprising a step of performing shaking culture. [4] The method according to any one of [1] to [3], comprising a step of culturing in a culture bag having multiple wells. [5-1] The method according to any one of [1] to [4], wherein the suspension culture is performed in a medium not containing hepatocyte growth factor and / or DAPT. [5-2] The method according to any one of [1] to [4], wherein the suspension culture is performed in a medium not containing hepatocyte growth factor and DAPT. [6] The method according to any one of [1] to [5-2], wherein at least one of the oncostatin M receptor agonists is oncostatin M. [7] The method according to any one of [1] to [6], wherein at least one of the ALK inhibitors is A83-01. [8] The method according to any one of [1] to [7], wherein at least one of the cAMP agonists is forskolin. [9] The method according to any one of [1] to [8], wherein at least one of the adrenergic receptor agonists is phenylephrine. [10-1] The method according to any one of [1] to [9], wherein the hepatoblasts are derived from pluripotent stem cells. [10-2] The method according to any one of [1] to [10-1], wherein the hepatoblasts are derived from humans.

[11] The method according to any one of [2] to [10-2], wherein the hepatoblast aggregates are obtained by suspension culture in a medium containing an oncostatin M receptor agonist, an ALK inhibitor, a cAMP agonist, and an adrenergic receptor agonist.

[12] Hepatocytes obtained by the method according to any one of [1] to

[11] .

[13] A cell transplantation therapeutic agent comprising the hepatocytes according to

[12] .

[14] The agent according to

[13] for treating or preventing liver disease.

[15] A method for evaluating the degree of metabolism of a test substance, comprising: (I) contacting the test substance with the hepatocytes described in

[12] , (II) measuring the activity level of a drug-metabolizing enzyme in the hepatocytes, and (III) evaluating the degree of metabolism of the test substance by the drug-metabolizing enzyme based on the activity level of the drug-metabolizing enzyme in step (II).

[16] A method for treating or preventing liver disease, comprising transplanting an effective amount of the hepatocytes described in

[12] into a mammal.

[17] The hepatocytes described in

[12] for use in the treatment or prevention of liver disease.

[18] Use of the hepatocytes described in

[12] in the manufacture of a medicament for the treatment or prevention of liver disease.

[0009] The present invention makes it possible to produce hepatocytes with excellent albumin-producing ability in vitro. The cells produced in this manner can have properties closer to those of in vivo hepatocytes, and are therefore expected to be useful for hepatocyte transplantation.

[0010] A schematic diagram of the branching pathway of hepatoblast differentiation into bile duct epithelial cells or hepatocytes is shown. Hepatoblasts were cultured in suspension under agitation in a medium containing oncostatin M and various compounds, and differentiated into hepatocyte clusters (hepatocyte-like cell (HLC)-aggregates). (a) Culture scheme. (b) Bright-field image of an HLC-aggregate on day 15 of induction. Scale bars, 500 μm. (c) Albumin secretion into the medium by an HLC-aggregate on day 15 of induction per 24 hours. Technical replicates, n=4; error bars, standard deviation. OSM, 20 ng / ml oncostatin M; HGF, 20 ng / ml hepatocyte growth factor (Peprotech, Cat# 100-39); DEX, 100 nM dexamethasone (Merck, Cat# D4902-25MG); Phe, 1 μM phenylephrine. Hepatoblasts were cultured in suspension under agitation in a medium containing oncostatin M and various compounds, and differentiated into HLC-aggregates. (a) Culture scheme. (b) Bright-field image of an HLC-aggregate on day 15 of induction. Scale bars, 500 μm. (c) Albumin secretion into the medium per 24 hours by an HLC-aggregate on day 15 of induction. OSM, 20 ng / ml oncostatin M; HGF, 20 ng / ml hepatocyte growth factor; A, 1 μM A83-01 (TGFβ inhibitor); D, 10 μM DAPT (NOTCH inhibitor, R&D, Cat# 2634 / 10); P, 1 μM phenylephrine (α1-adrenergic agonist); F, 10 μM forskolin (PKA agonist). Technical replicates, n=2; error bars, standard deviation. Hepatoblasts were cultured in suspension under agitation in a medium containing oncostatin M and various compounds, and differentiated into HLC-aggregates. (a) Culture scheme. (b) Bright-field image of HLC-aggregates on day 15 of induction. Scale bars, 500 μm.(c) Albumin secretion into the medium by HLC-aggregates on day 15 of induction per 24 hours. Technical replicates, n=2; error bars, standard deviation. (d) Bright-field image of HLC-aggregates on day 15 of induction. Scale bars, 500 μm. (e) Albumin secretion into the medium by HLC-aggregates on day 15 of induction per 24 hours. Biological replicates, n=2. OSM, 20 ng / ml oncostatin M; A, 1 μM A83-01; D, 10 μM DAPT; P, 1 μM phenylephrine; F, 10 μM forskolin. Hepatoblasts were cultured in suspension under stirring conditions in media containing oncostatin M and various compounds, and differentiated into HLC-aggregates. (a) Culture scheme. (b) Bright-field image of HLC-aggregates on day 15 of induction. Scale bars, 500 μm. (c) Albumin secretion into the medium per 24 hours by HLC-aggregates on day 15 of induction. Biological replicates, n=2. OSM, 20 ng / ml oncostatin M; A, 1 μM A83-01; D, 10 μM DAPT; P, 1 μM phenylephrine; F, 10 μM forskolin. Hepatoblast aggregates were formed from hepatoblasts by suspension culture in medium containing oncostatin M, A83-01, phenylephrine, and forskolin, and then cultured in suspension under agitation conditions to differentiate into HLC-aggregates. (a) Culture scheme. Forced aggregation refers to cell aggregates formed by culturing in a low-adhesion well bag, which causes cells to fall into the wells at the bottom of the device and forcibly aggregate. (b) Low-adhesion well bag used to form hepatoblast aggregates from hepatoblasts. (c) Brightfield image of an HLC-aggregate on day 17 of induction. Scale bars, 500 μm. (d) Diameter distribution of HLC-aggregates on day 17 of induction. (e) HE-stained image of HLC-aggregates. Scale bars, 50 μm. (f) Immunostained image of HLC-aggregates.Scale bar, 100 μm. Results of differentiation of hepatoblasts into HLC-aggregates in plate culture (2D) or suspension culture under agitation. (a) Culture scheme. (b) RT-qPCR. Technical replicates, n=5; error bars, standard deviation. Results of differentiation of hepatoblasts into HLC-aggregates in plate culture or suspension culture. (Top) Culture scheme. 3D indicates the conventional method (cell suspension culture in a 12-well plate with shaking naturally forms cell aggregates, which are then cultured under shaking. The same applies below). 3D, device, and 3D_d indicate the bag method (cell aggregates are formed using a low-adhesion well bag, then removed from the bag and transferred to a 12-well plate for shaking culture. The same applies below). (Bottom) RT-qPCR. Technical replicates, n=3; error bars, standard deviation. Results of differentiation of hepatoblasts into HLC-aggregates in plate culture or suspension culture. (Top) Culture scheme. (Bottom) RT-qPCR. Technical replicates, n=3; error bars, standard deviation. Results of differentiation from hepatoblasts to HLC-aggregates under plate or suspension culture conditions are shown. (Top) Culture scheme. (Bottom) RT-qPCR. Technical replicates, n=3; error bars, standard deviation. Results of differentiation from hepatoblasts to HLC-aggregates under plate or suspension culture conditions are shown. In the graph, Agi- indicates no shaking, and Agi+ indicates shaking. (Top) Culture scheme. (Bottom) RT-qPCR. Technical replicates, n=3; error bars, standard deviation. Results of differentiation from hepatoblasts to HLC-aggregates under plate or suspension culture conditions are shown. In the graph, Agi- indicates no shaking, and Agi+ indicates shaking. (Top) Culture scheme. (Bottom) RT-qPCR. Technical replicates, n=3; error bars, standard deviation. Results of differentiation from hepatoblasts to HLC-aggregates under plate or suspension culture conditions are shown. In the graph, Agi- indicates no shaking, and Agi+ indicates shaking. (Top) Culture scheme. (Bottom) RT-qPCR. Technical replicates, n=3; error bars, standard deviation.

[0011] 1. Method for Producing Hepatocytes The present invention provides a method for producing hepatocytes from hepatoblasts. Specifically, the present invention provides a method for producing hepatocytes (hereinafter sometimes referred to as the "production method of the present invention"), which comprises the step of culturing hepatoblasts in suspension in a medium containing an oncostatin M receptor (OSMR) agonist, an ALK inhibitor, a cAMP agonist, and an adrenergic receptor agonist (hereinafter, these substances may be collectively referred to as "hepatocyte induction-promoting substance") (in other words, culturing in suspension in the presence of the hepatocyte induction-promoting substance).

[0012] As used herein, the term "hepatoblast" refers to a cell that has the ability to differentiate into a hepatocyte (typically also has the ability to differentiate into a bile duct epithelial cell) and expresses at least one marker gene selected from the group consisting of AFP, Dlk, E-cadherin, Liv2, CD13, and CD133. Hepatoblasts are preferably cells that express AFP.

[0013] As used herein, "hepatocyte" refers to a cell that expresses albumin and typically has the ability to produce albumin. Hepatocytes typically express at least one (typically all) selected from the group consisting of AFP, HNF4α, FOXA2, PROX1, and A1AT. Hepatocytes may also have one or more of the known functions of hepatocytes, such as glycogen accumulation, low-density lipoprotein (LDL) uptake, albumin secretion, ammonia metabolism and urea synthesis, cytochrome P450 activity, lipid metabolism, and drug metabolism.

[0014] In this specification, unless otherwise specified, "cells" includes "cell populations." Furthermore, unless otherwise specified, "cells" refers to those obtained by cell culture. A cell population may be composed of one type of cell, or may be composed of two or more types of cells. Furthermore, unless otherwise specified, "cell populations" also include "cell aggregates" (also called "cell clumps").

[0015] Thus, the term "hepatoblast" includes cell populations containing hepatoblasts and cell aggregates containing hepatoblasts (also referred to as "hepatoblast cell clusters"). Similarly, the term "hepatocytes" includes cell populations containing hepatocytes and cell aggregates containing hepatocytes (also referred to as "hepatocellular clusters"). In one embodiment of the present invention, hepatoblasts are cultured in the form of cell aggregates. Also, in one embodiment of the present invention, hepatocytes are in the form of cell aggregates. The proportion of hepatoblasts or hepatocytes contained in the cell population or cell aggregate (number of hepatoblasts or stem cells / total number of cells) is not particularly limited, but is preferably 20% or more, and may be 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0016] As used herein, unless otherwise specified, the terms "expressing" or "positively expressing" a gene are used to mean at least "production of mRNA encoded by the gene," but preferably also "production of protein encoded by the mRNA." Therefore, if production of mRNA encoded by the gene is detected at least by RT-qPCR, it can be said that the gene is expressed.

[0017] As used herein, "oncostatin M receptor agonists" (hereinafter also referred to as "OSMR agonists") refer to substances that can directly bind to and act on the oncostatin M receptor (OSMR). OSMR is a cell surface receptor that binds to OSM and transmits signals to downstream factors. It forms a heterodimer with the OSM-specific oncostatin M receptor beta (OSMRβ) and gp130, which is common to receptors in the IL-6 family. OSM can bind with low affinity to both OSMRβ and gp130, but cannot transmit signals intracellularly when bound alone. OSMRβ is a single-transmembrane protein. Examples of OSMR agonists include OSM. Commercially available OSM, such as those available from Peprotech, can be used. Specific examples of human OSM include OSM isoform 1, designated NCBI Accession No: NP_065391.1 (SEQ ID NO: 2) (an example of the nucleotide sequence encoding this isoform is shown in SEQ ID NO: 1), and OSM isoform 2, designated NCBI Accession No: NP_001306037.1 (SEQ ID NO: 4) (an example of the nucleotide sequence encoding this isoform is shown in SEQ ID NO: 3). Multiple OSMR agonists may be used in combination.

[0018] When OSM is used as the OSMR agonist, its concentration in the medium is typically 0.1 ng / ml to 500 ng / ml, preferably 1 ng / ml to 100 ng / ml, and more preferably 10 ng / ml to 50 ng / ml (in one embodiment, 20 ng / ml). When an OSMR agonist other than OSM is used, the concentration of the OSMR agonist in the medium is selected appropriately.

[0019] Examples of the ALK inhibitor used in the present invention include SB431542 (4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide, 4-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide), A83-01 (3-(6-methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole), LDN193189 (4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), GW788388 (4-[4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl]-N-(tetrahydro-2H-pyran-4-yl)benzamide), SM16 (4-[4-(1,3-Benzodioxol-5-yl)-5-(6 [5-(6-Methyl-2-pyridinyl)-1H-imidazol-2-yl]-bicyclo[2.2.2]octane-1-carboxamide], IN-1130 (3-[[5-(6-Methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]-benzamide), GW6604 (2-Phenyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine), SB505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine), and A83-01 is preferred. Multiple ALK inhibitors may be used in combination.

[0020] When A83-01 is used as the ALK inhibitor, its concentration in the medium is typically 0.01 μM to 10 μM, preferably 0.05 μM to 5 μM, and more preferably 0.1 μM to 2.5 μM (1 μM in one embodiment). When an ALK inhibitor other than A83-01 is used, the concentration of the ALK inhibitor in the medium is selected appropriately.

[0021] As used herein, the term "cAMP agonist" refers to adenosine 3',5'-cyclic monophosphate (cAMP) or a substance with a function similar to cAMP, or a substance capable of increasing intracellular cAMP concentration upon contact with cells. Examples of cAMP agonists used in the present invention include adenylate cyclase activators and cAMP analogs. Examples of cAMP analogs include 8-Br-cAMP, dibutyryl-cAMP (Bucladesine), N6-benzoyl cAMP, and 8-thiomethyl cAMP. Examples of adenylate cyclase activators include forskolin, forskolin derivatives (e.g., NKH477 (colforsin dalopate)), PACAP-27, PACAP-38, and SKF83822, with forskolin being preferred. Multiple cAMP agonists may be used in combination.

[0022] When forskolin is used as the cAMP agonist, its concentration in the medium is typically 0.1 μM to 100 μM, preferably 0.5 μM to 50 μM, and more preferably 1 μM to 20 μM (10 μM in one embodiment). When a cAMP agonist other than forskolin is used, the concentration of the cAMP agonist in the medium is selected appropriately.

[0023] As used herein, the term "adrenergic receptor agonist" refers to a substance that can directly bind to and act on an adrenergic receptor. Preferably, the substance binds to at least an α1 receptor, and examples thereof include adrenaline, noradrenaline, etilefrine, naphazoline, phenylephrine ((R)-3-(1-hydroxy-2-(methylamino)ethyl)phenol), methoxamine, and midodrine. Preferred adrenergic receptor agonists in the present invention are etilefrine, phenylephrine, and methoxamine, with phenylephrine being particularly preferred. A combination of multiple adrenergic receptor agonists may be used.

[0024] When phenylephrine is used as the adrenergic receptor agonist, its concentration in the medium is typically 0.01 μM to 10 μM, preferably 0.05 μM to 5 μM, and more preferably 0.1 μM to 2.5 μM (1 μM in one embodiment). When an adrenergic receptor agonist other than phenylephrine is used, the concentration of the adrenergic receptor agonist in the medium is appropriately selected.

[0025] On the other hand, as shown in the Examples below, hepatocyte growth factor (HGF), which has been widely used in conventional methods, and DAPT (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester), a Notch signaling inhibitor, are not essential in the production methods of the present invention. Thus, in one embodiment of the present invention, the suspension culture in the production methods of the present invention is culture in a medium that does not contain HGF and / or DAPT (in other words, culture in the absence of HGF and / or DAPT).

[0026] When hepatoblasts are provided in an adherent state to a culture vessel, the method of the present invention can be carried out by exchanging the medium containing the hepatoblasts with a medium containing a hepatocyte induction promoter and culturing the cells in suspension. When hepatoblasts are provided in a suspended state in a medium, the medium containing the hepatoblasts may be exchanged with a medium containing a hepatocyte induction promoter and the cells may be cultured in suspension.

[0027] When hepatoblasts are provided as cell aggregates, they may be substantially separated (or dissociated) by any method to separate them into single cells, and then cultured. Examples of the separation method include mechanical separation and separation using a separation solution having protease activity and collagenase activity (e.g., Accutase, a solution containing trypsin and collagenase). TM , Accumax TM (Nacalai Tesque)) or separation using a separation solution having only collagenase activity. The separated or dissociated cells can be further isolated using methods such as flow cytometry or mass cytometry using surface antigens as an indicator, magnetic cell separation, or affinity columns on which the desired antigens are immobilized.

[0028] As used herein, "suspension culture" refers to culture performed under conditions that maintain cells or cell aggregates suspended in a culture medium, i.e., culture under conditions that do not allow the formation of strong cell-substratum junctions between the cells or cell aggregates and the culture vessel. Furthermore, as used herein, "adherent culture" (also referred to as "plate culture") refers to culture under conditions that allow the formation of strong cell-substratum junctions between the cells or cell aggregates and the cultureware, etc.

[0029] Incubators used for suspension culture include, but are not limited to, flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles. For agitation culture (also referred to as "agitation culture"), described below, sealed incubators are preferred. Examples of such incubators include tissue culture flasks, culture bags, and roller bottles. Furthermore, to enable culture under non-adhesive conditions, the incubator is preferably non-cell-adhesive. Examples of non-cell-adhesive incubators include those whose surfaces have not been artificially treated to improve cell adhesion (e.g., coated with an extracellular matrix, etc.) or those whose surfaces have been artificially treated to suppress cell adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA)).

[0030] Furthermore, as shown in the examples below, it has been shown that homogeneous hepatocyte clusters can be efficiently produced by using a culture bag having multiple wells (also referred to as a "multi-well device"). Therefore, in one aspect, the production method of the present invention includes a step of culturing cells in a multi-well device. From the viewpoint of reducing the risk of contamination during culture, a multi-well device that allows culture in a closed system is preferable. A multi-well device has multiple wells that are uniform in size and have been treated to make them non-adhesive to cells, allowing hepatocyte clusters to be formed in each well. This makes it possible to produce large quantities of uniformly sized hepatocyte clusters in a single culture.

[0031] The multi-well device may be a commercially available product (e.g., a product manufactured by Toyo Seikan Group Holdings, Ltd.) or may be prepared using known methods (e.g., the method described in JP 2017-184716 A). The multi-well device used in the present invention is not particularly limited as long as it has multiple wells, but the device described in JP 2017-184716 A is preferred. In one embodiment, the multi-well device used in the present invention comprises a container body made of a gas-permeable plastic film and an injection / exit port. The container body has a sealed periphery, a bulging top surface, and a plurality of recesses (wells) on the bottom surface of the container body that serve as cell culture areas. In such a multi-well device, the recesses typically have an opening diameter of 0.3 to 10 mm (0.5 mm in one embodiment) and a depth of 0.1 mm or more (0.2 mm in one embodiment), and the recesses occupy 30 to 90% of the area of ​​the bottom surface. The ratio d / D of the depth d to the diameter D of the recess is preferably 0.05 to 1. The oxygen permeability of the plastic film is typically 5000 mL / (m 2 ・day・atm) or more.

[0032] The material used to manufacture the container body of the multi-well device is not particularly limited as long as it has the desired gas permeability, and examples include thermoplastic resins such as polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyester, silicone-based elastomer, polystyrene-based elastomer, and tetrafluoroethylene-hexafluoropropylene copolymer (FEP). The number of wells can also be selected appropriately depending on the desired number of cell aggregates, but is typically 100 to 30,000 (18,000 in one embodiment).

[0033] As shown in the Examples below, shaking culture of hepatoblasts induced hepatocytes with superior albumin production ability compared to non-shaking cultures. Without being bound by any theory, it is speculated that the physical shaking of the medium during shaking culture allows for the supply of oxygen and nutrients and the efficient exchange of excreted waste products, thereby inducing hepatocytes with superior albumin production ability. Therefore, the shaking culture used in the present invention is not limited as long as it can physically shake the medium. Shaking culture can be performed, for example, by placing the culture vessel in which the hepatoblasts are cultured on a shaker, rotator, or the like. Shaking culture can also be performed by placing the hepatoblasts in an environment where a stirrer, impeller, or the like is rotating.

[0034] Shaking culture may be performed throughout the entire suspension culture period in the production method of the present invention, or only for a portion of the period. For example, static culture may be performed in a medium containing a hepatocyte induction promoter before shaking culture. The period of static culture is typically 6 hours to 3 days, preferably 0.5 days to 2 days, and more preferably 0.5 days to 1 day. Static culture allows the production of uniform hepatoblast cell clusters. In one embodiment of the present invention, the hepatoblast cell clusters used in the production method of the present invention are obtained by suspension culture (static culture) in the presence of an OSMR agonist, an ALK inhibitor, a cAMP agonist, and an adrenergic receptor agonist. In another embodiment, the production method of the present invention includes a step of suspension culture (static culture) of hepatoblasts in the presence of an OSMR agonist, an ALK inhibitor, a cAMP agonist, and an adrenergic receptor agonist prior to the step of suspension culture of the hepatoblast cell clusters in a medium containing a hepatocyte induction promoter.

[0035] Those skilled in the art can appropriately set parameters such as the speed of shaking culture. For example, when shaking culture is performed using a wave-type 3D shaker (e.g., Mini-Shaker 3D, manufactured by Biosan, etc.), the shaking speed range can typically be set between 5 and 60 rpm. When shaking culture is performed using a reciprocating shaker (e.g., NS-LR, manufactured by AS ONE, etc.), the shaking speed range can typically be set between 15 and 60 rpm. When shaking culture is performed using a seesaw shaker (e.g., NS-S, manufactured by AS ONE, etc.), the shaking speed range can typically be set between 5 and 50 rpm. Alternatively, for example, a spinner flask (e.g., 3152, manufactured by Corning, etc.) can be placed on a magnetic stirrer and cultured at a rotation speed that does not allow the hepatoblast cell clusters to settle visually. Culture can also be performed using a three-dimensional rotary suspension culture device (e.g., CellPet CUBE, manufactured by J-Tech; Clinostar, manufactured by Cellvivo, etc.). When shaking culture is performed using a three-dimensional rotary suspension culture device, the rotation speed can typically be set within the range of 15 to 60 rpm. Shaking culture may involve rotation perpendicular to the direction of gravity (horizontal rotation) or parallel to the direction of gravity (vertical rotation), with horizontal rotation being preferred.

[0036] The medium used in the present invention can typically be prepared by adding a hepatocyte induction promoter to a basal medium, such as IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and Lonza's HBM medium. TM Basal Medium, HCM TM SingleQuots TM Kit, HMM TM Basal Medium or HMM TM SingleQuots TMExamples include Hepatocyte Growth Medium or Hepatocyte Maintenance Medium from PromoCell, Hepatocyte Medium from Sigma-Aldrich, and a mixture of these media.

[0037] These basal media may contain serum (e.g., fetal bovine serum (FBS), horse serum, human serum, etc.) or may be serum-free. If serum-free, they may optionally contain one or more serum substitutes, such as albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. The media may also contain one or more substances, such as lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins (e.g., nicotinamide, ascorbic acid), growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, glucagon, hydrocortisone, epidermal growth factor (EGF), dexamethasone, and the like.

[0038] The duration of the process of producing hepatocytes from hepatoblasts of the present invention has no upper limit because it is assumed that long-term culture does not particularly affect the production efficiency of hepatocytes, but is typically 20 days or less (e.g., 15 days or less, 10 days or less, 9 days or less, 8 days or less, or 7 days or less). Examples of the culture period include 3 days or more, 4 days or more, 5 days or more, 6 days or more, and 7 days or more. Typically, the culture period is 3 to 9 days, preferably 5 to 8 days, and more preferably 5 to 7 days.

[0039] In the production method of the present invention, cells may be cultured under feeder-free conditions and / or xeno-free conditions. In the production method of the present invention, all steps may be performed under feeder-free and xeno-free conditions. As used herein, "feeder-free" refers to a medium or culture conditions that do not contain other cell types (i.e., feeder cells) that play a supporting role and are used to establish the culture conditions for the cells to be cultured. Furthermore, "xeno-free" refers to a medium or culture conditions that do not contain components derived from organisms other than the organism species of the cells to be cultured.

[0040] The hepatoblasts used in the present invention can be obtained by known methods. Examples include isolation from biological tissues (e.g., fetal liver, etc.) using known techniques, differentiation induction of endodermal cells (also referred to as "definitive endoderm cells"), and procurement from companies such as ATCC. Hepatoblasts can be isolated from biological tissues, for example, using surface antigens as an indicator, flow cytometry or mass cytometry, magnetic cell separation, or affinity columns immobilized with desired antigens. Preferably, the hepatoblasts used in the present invention are obtained by a method of differentiation induction of endodermal cells. Therefore, in one aspect, the production method of the present invention includes a step of culturing endodermal cells to produce hepatoblasts (also referred to as "differentiation induction") (hereinafter, also referred to as the "hepatoblast production step").

[0041] The hepatoblast production step can be performed by known methods (e.g., the methods described in Patent Document 1, Non-Patent Document 1, and Non-Patent Document 2). Specific examples include a method of culturing endoderm cells in a medium containing FGF2 and BMP4, a method of inducing hepatoblasts by culturing endoderm cells in a medium containing dimethyl sulfoxide (DMSO) (optionally further containing HGF), and a method of inducing posterior foregut cells by culturing endoderm cells in a medium containing FGF7 and SB431542 or a medium containing BMP4, TTNPB, A83-01, and FGF2, and then inducing hepatoblasts by culturing the cells in a medium containing FGF7, BMP4, and BMP3, or a medium containing forskolin, BMP4, Activin A, and C59 or CHIR99201.

[0042] Endodermal cells are cells that differentiate into cells that form tissues of organs such as the digestive tract, lung, thyroid gland, pancreas, and liver during mammalian development. As used herein, "endodermal cells" are cells that express at least one selected from the group consisting of SOX17, FOXA2, and CXCR4, and preferably cells that express at least SOX17.

[0043] When culturing endoderm cells, if the endoderm cells are provided as cell clusters, they may be cultured as single cells by substantially separating or dissociating them by any method, or they may be cultured in the state of cell clusters. Separation methods include, for example, mechanical separation, separation using a separation solution having protease activity and collagenase activity (for example, Accutase 1, a solution containing trypsin and collagenase), and the like. TM , Accumax TM (Nacalai Tesque) or separation using a separation solution having only collagenase activity.

[0044] FGF2 that can be used includes, for example, commercially available FGF2 from Fujifilm Wako Pure Chemical Industries, Ltd. When FGF2 is used in this step, the concentration of FGF2 in the medium is typically 0.1 ng / ml to 100 ng / ml, preferably 1 ng / ml to 50 ng / ml, and more preferably 5 ng / ml to 20 ng / ml (10 ng / ml in one embodiment).

[0045] BMP4 may be commercially available, for example, from Peprotech, Inc. When BMP4 is used in this step, the concentration of BMP4 in the medium is typically 0.2 ng / ml to 200 ng / ml, preferably 2 ng / ml to 100 ng / ml, and more preferably 10 ng / ml to 40 ng / ml (in one embodiment, 20 ng / ml).

[0046] The duration of the hepatoblast production process is not subject to an upper limit because it is believed that long-term culture does not have a particular effect on the efficiency of hepatoblast induction, but is typically 20 days or less (e.g., 15 days or less, 10 days or less, 9 days or less, 8 days or less, or 7 days or less). Examples of the culture period include 3 days or more, 4 days or more, 5 days or more, 6 days or more, and 7 days or more. Typically, the culture period is 3 to 9 days, preferably 3 to 8 days, and more preferably 4 to 7 days (5 days in one embodiment).

[0047] The endoderm cells used in the present invention can be obtained by known methods. Examples include isolation from biological tissue (e.g., intraembryonic tissue, etc.) using known techniques, differentiation induction of pluripotent stem cells, and procurement from companies such as ATCC. Endoderm cells can be isolated from biological tissue using, for example, flow cytometry or mass cytometry using surface antigens as an indicator, magnetic cell separation, or affinity columns immobilized with desired antigens. Preferably, the endoderm cells used in the present invention are obtained by a method of differentiation induction of pluripotent stem cells. Thus, in one aspect, the production method of the present invention includes a step of culturing pluripotent stem cells to produce endoderm cells (which can also be referred to as "differentiation induction") (hereinafter, also referred to as the "endodermal cell production step").

[0048] The endoderm cell production step can be performed by known methods (e.g., the methods described in Patent Document 1, Non-Patent Document 1, and Non-Patent Document 2). Specific examples include a method of activating activin A and the Wnt signaling pathway in pluripotent stem cells, a method of activating the Wnt signaling pathway in pluripotent stem cells under adherent culture conditions (WO2007 / 050043), and a method of co-culturing pluripotent stem cells with feeder cells (e.g., M15 cells) (WO2006 / 126574).

[0049] Preferably, the endoderm cell production step is a step of culturing pluripotent stem cells in a medium containing Activin A and a GSK-3β inhibitor. In such a step, the medium may further contain a histone deacetylase (HDAC) inhibitor.

[0050] Furthermore, the medium used in the endoderm cell production process may contain a ROCK inhibitor. Examples of such ROCK inhibitors include Y-27632 (see, e.g., Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), fasudil / HA1077 (see, e.g., Uenata et al., Nature 389: 990-994 (1997)), SR3677 (see, e.g., Feng Y et al., J Med Chem. 51: 6642-6645 (2008)), GSK269962 (see, e.g., Stavenger RA et al., J Med Chem. 50: 2-5 (2007) or WO2005 / 037197), GSK429286A, H1152 (see, e.g., Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (see, e.g., Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324 (2003)), thiazovivin and derivatives thereof. Among these, Y-27632 is preferred. When Y-27632 is used as a ROCK inhibitor, the concentration in the medium is typically 1 μM to 20 μM, preferably 5 μM to 15 μM (in one embodiment, 10 μM).

[0051] The term "pluripotent stem cells" refers to stem cells that can differentiate into various tissues and cells with different morphologies and functions in the body and have the ability to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm). Examples of pluripotent stem cells used in the present invention include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), multipotent germline stem cells (mGS cells), and embryonic germ stem cells (EG cells). Preferably, iPS cells (more preferably, human iPS cells) are used. When the pluripotent stem cells are ES cells or any cells derived from human embryos, they may be produced by or without the destruction of the embryo. However, from an ethical standpoint, cells produced without the destruction of the embryo are preferred.

[0052] ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in humans, monkeys, and other primates (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by isolating the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Alternatively, ES cells can be established using only a single blastomere from an embryo at the cleavage stage prior to the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or from a developmentally arrested embryo (Zhang X. et al. (2006), Stem Cells 24: 2669-2676).

[0053] Examples of ES cell lines that can be used in the present invention include mouse ES cell lines established by, for example, inGenious targeting laboratory, Inc., RIKEN (Riken), etc., and human ES cell lines established by, for example, the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, and Cellartis, Inc. Specific examples of human ES cell lines include CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 strains distributed by ESI Bio, H1 and H9 strains distributed by WiCell Research, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 strains distributed by RIKEN.

[0054] iPS cells are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of iPS cells, including iPSCs established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human cell-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872), Nanog-iPSCs established by selecting using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPSCs created using a method that does not include c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), iPSCs established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.), etc. can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells created by Sakurada et al. (JP Patent Publication No. 2008-307007) can also be used.In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7, 795-797), or patent publications (e.g., JP 2008-307007 A, JP 2008-283972 A, US 2008-2336610 A, US 2009-047263 A, WO 2007-069666 A, WO 2008-118220 A, WO 2008-124133 A, WO 2008-151058 A, WO 2009-006930 A, WO 2009-006997 A, WO 2009-007852 A) and known in the art can be used.

[0055] Available induced pluripotent stem cell lines include various iPSC lines established by the NIH, RIKEN, Kyoto University, etc. Examples of human iPSC lines include RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 lines, and Kyoto University's 253G1, 253G4, 1201C1, 1205D1, 1210B2, 1383D2, 1383D6, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A3, and FfI-01s04 lines.

[0056] The induced pluripotent stem cells used in the present invention may be cells derived from patients with hereditary diseases (e.g., patients with hereditary liver disease). Cells induced to differentiate from pluripotent stem cells derived from patients with hereditary liver disease can serve as disease models that reflect the pathology of the disease, and are therefore suitable for screening therapeutic or preventive drugs for the disease. Alternatively, pluripotent stem cells derived from patients with hereditary liver disease can be genetically repaired by genome editing using the CRISPR-Cas system or the like, and then differentiated into the desired cells, making it possible to use the cells as a therapeutic agent for the disease.

[0057] The species from which the pluripotent stem cells are derived is not particularly limited, and may be cells from, for example, rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, and sheep, carnivores such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. The preferred species is human.

[0058] Activin A that can be used includes, for example, commercially available products from R&D Systems, Inc. The concentration of Activin A in the medium used in this step is typically 1 ng / ml to 1000 ng / ml, preferably 10 ng / ml to 500 ng / ml, and more preferably 50 ng / ml to 200 ng / ml (100 ng / ml in one embodiment).

[0059] The GSK-3β inhibitor used in the present invention is not particularly limited as long as it can inhibit the function of GSK-3β, for example, kinase activity, and examples thereof include the indirubin derivative BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin 3'-oxime), the maleimide derivative SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), the phenyl alpha bromomethyl ketone compound GSK-3β inhibitor VII (4-dibromoacetophenone), the cell membrane-permeable phosphorylated peptide L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2), and the highly selective CHIR99021 (Nature (2008) 453: 519-523). Among these, CHIR99021 is preferred. The concentration of the GSK-3β inhibitor used in this step can be appropriately selected by those skilled in the art depending on the GSK-3β inhibitor used. For example, when CHIR99021 is used as the GSK-3β inhibitor, the concentration in the medium is typically 0.01 μM to 10 μM, preferably 0.05 μM to 5 μM, and more preferably 0.1 μM to 2.5 μM (in one embodiment, 1 μM).

[0060] Examples of HDAC inhibitors used in the present invention include valproic acid (VPA), trichostatin A, NaB, vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, apicidin, MGCD-0103, etc. The concentration of the HDAC inhibitor used in this step can be appropriately selected by those skilled in the art depending on the HDAC inhibitor used.

[0061] The duration of the endoderm cell production process has no upper limit because it is believed that long-term culture does not particularly affect the production efficiency of endoderm cells, but is typically 20 days or less (e.g., 15 days or less, 10 days or less, 9 days or less, 8 days or less, or 7 days or less). Examples of the culture period include 2 days or more, 3 days or more, 4 days or more, 5 days or more, and 6 days or more. Typically, the culture period is 2 to 8 days, preferably 2 to 7 days, and more preferably 3 to 6 days (4 days in one embodiment).

[0062] When the substance used in the present invention is a protein or peptide such as OSM or Activin A, its origin is not particularly limited, but is preferably mammalian (e.g., human, mouse, rat, monkey, bovine, equine, porcine, canine, etc.), with human origin being particularly preferred. In one embodiment, the protein or peptide used in the present invention is of human origin or an orthologue of a human protein or peptide in another mammalian species. Furthermore, the proteins or peptides used in the production methods of the present invention include not only wild-type proteins or peptides but also their variants that have similar functions (e.g., agonistic activity against OSMR). Examples of variants include proteins or peptides that have high identity (e.g., 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the amino acid sequence of a specific wild-type protein or peptide (e.g., the sequence set forth in SEQ ID NO: 2 or 4). Also preferred are variants of a particular wild-type protein or peptide (e.g., the sequence shown in SEQ ID NO: 2 or 4) in which one or several (2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids have been deleted, substituted, inserted, and / or added, and which have the same function as the wild-type protein or peptide.

[0063] When the substance used in the present invention is a compound, not only the free form but also its pharmacologically acceptable salts are included in the compound. The pharmacologically acceptable salts vary depending on the type of compound, but include, for example, inorganic base salts such as alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), aluminum salts, ammonium salts, etc., and organic base salts such as trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, etc., base addition salts, inorganic acid salts such as mesylate, hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, etc., and organic acid salts such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, etc. For example, phenylephrine also includes salts such as phenylephrine hydrochloride.

[0064] When the compound used in the present invention has isomers such as optical isomers, stereoisomers, positional isomers, and rotational isomers, either one of the isomers or a mixture thereof is encompassed by the compound. These isomers can be obtained as a single product by known synthesis methods, separation methods (e.g., concentration, solvent extraction, column chromatography, recrystallization, etc.), optical resolution methods (e.g., fractional recrystallization, chiral column method, diastereomer method, etc.), etc. The compound used in the present invention may be crystalline, and both a single crystalline form and a crystalline mixture are encompassed by the compound. Crystals can be produced by crystallization using known crystallization methods. The compound used in the present invention may be a solvate (e.g., hydrate, etc.) or a non-solvate (e.g., non-hydrate, etc.), and both are encompassed by the compound. Furthermore, the compound used in the present invention may contain isotopes (e.g., 3 H, 14 C. 35 S, 125 Also included are compounds labeled with iodine, i.e., iodine-1, iodine-2, iodine-3, iodine-4, iodine-5, iodine-6, iodine-7, iodine-8, iodine-9, iodine-10, iodine-11, iodine-12, iodine-13, iodine-14, iodine-15,

[0065] The culture used in the hepatoblast production process and / or endoderm cell production process may be either adherent culture or suspension culture, with adherent culture being preferred. Adherent culture can typically be performed by culturing cells using a coated culture vessel. Examples of coating agents include Matrigel (BD Biosciences), Synthemax (Corning), collagen, gelatin, heparan sulfate proteoglycan, entactin, and combinations thereof, with Matrigel, Synthemax, or gelatin being preferred.

[0066] Alternatively, from the viewpoint of xeno-free, laminin or a fragment thereof is also preferred. Examples of laminin or a fragment thereof include laminin-111 or a fragment thereof comprising its E8 region, laminin-211 or a fragment thereof comprising its E8 region (e.g., iMatrix-211), laminin-121 or a fragment thereof comprising its E8 region, laminin-221 or a fragment thereof comprising its E8 region, laminin-332 or a fragment thereof comprising its E8 region, laminin-3A11 or a fragment thereof comprising its E8 region, laminin-411 or a fragment thereof comprising its E8 region (e.g., iMatrix-411), laminin-421 or a fragment thereof comprising its E8 region, and laminin-511 or a fragment thereof comprising its E8 region (e.g., iMatrix-511, iMatrix-511). silk), laminin-521 or a fragment thereof containing its E8 region, laminin-213 or a fragment thereof containing its E8 region, laminin-423 or a fragment thereof containing its E8 region, laminin-523 or a fragment thereof containing its E8 region, laminin-212 / 222 or a fragment thereof containing its E8 region, and laminin-522 or a fragment thereof containing its E8 region.

[0067] The media used in the hepatoblast cell production process and the endoderm cell production process can typically be prepared by adding a differentiation inducer to the target cells, such as FGF2, BMP4, Activin A, or a GSK-3β inhibitor, to a basal medium. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), and KnockOut medium. TM Basal media include DMEM medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. Basal media may contain serum (e.g., fetal bovine serum (FBS), horse serum, human serum, etc.) or may be serum-free. Serum-free media may optionally contain one or more serum substitutes, such as albumin, transferrin, KSR (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. Basal media may also contain one or more substances, such as lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and the like. In one embodiment, the basal medium used in the hepatoblast production process is KnockOut medium containing KSR, L-glutamine, NEAA, 2-mercaptoethanol, and antibiotics. TM In one embodiment, the medium used in the hepatoblast production process is RPMI 1640 medium containing B27 supplement and antibiotics.

[0068] The hepatoblast production step and / or the endoderm cell production step may be a step of culturing cells under feeder-free conditions and / or xeno-free conditions.

[0069] The culture temperature in each step of the production method of the present invention is not particularly limited, but is 30°C to 40°C, preferably 37°C, and culture is performed in an atmosphere of CO2-containing air, with the CO2 concentration preferably being 2% to 5%.

[0070] The seeding density of cells in each step of the production method of the present invention is not particularly limited as long as the cells can grow. Typically, it is 1.0 × 10 2 ~1.0×10 7 cells / cm 2 , preferably 1.0 x 10 3 ~1.0×10 6 cells / cm 2 , more preferably 1.0 × 10 4 ~1.0×10 5 cells / cm 2 is.

[0071] Each substance used in the present invention may be synthesized by a known method, or a commercially available product may be used. Furthermore, when the compound is a protein or peptide such as a Wnt protein, the target protein or peptide may be obtained from a cell that expresses it. Cells that express the target protein or peptide can be produced by inserting DNA encoding the target protein or peptide into a known expression vector and then introducing the resulting expression vector into an appropriate host cell.

[0072] The production method of the present invention may include a step of recovering the target cells or tissues obtained in each step. The recovered cells may be cryopreserved using a cell cryopreservation solution. The obtained cells may be counted using a cell counter, or may be labeled with an antibody against a cell surface marker and then selected or purified by flow cytometry, mass cytometry, magnetic cell sorting, or the like.

[0073] 2. Uses of Hepatocytes In another aspect of the present invention, hepatocytes (hereinafter sometimes referred to as "hepatocytes of the present invention") obtained by the production method of the present invention ("obtained" can be read as "obtained" as appropriate) are also provided. The hepatocytes of the present invention may be in the form of hepatocyte clusters, or may be single cells isolated by flow cytometry, mass cytometry, magnetic cell separation, or the like.

[0074] Because the hepatocytes of the present invention can be used in medical treatments such as regenerative medicine, in another aspect, a transplantation therapeutic agent (hereinafter, sometimes referred to as the "transplantation therapeutic agent of the present invention") containing the hepatocytes of the present invention is provided. Also encompassed by the present invention is a method for treating or preventing liver disease, in which an effective amount of the hepatocytes of the present invention is transplanted into a mammal (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.) that is the target of treatment or prevention. Unless otherwise specified herein, the term "disease therapeutic or preventive agent (or therapeutic or preventive method)" also encompasses a pharmaceutical agent (or method) that can both treat and prevent the disease.

[0075] Examples of liver diseases that can be treated or prevented include hemochromatosis, Wilson's disease, glycogen storage disease, amino acid metabolism disorders, urea cycle metabolism disorders, porphyria, constitutional jaundice, fibrotic polycystic liver disease, nonalcoholic steatohepatitis, liver cancer, hepatitis B, hepatic fibrosis, liver cirrhosis, hereditary ATTR amyloidosis (familial amyloidotic polyneuropathy (FAP)), alpha-1 antitrypsin deficiency (AATD), etc. The hepatocytes of the present invention exert a therapeutic or preventive effect on liver diseases when transplanted into the affected area or into a site resected for disease treatment.

[0076] The transplantation therapy agent of the present invention can be used by transplanting it into the living body of a subject in need thereof. The amount of hepatocytes to be transplanted should be a therapeutically or prophylactically effective amount, which may vary depending on factors such as the age, weight, size of the transplant site, and severity of the disease of the transplant subject, and is not particularly limited. For example, the number of cells may be 10 × 10 4 Cell ~10×10 11More specifically, transplantation of the transplant agent of the present invention into a patient can be performed, for example, by forming the hepatocytes of the present invention into a sheet and attaching it to the patient's liver, by suspending the obtained hepatocytes in physiological saline or the like and directly transplanting them into the patient's liver, or by three-dimensionally culturing the hepatocytes on a scaffold made of Matrigel or the like and transplanting the obtained hepatocyte cell mass.

[0077] When the hepatocytes of the present invention are used as transplantation therapeutic agents, it is desirable to use hepatocytes derived from iPS cells established from somatic cells with the same or substantially the same HLA genotype as the recipient individual, in order to avoid rejection. Here, "substantially the same" means that the HLA genotype is identical to that of the transplanted cells to an extent that immune responses can be suppressed with immunosuppressants, e.g., somatic cells with an HLA type that matches the three HLA loci (HLA-A, HLA-B, and HLA-DR) or the four HLA loci (HLA-C). If sufficient cells cannot be obtained due to age, constitution, or other reasons, they can be transplanted in a state that avoids rejection by embedding them in capsules or porous containers made of polyethylene glycol or silicone.

[0078] The hepatocytes of the present invention are prepared as parenteral formulations such as injections, suspensions, and infusions by mixing with a pharmaceutically acceptable carrier according to conventional methods. Therefore, in one embodiment, a method for producing a transplantation therapy agent is also provided, which includes a step of formulating the hepatocytes of the present invention. Such a method may include a step of preparing the hepatocytes of the present invention. Furthermore, the method may include a step of preserving the hepatocytes of the present invention.

[0079] Pharmaceutically acceptable carriers that can be contained in such parenteral formulations include aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), etc. The hepatocytes of the present invention may be formulated with, for example, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc.

[0080] The transplantation therapy agent of the present invention is provided in a cryopreserved state under conditions typically used for cryopreserving cells and can be thawed immediately before use. In this case, it may further contain serum or a serum substitute, an organic solvent (e.g., DMSO), etc. In this case, the concentration of the serum or serum substitute is not particularly limited, but may be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, but may be 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0081] The hepatocytes of the present invention can also be used in methods for screening candidate drugs useful for treating or preventing liver disease. Thus, in yet another aspect of the present invention, a method for screening therapeutic or preventive drugs for liver disease is provided, comprising culturing the hepatocytes of the present invention in the presence or absence of a test substance. For example, a disease model reflecting the pathology of liver disease is used as the hepatocytes, and if the pathology improves in the presence of the test substance, the test substance can be selected as a candidate drug for treating or preventing liver disease. Examples of such liver diseases include those similar to the liver diseases described above that are the target of treatment or prevention using the transplantation therapy agent of the present invention.

[0082] The hepatocytes of the present invention can also be used to evaluate the pharmacokinetics and toxicity of drugs. Specifically, the hepatocytes of the present invention can be used to test, for example, drug interactions of test substances, induction of drug-metabolizing enzymes, induction of drug transporters, toxicity, etc.

[0083] Because the hepatocytes of the present invention can express drug-metabolizing enzymes, the degree of metabolism of a drug (test substance) can be evaluated using the activity of the drug-metabolizing enzyme as an indicator. Evaluating the degree of drug metabolism also makes it possible to evaluate the effects, toxicity, etc. of the drug in vivo. Furthermore, analyzing (e.g., detecting, quantifying, identifying, etc.) drug metabolites produced as a result of an enzymatic reaction using the drug as a substrate by the drug-metabolizing enzyme also makes it possible to evaluate the effects, toxicity, etc. of the drug in vivo. Therefore, in another aspect of the present invention, there is provided a method for evaluating the degree of metabolism of a test substance, comprising: (I) contacting the test substance with the hepatocytes of the present invention; (II) measuring the activity level of the drug-metabolizing enzyme in the hepatocytes; and (III) evaluating the degree of metabolism of the test substance by the drug-metabolizing enzyme based on the activity level of the drug-metabolizing enzyme in step (II).

[0084] Step (II) is typically a step of measuring the expression level of a drug-metabolizing enzyme. The expression level of a drug-metabolizing enzyme can be evaluated at the mRNA level or protein level. For example, if the mRNA level of the drug-metabolizing enzyme increases compared to before contact with the test substance or compared to contact with a negative control, it can be determined that at least a portion of the test substance has been metabolized. The expression level can also be used to evaluate the degree of drug metabolism (e.g., the proportion of drug metabolized, metabolic rate, etc.). Examples of metabolism of the test substance include hydrolysis of esters, oxidation reactions (especially oxidation by cytochrome P450), chemical modification of the test substance by reduction reactions, etc.

[0085] Furthermore, in step (I), the hepatocytes of the present invention can also be contacted with substances known to be metabolized by drug-metabolizing enzymes (e.g., triazolam (metabolite; α-hydroxy-triazolam), diclofenac (metabolite; 4-hydroxydiclofenac), 7-hydroxycoumarin (metabolite; 7-hydroxycoumarin glucuronide), etc.), and the amount of the metabolite of the substance can be measured. Alternatively, the measurement can be performed using a commercially available kit for measuring the activity of drug-metabolizing enzymes (e.g., P450-Glo TMIt can also be measured using CYP3A4 assays.

[0086] Examples of drug-metabolizing enzymes used in the present invention include cytochrome P450 (e.g., CYP3A4, CYP2C9, etc.), uridine diphosphate-glucuronosyltransferase (e.g., UGT1A1, UGT1A8, UGT1A10, etc.), sulfotransferase (e.g., SULT1A3, etc.), etc. Multiple types of drug-metabolizing enzymes may be used in combination.

[0087] In yet another aspect, the present invention provides a method for evaluating the efficacy or toxicity of a drug in a living body, comprising: (i) contacting a test substance with a hepatocyte of the present invention; and (ii) analyzing metabolic products of the test substance.

[0088] For example, if the amount of metabolites of a test substance is small, the test substance can be determined to be less susceptible to metabolic effects in the liver and to be highly effective in the living body.Furthermore, if the metabolites are toxic, the test substance can be determined to be toxic in the living body.

[0089] The method for analyzing metabolites can be selected appropriately depending on the test substance and its predicted metabolic products. For example, mass spectrometry, liquid chromatography, immunological techniques (e.g., fluorescence immunoassay (FIA) and enzyme immunoassay (EIA)), etc. can be used.

[0090] Furthermore, a method for evaluating the toxicity of a drug in a living body is also provided, which includes the steps of contacting a test substance with hepatocytes of the present invention and evaluating the state of the hepatocytes. The state of such hepatocytes can be evaluated by measuring viability, observing cell morphology, measuring liver damage markers (GOT, GPT, etc.) in the culture medium, and the like. For example, if a decrease in viability is observed upon contact with the test substance, the test substance can be determined to be hepatotoxic. Similarly, if abnormalities in cell morphology are observed upon contact with the test substance or the amount of liver damage markers in the culture medium is increased, the test substance can be determined to be hepatotoxic. Quantitative evaluation may also be performed depending on the degree of decrease in viability or the amount of liver damage markers.

[0091] The step of contacting the test substance with the hepatocytes of the present invention can typically be carried out by adding the test substance to a culture medium in which the hepatocytes of the present invention are cultured, or by transferring the hepatocytes of the present invention to a culture medium to which the test substance has been added in advance.

[0092] The period of time for contacting the test substance with the hepatocytes of the present invention is not particularly limited, but is typically 1 minute to 5 days, preferably 1 hour to 1 day.

[0093] Test substances used in the present invention include, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low-molecular-weight compounds, and natural compounds. Test substances may also be existing or candidate components of pharmaceuticals, nutritional foods, etc. Furthermore, by simultaneously contacting two or more test substances with cells, interactions, synergistic effects, etc. between the test substances can be verified.

[0094] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0095] Hepatocyte clusters were prepared according to the following procedure: In this example, human pluripotent stem cells (1231A3 strain) were maintained and cultured as starting cells by the feeder-free method, and were used in the subsequent differentiation induction experiments.

[0096] Each well of human pluripotent stem cells was washed with 0.5 mM EDTA (UltraPure 0.5 M EDTA, Thermo Fisher Scientific, Cat# 15575020) diluted with Stage 1 PBS(-) (Nacalai Tesque, Cat# 14249-24), and then the cells were dispersed using TrypLE Select CTS (Thermo Fisher Scientific, Cat# A1285901) diluted 2-fold with 0.5 mM EDTA. The cells were counted and suspended in Stage 1 medium (48.5 ml RPMI1640 (Nacalai Tesque, Cat# 30264-50), 1 ml B-27 supplement (Thermo Fisher Scientific, Cat# 17504044), 500 μl Penicillin-Streptomycin (Thermo Fisher Scientific, Cat# 15140122), 100 ng / ml activin A (R&D, Cat# 338-AC-01M), 1 μM CHIR99021 (Axon Medchem, Cat# Axon 1386)) containing 10 μM Y-27632 (Fujifilm-Wako, Cat# 253-00513), and then cultured in Matrigel (BD Matrigel Matrix Growth Factor Reduced, BD Biosciences, Cat# 4 × 10 cells were placed on a 12-well plate (Greiner) coated with 300 μL of PBS (Product No. 354230). 5 Cells were seeded at a density of 1000 cells / well. The medium was replaced with Stage 1 medium every day until day 4 of induction.

[0097] On the fourth day after Stage 2 induction, the medium was replaced with Stage 2 medium. Stage 2 medium (44 ml KnockOut DMEM (Thermo Fisher Scientific, Cat# 10829018), 5 ml KnockOut Serum Replacement (Thermo Fisher Scientific, Cat# 10828028), 500 μl L-glutamine 200 mM (Thermo Fisher Scientific, cat# 25030081), 500 μl MEM Non-Essential Amino Acids Solution x100 (Thermo Fisher Scientific, cat# 11140050), 100 μl 2-Mercaptoethanol (Thermo Fisher Scientific, cat# 21985023), 250 μl Penicillin-Streptomycin (Thermo Fisher Scientific, cat# 15140122), 20 ng / ml BMP4 (Peprotech, Cat# AF-120-05ET), 10 The medium was replaced with 200 ng / ml FGF2 (Fujifilm-Wako, Cat# 060-04543) every two days.

[0098] On day 9 of Stage 3 induction, cells were dispersed using Accumax (Nacalai Tesque, Cat# 1708754) or TrypLE Select CTS and suspended in Stage 3 medium for spinner culture containing 10 μM Y-27632 (HCM BulletKit (Lonza, Cat# CC-3198, EGF provided with the kit was not used), candidate compound). (1) Without a well bag (conventional method): The cell suspension was seeded into an uncoated 12-well plate. The cell seeding density was adjusted as appropriate, for example, by dispersing cells collected from one well and seeding them in one well, or by dispersing cells collected from two wells and seeding them in one well. The 12-well plate was placed on a shaker in a 37°C, 5% CO2 incubator and cultured by reciprocal shaking. The following day, numerous cell clusters had formed, and the medium was replaced with Stage 3 medium for spinner culture without Y-27632. The medium was then replaced every two days, and HLC-aggregates obtained on day 6 of spinning culture were used for assays. (2) Using well bags (bag method): Induced cells from two 12-well plates were suspended in 20 ml of Stage 3 spinning culture medium containing 10 μM Y-27632 and placed in a low-adhesion well bag (18,000 wells, Toyo Seikan) and cultured overnight in a 37°C, 5% CO2 incubator. The formed cell aggregates were replaced with Stage 3 spinning culture medium without Y-27632 and replated onto two 12-well plates for spinning culture. The medium was replaced on day 6 of spinning culture, and HLC-aggregates obtained on day 7 of spinning culture were used for assays.

[0099] Example 1: Verification of the Effect of Compound Cocktails on Hepatocyte Differentiation. It has been reported that NOTCH signaling and TGFβ signaling promote the differentiation of hepatoblasts into bile duct epithelial cells, while inhibiting their differentiation into hepatocytes. Furthermore, it has been reported that PKA agonists promote the functional maturation of induced hepatocytes (Lay Teng Ang et al., Cell Rep. 22(8):2190-2205 (2018)) (Figure 1). Based on these previous reports on plate culture, we investigated compound combinations that may be useful for inducing hepatocyte differentiation. Unless otherwise noted, the abbreviations for the compounds refer to the following: OSM: 20 ng / ml oncostatin M HGF: 20 ng / ml hepatocyte growth factor DEX: 100 nM dexamethasone Phe or P: 1 μM phenylephrine A: 1 μM A83-01 D: 10 μM DAPT (n-[n-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycine t-butyl ester) F: 10 μM forskolin

[0100] Figure 2(a) shows the culture scheme used in Example 1. Starting on day 9 of induction, hepatoblasts were cultured in stage 3 medium using a spinner. Candidate compounds (HGF + OSM + DEX, HGF + OSM, OSM, or OSM + Phe) were added to the stage 3 medium, and evaluation was performed on day 15 of induction. The results showed no significant differences in the morphology of the HLC-aggregates (Figure 2(b)). Furthermore, the amount of albumin secreted into the medium by each HLC-aggregate per 24 hours was measured by ELISA (Figure 2(c)). The results showed that HGF, DEX, and Phe did not have an additive effect on HLC-aggregate maturation with OSM. In the following examples, the evaluation of candidate compound combinations in stage 3 medium during spinner culture was based on whether or not they had an additive effect on OSM.

[0101] Example 2: Verification of the Effect of the Combination of OSM and ADPF Cocktail Figure 3(a) shows the culture scheme for Example 2. As in Example 1, hepatoblasts were cultured in spinner culture in stage 3 medium from day 9 of induction. Candidate compounds (HGF + OSM, OSM, OSM + ADP, or OSM + ADPF) were added to stage 3 medium, and evaluation was performed on day 15 of induction. The addition of the ADPF cocktail to the medium clearly altered the morphology of HLC-aggregates (Figure 3(b)). Furthermore, the amount of albumin secreted into the medium by each HLC-aggregate per 24 hours was measured by ELISA (Figure 3(c)). The addition of the ADPF cocktail to Oncostatin M significantly increased albumin secretion.

[0102] Example 3: Minus-one assay of ADPF cocktail and plus-one assay of OSM. Figure 4(a) shows the culture scheme for Example 3. As in Example 1, hepatoblasts were cultured in spinner culture in stage 3 medium from day 9 of induction. The OSM+ADPF cocktail or a cocktail omitting one compound from the above (OSM+ADPF, OSM+DPF, OSM+APF, OSM+ADF, or OSM+ADP) was added to stage 3 medium, and evaluation was performed on day 15 of induction. The morphological changes in HLC-aggregates induced by the ADPF cocktail were abolished when forskolin was omitted (Figure 4(b)). Furthermore, the amount of albumin secreted into the medium by each HLC-aggregate per 24 hours was measured by ELISA (Figure 4(c)). The removal of forskolin from the ADPF cocktail significantly reduced albumin secretion. Therefore, forskolin is considered to be the primary factor in the maturation of HLC-aggregates among the compound cocktails.

[0103] Furthermore, OSM plus one compound cocktail (OSM+A, OSM+D, OSM+P, OSM+F, OSM+ADPF, or OSM+APF) was added to the stage 3 medium and evaluated on day 15 of induction. The addition of forskolin to OSM induced morphological changes in HLC-aggregates (Fig. 4(d)). Furthermore, the amount of albumin secreted into the medium by each HLC-aggregate per 24 h was measured by ELISA (Fig. 4(e)). The additive effect of forskolin on OSM was clear. On the other hand, DAPT did not exhibit any additive effect with OSM. In fact, omitting DAPT from the ADPF cocktail increased albumin secretion, indicating that DAPT was ineffective in maturing HLC-aggregates.

[0104] Example 4: Verification of the Effect of the Combination of OSM and APF Cocktail Figure 5(a) shows the culture scheme for Example 4. As in Example 1, hepatoblasts were cultured in spinner culture in stage 3 medium from day 9 of induction. Candidate compounds (OSM+F, OSM+AF, OSM+DF, OSM+PF, or OSM+APF) were added to stage 3 medium, and evaluation was performed on day 15 of induction. Cell aggregates were generated from roughly the same number of cells under each condition, but HLC-aggregates were most efficiently obtained under the OSM+APF condition (Figure 5(b)). Furthermore, the amount of albumin secreted into the medium by each HLC-aggregate per 24 hours was measured by ELISA (Figure 5(c)). OSM+APF showed the highest albumin secretion. Therefore, OSM+APF was demonstrated to be the optimal compound combination for spinner culture of hepatoblasts and the generation of hepatocyte aggregates.

[0105] Example 5: Verification of HLC-aggregate production using a multiwell device We investigated whether homogeneous HLC-aggregates could be produced using a multiwell device (also referred to as a well bag). Figure 6(a) shows the culture scheme for Example 5. On day 9 of induction, hepatoblasts suspended in stage 3 medium were injected into a well bag (Figure 6(b)). Forced aggregation was allowed to occur overnight in suspension (static culture). The cell aggregates were then replated in a 12-well plate and cultured in agitated culture. OSM+APF was added to the stage 3 medium, and evaluation was performed on day 17 of induction. The results demonstrated that homogeneous HLC-aggregates could be efficiently produced using a multiwell device (Figures 6(b)-(f)).

[0106] Example 6: Comparison of Spinner Culture and Plate Culture Using OSM+APF The maturity of HLCs was compared between spinner culture and plate culture. Figure 7(a) shows the culture scheme for Example 6. For plate culture, HLCs were generated by adding 20 ng / ml OSM and 20 ng / ml HGF to the medium from day 9 of induction onward. For spinner culture, HLC-aggregates were generated according to the method described in Example 5. Evaluation was performed on day 14 of induction for plate culture and day 17 for spinner culture. The results showed that the HLC-aggregates generated by OSM+APF and spinner culture were more mature in terms of gene expression than HLCs obtained by conventional plate culture (Figure 7(b)).

[0107] Example 7: Evaluation of HLCs prepared under various culture conditions HLCs prepared by varying the timing of RNA collection, the type of culture (suspension culture or plate culture), and the presence or absence of shaking were evaluated. The culture schemes and results are shown in Figures 8 to 12. These results demonstrate that suspension culture, regardless of whether shaking is performed, yields more mature HLCs than plate culture, but that shaking yields even more mature HLCs.

[0108] The present invention makes it possible to produce hepatocytes in vitro. The cells produced in this manner can have properties closer to those of living hepatocytes, and therefore can be used in drug screening, drug toxicity evaluation, regenerative medicine for liver diseases, and other fields, and are therefore expected to be applied in the medical field, particularly in the field of regenerative medicine.

[0109] This application is based on patent application No. 2024-038486 filed in Japan (filing date: March 12, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing hepatocytes, comprising the step of culturing hepatoblasts in suspension in a medium containing an oncostatin M receptor agonist, an ALK inhibitor, a cAMP agonist, and an adrenergic receptor agonist.

2. The method of claim 1, wherein the hepatocytes are in the form of cell aggregates.

3. The method according to claim 1 or 2, which comprises a step of performing shaking culture.

4. The method according to any one of claims 1 to 3, which comprises a step of culturing in a culture bag having a plurality of wells.

5. The method according to any one of claims 1 to 4, wherein the suspension culture is carried out in a medium that does not contain hepatocyte growth factor and / or DAPT.

6. The method of any one of claims 1 to 5, wherein at least one of the oncostatin M receptor agonists is oncostatin M.

7. The method of any one of claims 1 to 6, wherein at least one of the ALK inhibitors is A83-01.

8. The method according to any one of claims 1 to 7, wherein at least one of the cAMP agonists is forskolin.

9. The method of any one of claims 1 to 8, wherein at least one of the adrenergic receptor agonists is phenylephrine.

10. The method of any one of claims 1 to 9, wherein the hepatoblasts are derived from pluripotent stem cells.

11. The method according to any one of claims 2 to 10, wherein the hepatoblast aggregates are obtained by suspension culture in a medium containing an oncostatin M receptor agonist, an ALK inhibitor, a cAMP agonist, and an adrenergic receptor agonist.

12. Hepatocytes obtained by the method according to any one of claims 1 to 11.

13. A cell transplantation therapy comprising the hepatocytes described in claim 12.

14. The agent according to claim 13 for the treatment or prevention of liver disease.

15. A method for evaluating the degree of metabolism of a test substance, comprising: (I) contacting the test substance with the hepatocytes described in claim 12; (II) measuring the activity level of a drug-metabolizing enzyme in the hepatocytes; and (III) evaluating the degree of metabolism of the test substance by the drug-metabolizing enzyme based on the activity level of the drug-metabolizing enzyme in step (II).

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