Method for purifying hepatic parenchymal cells

Abstract

To provide a method that enables purification of hepatic parenchymal cells at high purity, utilizing a hepatic parenchymal cell-specific metabolic pathway, in particular, a method that enables purification of hepatic parenchymal cells without employing cell sorting, and also to provide a cell population comprising hepatic parenchymal cells obtained by the method at high purity.SOLUTION: The present invention provides a method for producing a cell population comprising enriched hepatic parenchymal cells, the method involving a step of culturing a cell population comprising hepatic parenchymal cells in the absence of glucose, fructose, and galactose.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing a cell population enriched with hepatocytes, etc. More specifically, the present invention relates to a method for producing a cell population enriched with hepatocytes using a metabolic pathway specific to hepatocytes, and to the use of the cell population produced by the method, etc.

Background Art

[0002] Hepatocytes are cells responsible for important physiological functions of the liver, such as metabolism of nutrients, detoxification of drugs and additives, bile production, and albumin synthesis, and are distinguished from non-parenchymal cells (bile duct epithelial cells, hepatic stellate cells, sinusoidal endothelial cells, etc.). Since the liver has the ability to regenerate, even when a disease develops in the liver, it can be repaired, regenerated, and its function can be maintained. However, in cases of chronic hepatitis, etc., the regenerative ability decreases, leading to progression to fibrosis and cirrhosis, and ultimately there is also a possibility of causing liver cancer. For such severe liver diseases, liver transplantation is the only fundamental treatment method, but in Japan, there is a problem of a serious shortage of donors. Although living donor liver transplantation is often performed instead of liver transplantation, there is also a risk of the donor's death or developing complications. Since hepatocytes derived from pluripotent stem cells that can proliferate infinitely can be a useful source of hepatocytes, attention has been focused on liver regenerative medicine using pluripotent stem cells, particularly induced pluripotent stem cells (iPS cells). However, in regenerative medicine using pluripotent stem cells, there is a risk of tumorigenesis due to contamination of undifferentiated cells or accidentally differentiated proliferative cells in the transplanted organ.

[0003] Therefore, in order to obtain more uniform and safer hepatocytes, the development of a technique for highly purifying hepatocytes derived from pluripotent stem cells from a cell population is desired. As a technique for purifying such hepatocytes, for example, there is a gene modification method (for example, Non-Patent Document 1, etc.), but in this method, toxicity and side effects due to gene modification become problems, and furthermore, there are also problems that the uncertainty of gene modification, the purification purity, and the yield are not sufficient. There is also an mRNA switch method (Non-Patent Document 2), but in this method, there is a risk that exogenous mRNA remains. There is also an anti-Asialoglycoprotein receptor 1 (ASGR1) antibody method (Non-Patent Document 3), but in this method, when using a cell population derived from pluripotent stem cells, since ASGR1, which is expressed only in a very small part of hepatocytes (about 30% at most), is used as an index, there is a problem that the yield is low and only special cells are extracted. The present inventors have previously reported a mitochondrial staining and anti-Activated leukocyte cell adhesion molecule (ALCAM) antibody method (Non-Patent Document 4). However, all of these methods are purification methods by cell sorting, and due to enzyme treatment and mechanical stress, the survival recovery rate of target cells is low, and it is difficult to prepare a sufficient number of cells applicable to clinical applications.

[0004] As a method for purifying hepatocytes without using cell sorting, there is also a density gradient centrifugation method (Non-Patent Document 5), but in this method, there is a problem that high purification is impossible.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0006] Therefore, an object of the present invention is to provide a method for purifying hepatocytes with high purity by utilizing the energy metabolism pathway specifically possessed by hepatocytes, particularly a method for purifying hepatocytes without using cell sorting. Another object is to provide a cell population containing hepatocytes obtained by the above method with high purity.

Means for Solving the Problems

[0007] As a result of intensive research, the present inventors conceived that hepatocytes could be purified by culturing cells under culture conditions in which only hepatocytes can survive, taking advantage of the metabolic characteristics of hepatocytes. Since all cells survive depending on glucose metabolism, the inventors conceived of allowing only hepatocytes to survive by replacing glucose, which is the energy source in the culture medium, with an energy source that conforms to the metabolic characteristics of hepatocytes. Therefore, the inventors first focused on ketohexokinase (KHK), which is specifically expressed in hepatocytes. KHK is an enzyme that catalyzes the phosphorylation of fructose to produce fructose 1-phosphate. Then, the inventors conceived of using fructose instead of glucose, which is commonly used, as the sugar that is the energy source in the culture medium. On the other hand, since hexokinase (HK), which catalyzes the reaction of converting fructose to fructose 6-phosphate, exists in cells other than hepatocytes (non-hepatocytes), it was decided to inhibit HK in parallel. As a result, they succeeded in selectively killing non-hepatocytes from a cell population containing hepatocytes and concentrating hepatocytes.

[0008] Secondly, the inventors focused on the fructose metabolic pathway in naked mole-rats growing underground. In general mammalian animals, fructose metabolism occurs only in the liver. On the other hand, naked mole-rats growing underground in a hypoxic state can metabolize fructose by KHK in all organs. In the normal glycolytic metabolic pathway, there is a feedback inhibition mechanism for phosphofructokinase (PFK) that catalyzes the production of fructose 1,6-bisphosphate from fructose 6-phosphate produced by HK. Therefore, there is a possibility that the glycolytic system may be suppressed even under hypoxia. Therefore, it is known that naked mole-rats enable KHK metabolism of fructose, which is not regulated by feedback inhibition, in all organs to avoid anoxia (Park et al., Science 356, 307-311 (2017)). Therefore, the inventors thought that in human cells, since cells other than hepatocytes cannot metabolize by KHK, culturing under hypoxic conditions can function the feedback inhibition on PFK, and thereby kill non-parenchymal liver cells.

[0009] Therefore, when experiments were actually conducted, non-parenchymal liver cells could not be killed. When investigating the cause, it was unexpectedly found that in non-parenchymal liver cells, the gene expression of an enzyme (PFKFB3) related to the activation of PFK was increased under hypoxic conditions. Therefore, under hypoxic conditions, the idea was conceived to suppress the glycolytic metabolism of non-parenchymal liver cells by inhibiting PFK. As a result, non-parenchymal liver cells were selectively killed from a cell population including parenchymal liver cells, and successful enrichment of parenchymal liver cells was achieved.

[0010] Thirdly, the inventors focused on the property of hepatocytes to store glycogen. Hepatocytes break down glycogen to obtain an energy source when sugars such as glucose are not present in the culture medium, while non-hepatocytes store less glycogen and it is difficult to secure an energy source. Therefore, when a cell population containing hepatocytes was cultured in a medium lacking sugars such as glucose and fructose, they succeeded in selectively killing the hepatocytes. Furthermore, when an HK inhibitor was used in combination, they found that non-hepatocytes could be killed more efficiently.

[0011] Furthermore, the present inventors considered that stress was also imposed on hepatocytes by removing or replacing glucose from the medium, or removing all sugars, or by combining hypoxia and inhibitors. Therefore, in order to make this purification method more easily applicable, the inventors conceived of specifically protecting only hepatocytes during the purification process. When comprehensively investigating the metabolic characteristics of hepatocytes in a sugar-free medium by metabolome analysis, the present inventors found that hepatocytes were exposed to oxidative stress, changes in energy storage substances (creatine phosphate), and changes in amino acid degradation and synthesis. Therefore, the present inventors devised a method of adding reduced glutathione and N-acetylcysteine to suppress oxidative stress. In addition, the present inventors devised a method of adding creatine, arginine, glycine, guanidinoacetic acid, and methionine, which are the sources of creatine synthesis, to the medium in order to assist the changes in creatine phosphate. Furthermore, since fluctuations in amino acid utilization were observed by metabolome analysis, the present inventors focused on alanine, which is not contained in the sugar-free medium. Therefore, when alanine was added to the sugar-free medium, non-hepatocytes died, and only hepatocytes survived. Subsequently, the inventors succeeded in extending the period until the hepatocytes died. In addition, ornithine in the urea cycle (also called the ornithine cycle), which is unique to hepatocytes, increased under sugar-free conditions, and it was considered that this was due to the increase in ammonia at the end of amino acid degradation. Therefore, when ornithine was added to the sugar-free medium, the inventors succeeded in extending the survival period of hepatocytes. As a result of further research based on these findings, the present inventors completed the present invention.

[0012] That is, the present invention is as follows. [1] A method for producing a cell population enriched in hepatocytes, comprising the step of culturing a cell population containing hepatocytes in the absence of glucose, fructose, and galactose. [2] The method according to [1], wherein the culturing is carried out in the presence of a hexokinase inhibitor. [3] A method for producing a cell population enriched in hepatocytes, comprising the step of culturing a cell population containing hepatocytes in the absence of glucose and in the presence of a hexokinase inhibitor. [4] The method according to [3], wherein the culturing is culturing in the presence of fructose. [5] The method according to [2], wherein at least one of the hexokinase inhibitors is 3-bromopyruvic acid. [6] The method according to [3] or [4], wherein at least one of the hexokinase inhibitors is 3-bromopyruvic acid. [7-1] A method for producing a cell population enriched in hepatocytes, comprising the step of culturing a cell population containing hepatocytes in the absence of glucose, in the presence of fructose and a phosphofructokinase inhibitor, and under conditions where the oxygen concentration is 5% or less. [7-2] The method according to [7-1], wherein the culturing is culturing under conditions where the oxygen concentration is 1% or less. [8] The method according to any one of [3] to [7-2], wherein the culturing is culturing in the absence of galactose. [9] The method according to any one of [1] to [8], wherein the culturing is culturing in the presence of a glycogenic amino acid.

[10] The method according to any one of [1] to [9], wherein the culturing is culturing in the presence of an intermediate metabolite of the urea cycle.

[11] The method according to any one of [1] to

[10] , wherein the culturing is culturing in the presence of a glycogenic amino acid and an intermediate metabolite of the urea cycle.

[12] The method according to any one of [9] to

[11] , wherein at least one of the glycogenic amino acids is alanine.

[13] The method according to any one of

[10] to

[12] , wherein at least one of the intermediate metabolites of the urea cycle is ornithine.

[14] The method according to any one of [1] to

[13] , wherein the culturing is culturing in the presence of alanine and ornithine.

[15] The method according to

[14] , wherein the culturing is culturing in the presence of 3-bromopyruvic acid.

[16] The method according to any one of [1] to

[15] , wherein the culturing is culturing in the presence of one or more compounds selected from the group consisting of creatine, arginine, glycine, guanidinoacetic acid, methionine, reduced glutathione, and N-acetylcysteine. [17-1] The method according to any one of [1] to

[16] , wherein the cell population containing the hepatocytes is derived from pluripotent stem cells. [17-2] The method according to any one of [1] to [17-1], wherein the cells are of human origin. [18-1] The method according to any one of [1] to [17-2], wherein the period of the culturing is from 6 hours to 7 days. [18-2] The method according to any one of [1] to [18-1], wherein the period of the culturing is from 12 hours to 5 days.

[19] A method for culturing hepatocytes, comprising culturing a cell population produced by the method according to any one of [1] to [18-2] in the presence of glucose.

[20] A cell population obtained by the method according to any one of [1] to

[19] . [21-1] A cell population in which 80% or more of all cells are hepatocytes, which does not contain cells labeled with an antibody, and which does not contain cells having exogenous mRNA that expresses a protein in response to miRNA. [21-2] The cell population according to [21-1], wherein 90% or more of all cells are hepatocytes.

[22] A cell transplantation therapeutic agent containing the cell population according to

[20] .

[23] A cell transplantation therapy agent comprising the cell population described in [21-1] or [21-2].

[24] An agent described in

[22] or

[23] for the treatment or prevention of liver diseases.

[25] A method for treating or preventing liver diseases, comprising transplanting an effective amount of the cell population described in any one of

[20] to [21-2] into a subject.

[26] The cell population described in any one of

[20] to [21-2] for use in the treatment of liver diseases.

[27] Use of the cell population described in any one of

[20] to [21-2] in the manufacture of an agent for the treatment or prevention of liver diseases. [Effect of the Invention]

[0013] According to the present invention, a large amount of hepatocytes can be purified at low cost by simply changing the medium. In one aspect, hepatocytes can be purified without generating residues such as antibodies and mRNA caused by cell sorting methods or mRNA switch methods. Further, according to the present invention, remaining undifferentiated cells can be removed. Thereby, since the safety of transplantation medicine using hepatocytes derived from human pluripotent stem cells is ensured, the cells purified by the present invention can be preferably used particularly in the medical field. [Brief Explanation of Drawings]

[0014]

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Modes for Carrying Out the Invention

[0015] 1. Method for purifying hepatocytes The present invention provides a method for concentrating (which can also be paraphrased as "purifying") hepatocytes in a cell population containing hepatocytes. Specifically, the present invention provides a method for producing a cell population in which hepatocytes are concentrated (hereinafter sometimes referred to as "Production Method 1 of the present invention"), which includes a step of culturing a cell population containing hepatocytes in the absence of glucose, fructose, and galactose. In one aspect, the culturing in such a method is culturing under sugar-free conditions.

[0016] In another aspect, the present invention provides a method for producing a cell population enriched in hepatocytes (hereinafter sometimes referred to as "Production Method 2 of the present invention"), which includes a step of culturing a cell population containing hepatocytes in the absence of glucose and in the presence of a hexokinase inhibitor. In one aspect, the culturing in such a method is further culturing in the presence of fructose. As shown in the following examples, in culturing in the absence of glucose, by using a hexokinase inhibitor, an effect of suppressing the production of reactive oxygen species in hepatocytes can also be expected.

[0017] In yet another aspect, the present invention provides a method for producing a cell population enriched in hepatocytes (hereinafter sometimes referred to as "Production Method 3 of the present invention"), which includes a step of culturing a cell population containing hepatocytes in the absence of glucose, in the presence of fructose and a phosphofructokinase inhibitor, and under a condition where the oxygen concentration is 5% or less. Also, in one aspect of Production Methods 2 and 3 of the present invention, the culturing is performed in the absence of galactose.

[0018] As used herein, the term "hepatocyte" means a cell that expresses HNF4α and has the ability to metabolize fructose and / or the ability to store glycogen. "Having the ability to metabolize fructose" means at least expressing KHK-C, and may further express KHK-A and / or GLUT5. Also, "having the ability to store glycogen" means that when cells are cultured in the presence of glucose, accumulation of glycogen is observed intracellularly.

[0019] As used herein, the term "expressing" a gene, unless otherwise specified, is used in the sense that it includes at least "production of mRNA encoded by the gene", but preferably is used in the sense that it further includes "production of a protein encoded by the mRNA". Therefore, when the production of mRNA encoded by a gene is detected by at least the method (RT-qPCR) described in the following examples, it can be said that the gene is expressed.

[0020] As used herein, the "cell population enriched in hepatocytes" means that the proportion of hepatocytes (number of hepatocytes / total number of cells constituting the cell population) is higher compared to the cell population before performing the production method of the present invention (in other words, the cell population before culturing in the absence of a specific saccharide such as glucose). The proportion of hepatocytes can be measured by the method described in the Examples below (flow cytometry using an anti-HNF4α antibody). Since hepatocytes can be purified by the production method of the present invention, in this specification, the "method for producing a cell population enriched in hepatocytes" can also be read as the "method for purifying hepatocytes" or the "method for enriching hepatocytes". Hereinafter, as a general term for Production Methods 1 to 3 of the present invention, the term "production method of the present invention" may be simply used.

[0021] In this specification, in the absence of sugars such as glucose, it is preferable that the sugar in the medium is at a concentration below the detection limit by liquid chromatography-mass spectrometry method, but it may be contained in trace amounts (at a concentration of 1 / 100 or less of the concentration used in normal culture) in the medium. Also, in this specification, the "sugar-free condition" means a condition in which mammalian cells do not contain sugars that can be metabolized as an energy source, and may contain sugars that cannot be converted into energy. Examples of sugars that can be metabolized as an energy source by mammalian cells include glucose, fructose, and galactose in the case of cells of primates such as humans. Furthermore, the "sugar-free condition" may be a condition in which monosaccharides are not contained, a condition in which monosaccharides and disaccharides are not contained in the medium, or a condition in which monosaccharides, disaccharides, and polysaccharides are not contained.

[0022] In the present invention, the hexokinase inhibitor is not particularly limited as long as it can inhibit the function of hexokinase. For example, 3-bromopyruvic acid, 2-deoxy-D-glucose, lonidamine, metrizamide, trehalose-6-phosphate, benitrobenzide, benserazide, etc. can be mentioned. The hexokinase inhibitors may be used in combination of two or more. Further, the hexokinase inhibitor used in the present invention preferably can inhibit at least hexokinase 2, and more preferably can inhibit all of hexokinases 1 to 3. Alternatively, it is also preferable to inhibit all of hexokinases 1 to 3 by combining a plurality of inhibitors. When 3-bromopyruvic acid is used as the hexokinase inhibitor, the concentration in the medium is typically 1 to 500 μM, preferably 20 to 200 μM, more preferably 50 to 80 μM (in one embodiment, 60 μM).

[0023] The phosphofructokinase inhibitor is not particularly limited as long as it can inhibit the function of phosphofructokinase 1. For example, PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), YZ-9, 3PO, etc. can be mentioned. The phosphofructokinase inhibitors may be used in combination of two or more. When PFK15 is used as the phosphofructokinase inhibitor, the concentration in the medium is typically 1 to 500 μM, preferably 5 to 100 μM, more preferably 20 to 40 μM (particularly, 35 μM).

[0024] The hexokinase inhibitor may be an inhibitor of hexokinase gene expression. The same applies to the phosphofructokinase inhibitor. These expression inhibitors may be nucleic acids, for example, antisense nucleic acids (e.g., antisense oligonucleotides (ASO), etc.) (including nucleic acids encoding the nucleic acids), siRNA (including nucleic acids encoding the siRNA), heteroduplex oligonucleotide (HDO), shRNA (including nucleic acids encoding the shRNA), miRNA (microRNA) (including nucleic acids encoding the miRNA), antigene nucleic acids, CRISPR-Cas systems, and the like.

[0025] The expression inhibitor contains a base sequence complementary to a partial sequence of the transcript of hexokinase II or phosphofructokinase 1 encoding the full-length protein. Such a base sequence can be designed, for example, based on the cDNA base sequences of the full-length human hexokinase II transcript and the full-length human phosphofructokinase 1 transcript, which are registered as Genbank Accession No. NM_000189 and NM_000289, respectively. Further, when the expression inhibitor targets a gene such as an antigene nucleic acid or a CRISPR / Cas system, the expression inhibitor (in the case of the CRISPR / Cas system, the guide RNA constituting the system) contains a base sequence complementary to a partial sequence of the gene.

[0026] In Production Method 3 of the present invention, since hepatocytes can generate ATP through an anaerobic metabolic pathway in the presence of fructose, oxygen is not essential. Therefore, the oxygen concentration in Production Method 3 of the present invention is 5% (v / v) or less (for example, 4% (v / v), 3% (v / v), 2% (v / v), 1% (v / v) or less), and 0% (v / v) or more.

[0027] The medium used in the production method of the present invention is a medium that does not contain undesirable saccharides such as glucose (for example, saccharides that mammalian cells can metabolize as an energy source) according to the purpose of the present invention, or a medium that has been subjected to a treatment for removing such saccharides. Examples of the basal medium include, but are not limited to, Minimum Essential Medium (MEM), Eagle MEM medium, αMEM medium, Dulbecco's Modified Eagle Medium (DMEM), Glasgow MEM medium, Improved MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, DMEM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, L-15 medium, StemFit AK02 medium, StemFit AK03 medium, StemFit Basic03 medium, and mixed media thereof. Preferably, it is DMEM.

[0028] The medium may be a serum-containing medium (e.g., fetal bovine serum (FBS), equine serum, human serum, etc.) or a serum-free medium. A serum-free medium (SFM) means a medium that does not contain any untreated or unpurified serum. When using serum, its concentration in the medium can be 5 - 30%, preferably 10 - 20%. The SFM may or may not contain any serum substitute. Examples of serum substitutes include substances that appropriately contain albumin (e.g., albumin substitutes such as lipid-rich albumin, recombinant albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron carriers), insulin, collagen precursors, trace elements, sodium selenite, 2-mercaptoethanol, ethanolamine, 3'-thioglycerol, or their equivalents, etc. Such serum substitutes can be prepared, for example, by the method described in WO 98 / 30679. Also, for simplicity, commercially available ones can be used. Examples of commercially available substances include Knockout(™) Serum Replacement (KSR), Glutamax (Invitorogen), ITS supplement (e.g., ITS-G, ITS-A, ITS-X, all from Fujifilm Wako Pure Chemical Industries). Since serum usually contains saccharides, when using serum containing undesirable saccharides such as glucose (e.g., saccharides that mammalian cells can metabolize as an energy source) according to the purpose of the present invention, typically, a treatment for removing the saccharides is performed. Also, when the serum substitute is formulated with saccharides such as glucose, in this case, typically, a treatment for removing the saccharides is performed according to the purpose of the present invention.

[0029] The medium may contain other additives known per se. The additives are not particularly limited, and examples thereof include growth factors (e.g., insulin, etc.), polyamines (e.g., putrescine, etc.), minerals (e.g., sodium selenate, etc.), amino acids (e.g., L-glutamine, etc.), reducing agents (e.g., 2-mercaptoethanol, etc.), vitamins (e.g., ascorbic acid, d-biotin, etc.), steroids (e.g., β-estradiol, progesterone, etc.), antibiotics (e.g., streptomycin, penicillin, gentamicin, etc.), buffers (e.g., HEPES, etc.). Each additive is preferably contained within a concentration range known per se. When an undesirable saccharide such as glucose (e.g., a saccharide that mammalian cells can metabolize as an energy source) is formulated in each additive, typically, depending on the object of the present invention, a treatment for removing the saccharide is performed.

[0030] The basal medium usually contains, in addition to sugar, pyruvic acid which is the final product in the glycolysis system. Pyruvic acid can be taken into cells and serve as an energy source. Also, lactic acid which can be easily converted into pyruvic acid can be taken into cells. Therefore, in the production method of the present invention, it is desirable to use a medium that does not contain pyruvic acid and / or lactic acid. When a medium containing pyruvic acid and / or lactic acid is used, it is preferable to perform a treatment for removing the product. It is preferable not to externally add pyruvic acid and / or lactic acid to the medium during the culture, and pyruvic acid and / or lactic acid synthesized by cells may be present.

[0031] As shown in the examples described below, metabolome analysis of hepatocytes derived from human iPS cells revealed that culturing under sugar-free conditions caused fluctuations in the utilization of amino acids as compared to culturing in the presence of glucose and galactose. In addition, it was shown that adding alanine to the sugar-free medium could extend the survival period of hepatocytes. Without being bound by any theory, it is presumed that the promoting effect of alanine addition on hepatocyte survival is due to alanine being a major glucogenic amino acid for hepatocytes. Therefore, it is considered that glucogenic amino acids other than alanine can similarly exert a promoting effect on hepatocyte survival. Thus, the medium used in the present invention may contain glucogenic amino acids such as alanine. Examples of such glucogenic amino acids include alanine, glycine, serine, threonine, cysteine, tryptophan, glutamic acid, glutamine, arginine, histidine, proline, isoleucine, methionine, valine, phenylalanine, tyrosine, aspartic acid, asparagine, and the like. When using glucogenic amino acids, it is preferable that the medium contains at least alanine. Glucogenic amino acids may be used in combination of two or more kinds.

[0032] As shown in the examples described below, metabolome analysis of hepatocytes derived from human iPS cells revealed that culturing under sugar-free conditions resulted in a significantly higher amount of creatine phosphate in the cells as compared to culturing in the presence of glucose and galactose. Creatine phosphate transfers phosphate to ADP to resynthesize ATP, the energy currency. Creatine (1-methylguanidinoacetic acid) is synthesized by the reaction of L-arginine and glycine in the kidney and liver, and also in the liver by the reaction of guanidinoacetic acid and S-adenosylmethionine. Therefore, in the production method of the present invention, it is also preferable to use creatine, and at least any one of L-arginine, glycine, guanidinoacetic acid, and methionine, which are the sources of creatine synthesis. A combination of L-arginine and glycine or a combination of guanidinoacetic acid and methionine is more preferable, and it is even more preferable to use all of creatine, arginine, glycine, guanidinoacetic acid, and methionine.

[0033] In addition, metabolome analysis showed that the amount of intracellular reduced glutathione decreased by culturing under sugar-free conditions as compared with culturing in the presence of glucose and galactose. This is presumably due to the effect of reactive oxygen species caused by starving the cells. Therefore, in the production method of the present invention, it is also preferable to use reduced glutathione or N-acetylcysteine (NAC), which is a precursor of glutathione that is easily taken up by cells.

[0034] Furthermore, metabolome analysis showed that the amount of intracellular ornithine increased significantly by culturing under sugar-free conditions as compared with culturing in the presence of glucose and galactose. Also, as shown in the following examples, it was shown that adding ornithine to the sugar-free medium extended the survival period of hepatocytes. In order for cells to metabolize amino acids, they need to break down the amino acids to remove the amino groups, and ammonia is generated in the process. Since ammonia is toxic, it is decomposed into urea in the urea cycle of hepatocytes. Since ornithine is an intermediate metabolite of the urea cycle, it is also preferable to add this to the medium to promote the urea cycle. Therefore, it is also preferable to use at least any one of citrulline, aspartic acid, argininosuccinic acid, and arginine, which are intermediate metabolites of the urea cycle other than ornithine, and it is also preferable to use all of ornithine, citrulline, aspartic acid, argininosuccinic acid, and arginine.

[0035] Therefore, in one aspect of the production method of the present invention, the culture is carried out in the presence of one or more compounds selected from the group consisting of creatine, arginine, glycine, guanidinoacetic acid, methionine, reduced glutathione, N-acetylcysteine, ornithine, citrulline, aspartic acid, argininosuccinic acid, alanine, glycine, serine, threonine, cysteine, tryptophan, glutamic acid, glutamine, histidine, proline, isoleucine, methionine, valine, phenylalanine, tyrosine, aspartic acid, and asparagine. In the production method of the present invention, it is also preferable to carry out the cell culture in the presence of alanine and / or ornithine, particularly in the presence of alanine and ornithine. Furthermore, in the production method of the present invention, it is also preferable to culture the cells in the presence of a glycogenic amino acid (for example, alanine), an intermediate metabolite of the urea cycle (for example, ornithine), and a hexokinase inhibitor (for example, 3-bromopyruvic acid).

[0036] The culture in each step of the production method of the present invention may be adherent culture or suspension culture, but preferably adherent culture. In the case of adherent culture, it may be carried out using a culture vessel coated with an extracellular matrix component, or co-cultured with feeder cells. The feeder cells are not particularly limited, and examples include fibroblasts (such as mouse embryonic fibroblasts (MEF), mouse fibroblasts (STO), etc.). The feeder cells are preferably inactivated by a method known per se, such as irradiation with radiation (such as gamma rays) or treatment with an anticancer agent (such as mitomycin C). Examples of the extracellular matrix component include fibrous proteins such as Matrigel (Niwa A, et al. PLoS One.6(7):e22261, 2011), gelatin, collagen, and elastin, glucosaminoglycans and proteoglycans such as hyaluronic acid and chondroitin sulfate, and cell adhesion proteins such as fibronectin, vitronectin, and laminin. In one aspect, in the production method of the present invention, a culture vessel coated with Matrigel is used.

[0037] The incubator used for culturing is not particularly limited. For example, it includes flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multiwell plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles, etc.

[0038] The culture temperature is not particularly limited, but is about 30 to about 40 °C, preferably about 37 °C. The culture is carried out in the presence of air containing CO2, and the CO2 concentration is preferably about 2 to 5%.

[0039] The culture period of the production method of the present invention is not particularly limited, but is typically 6 hours to 7 days, preferably 8 hours to 6 days, more preferably 12 hours to 5 days.

[0040] The cell population containing hepatocytes used in the present invention can be obtained by known methods. For example, methods of isolating from biological tissues (such as the liver, etc.) by known techniques, methods of inducing differentiation of pluripotent stem cells, methods of obtaining from companies such as ATCC, etc. can be mentioned. From biological tissues, for example, using surface antigens (such as E-cadherin, CXCR4, CD55, etc.) as indicators, methods using flow cytometry or mass cytometry, magnetic cell separation methods, affinity columns immobilized with desired antigens, etc. can be used to isolate hepatocytes. Also, as described in Non-Patent Document 5, hepatocytes can also be isolated by density gradient centrifugation. The cell population containing hepatocytes used in the present invention is preferably obtained by inducing differentiation of pluripotent stem cells.

[0041] Induction of differentiation from pluripotent stem cells into hepatocytes can be carried out by known methods such as those described in Non-Patent Documents 1 to 4, Siller R. et al., Stem Cell Reports. 4(5): 939-952 (2015), for example. Specifically, for example, it can be carried out by a method including (1) a step of inducing differentiation of pluripotent stem cells into definitive endoderm, (2) a step of inducing differentiation of the definitive endoderm into hepatic progenitor cells, and (3) a step of inducing differentiation of the hepatic progenitor cells into hepatocytes. Therefore, the production method of the present invention may include at least one of the above steps (1) to (3) before the step of culturing a cell population containing hepatocytes.

[0042] In this specification, unless otherwise specified, "cells" shall include "cell populations". A cell population may be composed of one type of cell or may be composed of two or more types of cells.

[0043] Any undifferentiated cell having "self-renewal ability" to proliferate while maintaining an undifferentiated state and "pluripotency to differentiate into all three primary germ layers" may be used as the pluripotent stem cell used in the present invention. Examples of such pluripotent stem cells include, for example, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (nuclear transfer Embryonic stem cell: ntES cells), multipotent germline stem cells (referred to as "mGS cells"), embryonic germ cells (EG cells), etc., but preferably iPS cells (more preferably human iPS cells). When the above pluripotent stem cell is an ES cell or any cell derived from a human embryo, the cell may be a cell prepared by destroying an embryo or a cell prepared without destroying an embryo, but from an ethical perspective, preferably, it is a cell prepared without destroying an embryo.

[0044] iPS cells are artificial stem cells derived from somatic cells that can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or proteins, and have properties almost equivalent to those of ES cells, such as pluripotency and the ability to proliferate by self-renewal (Takahashi K. and Yamanaka S. (2006) Cell, 126:663-676; Takahashi K. et al. (2007), Cell, 131:861-872; Yu J. et al. (2007), Science, 318:1917-1920; Nakagawa M. et al., Nat. Biotechnol26:101 - 106(2008); WO 2007 / 069666). When using iPS cells, the iPS cells may be prepared from somatic cells by methods known per se, or established and stocked iPS cells may be used. The reprogramming factors may be composed of genes specifically expressed in ES cells, their gene products or non-coding RNAs, or genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products or non-coding RNAs, or small molecules. Examples of genes included in the reprogramming factors are Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1, etc. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors are described in WO 2007 / 069666, WO 2008 / 118820, WO 2009 / 007852, WO 2009 / 032194, WO 2009 / 058413, WO 2009 / 057831, WO 2009 / 075119, WO 2009 / 079007, WO 2009 / 091659, WO 2009 / 101084, WO 2009 / 101407, WO 2009 / 102983, WO 2009 / 114949, WO 2009 / 117439, WO 2009 / 126250, WO 2009 / 126251, WO 2009 / 126655, WO 2009 / 157593, WO 2010 / 009015, WO 2010 / 033906, WO 2010 / 033920, WO 2010 / 042800, WO 2010 / 050626, WO 2010 / 056831, WO 2010 / 068955, WO 2010 / 098419, WO 2010 / 102267, WO 2010 / 111409, WO 2010 / 111422, WO 2010 / 115050, WO 2010 / 124290, WO 2010 / 147395, WO 2010 / 147612, Huangfu D, et al. (2008), Nat.The combinations described in Biotechnol., 26:795-797, Shi Y, et al. (2008), Cell Stem Cell, 2:525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, R.L. Judson et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9 are exemplified.

[0045] As human pluripotent stem cell lines, various iPSC lines established by NIH, RIKEN, Kyoto University, etc. can be used. For example, in the case of human iPSC lines, there are RIKEN's HiPS-RIKEN-1A line, HiPS-RIKEN-2A line, HiPS-RIKEN-12A line, Nips-B2 line, etc., and Kyoto University's 253G1 line, 253G4 line, 1201C1 line, 1205D1 line, 1210B2 line, 1383D2 line, 1383D6 line, 201B7 line, 409B2 line, 454E2 line, 606A1 line, 610B1 line, 648A1 line, 1231A3 line, FfI-01s04 line, and so on.

[0046] ES cells are stem cells established from the inner cell mass of early embryos (e.g., blastocysts) of mammals such as humans and mice, and have the ability of pluripotency and proliferation by self-renewal. ES cells were discovered in mice in 1981 (M.J. Evans and M.H. Kaufman (1981), Nature 292:154-156), and thereafter, ES cell lines have also been established in primates such as humans and monkeys (J.A. Thomson et al. (1998), Science 282:1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; J.A. Thomson et al. (1996), Biol. Reprod., 55:254-259; J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by removing the inner cell mass from the blastocyst of a fertilized egg of the target animal and culturing the inner cell mass on a fibroblast feeder. Methods for establishing and maintaining human and monkey ES cells are described, for example, in USP5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. U S A. 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1145-1147; Suemori H. et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; Ueno M. et al. (2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559; Suemori H. et al. (2001), Dev. Dyn., 222:273-279; Kawasaki H. et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I. et al. (2006), Nature. 444:481-485, etc.Alternatively, ES cells can be established using only a single blastomere of an embryo at the cleavage stage prior to the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or can also be established using embryos with developmental arrest (Zhang X. et al. (2006), Stem Cells 24: 2669-2676.).

[0047] ntES cells are ES cells derived from cloned embryos produced by nuclear transfer technology and have almost the same characteristics as ES cells derived from fertilized eggs (Wakayama T. et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; Byrne J. et al. (2007), Nature, 450:497-502). That is, ES cells established from the inner cell mass of a blastocyst derived from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell are ntES (nuclear transfer ES) cells. For the production of ntES cells, a combination of nuclear transfer technology (Cibelli J.B. et al. (1998), Nature Biotechnol., 16:642-646) and ES cell production technology (described above) is utilized (Kiyoshi Wakayama et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Extra Issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell can be injected into an enucleated unfertilized egg of a mammal and initialized by culturing for several hours.

[0048] mGS cells are pluripotent stem cells derived from the testis and are the origin cells for spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various cell lineages. For example, when transplanted into a mouse blastocyst, they can produce chimeric mice (Kanatsu-Shinohara M. et al. (2003) Biol. Reprod., 69:612-616; Shinohara K. et al. (2004), Cell, 119:1001-1012). They can self-renew in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and germ stem cells can be obtained by repeating subculturing under the same culture conditions as ES cells (Masanori Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Extra Issue), pp. 41-46, Yodosha (Tokyo, Japan)).

[0049] EG cells are cells with pluripotency similar to ES cells, established from primordial germ cells in the embryonic period. They can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factor (Matsui Y. et al. (1992), Cell, 70:841-847; J.L. Resnick et al. (1992), Nature, 359:550-551).

[0050] The origin species of pluripotent stem cells are not particularly limited. For example, they can be cells of rodents such as rats, mice, hamsters, guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, sheep, carnivores such as dogs, cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, etc. A preferred origin species is human.

[0051] The induced pluripotent stem cells used in the production method of the present invention may be cells derived from a patient with a genetic disease (for example, a patient with a genetic liver disease, etc.). Since cells induced to differentiate from pluripotent stem cells derived from a patient with a genetic liver disease can be a disease model reflecting the pathological condition of the disease, they are suitable for screening for a therapeutic or preventive drug for the disease. Alternatively, by repairing a gene by genome editing using a CRISPR-Cas system or the like on pluripotent stem cells derived from a patient with a genetic liver disease and then differentiating them into target cells, it becomes possible to use the cells or tissues as a therapeutic drug for the disease.

[0052] In the present specification, the "definitive endoderm cell" means a cell that expresses SOX17 and FOXA2 and has the ability to differentiate into hepatic progenitor cells. The definitive endoderm cells used in the present invention can be obtained by known methods. For example, methods of isolating from a biological tissue by a known technique, methods of inducing differentiation of pluripotent stem cells, methods of obtaining from companies such as ATCC, etc. can be mentioned. From a biological tissue, for example, using a surface antigen (for example, E-cadherin, CXCR4, CD55, etc.) as an index, a method using flow cytometry or mass cytometry, a magnetic cell separation method, an affinity column immobilized with a desired antigen, etc. can be used to isolate the definitive endoderm. The definitive endoderm cells used in the present invention are preferably obtained by a method of inducing differentiation of pluripotent stem cells.

[0053] In this specification, the "hepatic progenitor cell" means a cell that expresses α-fetoprotein (AFP) and has the ability to differentiate into hepatocytes and cholangiocytes. The hepatic progenitor cells used in the present invention can be obtained by known methods. For example, methods of isolating from a biological tissue (such as the liver, etc.) by known techniques, methods of inducing differentiation of embryonic endoderm, methods of obtaining from companies such as ATCC, etc. can be mentioned. From a biological tissue (such as liver tissue), for example, using flow cytometry or mass cytometry with surface antigens (such as CD13, CD133, etc.) as indicators, magnetic cell separation method, affinity column immobilized with a desired antigen, etc., hepatic progenitor cells can be isolated. Isolation of hepatic progenitor cells from a biological tissue can use, for example, the method described in Hepatology Research 2015 Jan;45(1):29-37. The hepatic progenitor cells used in the present invention are preferably obtained by a method of inducing differentiation of embryonic endoderm.

[0054] The basal medium and medium additives used in steps (1) to (3) can be the same as those used in the production method of the present invention. As the basal medium used in steps (1) and (2), RPMI 1640 medium is preferred. Also, as the basal medium used in step (3), L-15 medium is preferred. The culture temperature in steps (1) to (3) is typically about 30 to 40°C, preferably about 37°C, and the culture is carried out in an atmosphere of air containing CO2, and the CO2 concentration is preferably about 2 to 5%. Also, steps (1) to (3) are preferably carried out by adherent culture.

[0055] Differentiation of pluripotent stem cells into embryonic endoderm cells in step (1) can be carried out, for example, by culturing pluripotent stem cells in a medium containing a low dose of activin A. The medium may further contain a ROCK inhibitor and a GSK3β inhibitor. The culture period is typically 2 to 8 days, preferably 2 to 4 days.

[0056] The concentration of activin A used in the project (1) in the medium is, for example, 5 to 500 ng / mL, preferably 20 to 200 ng / mL, more preferably 30 to 100 ng / mL (particularly 50 ng / mL).

[0057] Examples of the GSK3β inhibitor used in Project (1) include, for example, CHIR98014 (2-[[2-[(5-nitro-6-aminopyridin-2-yl)amino]ethyl]amino]-4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidine), CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile), CP21R7 (3-(3-aminophenyl)-4-(1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione), LY2090314 (3-[9-Fluoro-1,2,3,4-tetrahydro-2-(1-piperidinylcarbonyl)pyrrolo[3,2,1-jk][1,4]benzodiazepin-7-yl]-4-imidazo[1,2-a]pyridin-3-yl-1h-pyrrole-2,5-dione), TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), TWS-119 (3-[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yloxy]phenol), Kenpaullone, 1-Azakenpaullone, SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione), AR-AO144-18 (1-[(4-methoxyphenyl)methyl]-3-(5-nitro-1,3-thiazol-2-yl)urea), CT99021, CT20026, BIO ((2'Z,3'E)-6-bromoindirubin-3'-oxime), BIO-acetoxime, pyridocarbazole-cyclopentadienylruthenium complex, OTDZT, alpha-4-dibromoacetophenone, lithium, and the like. Among them, CHIR99021 is preferred. The GSK3β inhibitor may be used in combination of two or more kinds.When using CHIR99021 as a GSK3β inhibitor, the concentration in the medium is typically 0.5 - 10 μM, preferably 3 - 6 μM.

[0058] Examples of the ROCK inhibitor used in step (1) include, for example, 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 their salts or derivatives, etc. Among them, Y-27632 is preferred. The ROCK inhibitor may be used in combination of two or more. When using Y-27632 as the ROCK inhibitor, the concentration in the medium is typically 1 - 20 μM, preferably 5 - 15 μM.

[0059] Insulin can be further added to the medium. The concentration of insulin in the medium is typically 0.01 - 20 μM, preferably 0.1 - 10 μM, more preferably 0.5 - 5 μM. Insulin may be contained in a medium additive such as B-27 supplement.

[0060] The differentiation of the embryonic endoderm cells in step (2) into hepatic progenitor cells can be carried out, for example, by culturing the embryonic endoderm cells in a medium containing dimethyl sulfoxide (DMSO). The culture period is typically 2 to 8 days, preferably 3 to 5 days.

[0061] The concentration of DMSO in the medium used in step (2) is typically 0.1 to 10% (v / v), preferably 0.5 to 5% (v / v), more preferably 0.7 to 3% (v / v) (especially 1% (v / v)).

[0062] The differentiation of the hepatic progenitor cells in step (3) into hepatocytes can be carried out, for example, by culturing the hepatic progenitor cells in a medium containing Dexamethasone and Dihexa. The culture period is not particularly limited because hepatocytes are maintained over a long period by the above culture, but it is typically 3 to 25 days, preferably 3 to 14 days, more preferably 4 to 12 days.

[0063] The concentration of Dexamethasone in the medium used in step (3) is typically 10 to 1000 nM, preferably 20 to 500 nM, more preferably 50 to 200 nM (especially 100 nM). The concentration of Dihexa in the medium used in step (3) is typically 10 to 1000 nM, preferably 20 to 500 nM, more preferably 50 to 200 nM (especially 100 nM).

[0064] Before the above step (1), a step of dispersing and culturing pluripotent stem cells in a medium containing a ROCK inhibitor may be carried out. The period of such a step is not particularly limited, but it is typically 12 hours to 3 days, preferably 1 day. Further, the pluripotent stem cells may be maintained in a medium not containing a ROCK inhibitor thereafter. Examples of the ROCK inhibitor include the same ones as those listed for step (1) above.

[0065] In addition, the cell population enriched with hepatocytes after performing the production method of the present invention may be cultured in a medium containing glucose (for example, DMEM). The period of such a step is not particularly limited, but is typically 12 hours to 7 days, preferably 2 days to 5 days.

[0066] 2. Cell population containing hepatocytes In another aspect of the present invention, there is also provided a cell population containing hepatocytes obtained by the production method of the present invention (which may also be read as "obtained"). In one aspect, hepatocytes can be purified without using the cell sorting method and the mRNA switch method. Therefore, the cell population of the present invention can be a cell population that does not contain cells labeled with an antibody and does not contain cells having exogenous mRNA that expresses a protein in response to miRNA.

[0067] The ratio in the cell population of hepatocytes can be calculated by dividing the number of hepatocytes by the total number of cells. The cell population of the present invention typically has 80% or more of all cells being hepatocytes. In this specification, "80% or more are hepatocytes" means that in the method described in the examples below (flow cytometry using an anti-HNF4α antibody), the ratio of hepatocytes is evaluated to be 80% or more (for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%).

[0068] "Cells labeled with an antibody" refers to cells to which an exogenous primary antibody and / or secondary antibody used in cell sorting is bound to a surface antigen. Further, "exogenous mRNA that expresses a protein in response to miRNA" refers to exogenous mRNA having an endogenous miRNA binding site, which is used in the mRNA switch method described in Non-Patent Document 2, etc., and when a miRNA corresponding to the binding site binds, a downstream protein is expressed. The cell population of the present invention typically does not include cells having exogenous mRNA that expresses a protein in response to miRNA, but preferably does not include any exogenous mRNA. An exogenous substance means a substance that does not exist in a cell unless introduced from the outside.

[0069] 3. Cell transplantation therapy agent The cell population of the present invention can be suitably used for cell transplantation therapy. Therefore, in another aspect of the present invention, there is provided a cell transplantation therapeutic agent (hereinafter sometimes referred to as "the cell transplantation therapeutic agent of the present invention") comprising the cell population of the present invention. The cell transplantation therapeutic agent of the present invention is typically used as a therapeutic or prophylactic agent for liver diseases. Further, a method for treating or preventing liver diseases, in which an effective amount of the cell population of the present invention is administered or transplanted to a mammal (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.) to be treated, is also included in the present invention.

[0070] Examples of liver diseases to be treated or prevented include hemochromatosis, Wilson's disease, glycogenosis, amino acid metabolism disorders, urea cycle metabolism disorders, porphyria, constitutional jaundice, fibrocystic liver disease, non-alcoholic steatohepatitis, liver cancer, hepatitis B, liver fibrosis, liver cirrhosis, hereditary ATTR amyloidosis (familial amyloid polyneuropathy (FAP)), α-1 antitrypsin deficiency (AATD), and the like.

[0071] When using the cell population of the present invention in cell transplantation therapy, from the viewpoint of avoiding rejection reactions, it is desirable to use cells derived from iPS cells established from somatic cells having the same or substantially the same HLA genotype as the individual at the transplantation site. Here, "substantially the same" means that the HLA genotypes match to the extent that the immune response can be suppressed by an immunosuppressant against the transplanted cells. For example, somatic cells having an HLA type in which three loci of HLA-A, HLA-B, and HLA-DR or four loci including HLA-C match. When sufficient cells cannot be obtained due to reasons such as age and constitution, it is also possible to embed them in capsules such as polyethylene glycol or silicone, porous containers, etc. and transplant them in a state where rejection reactions are avoided.

[0072] The cell population of the present invention is produced as a parenteral preparation such as an injection, a suspension, or a drip by mixing it with a pharmaceutically acceptable carrier according to conventional means. Therefore, in one aspect, a method for producing a cell transplantation therapeutic agent including the step of formulating the cell population of the present invention is also provided. Such a production method may include the step of preparing the cell population of the present invention. Furthermore, it can also include the step of storing the cell population of the present invention.

[0073] Examples of pharmaceutically acceptable carriers that can be included in the parenteral preparation include aqueous liquids for injection such as physiological saline, isotonic solutions containing glucose and other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). The cell transplantation therapeutic agent of the present invention may be formulated with, for example, a buffer (e.g., phosphate buffer, sodium acetate buffer), a pain reliever (e.g., benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), a preservative, an antioxidant, etc. When formulating the cell transplantation therapeutic agent of the present invention as an aqueous suspension, for example, about 1×10 6 ~about 1×10 8The cells may be suspended so as to be at a cell density of cells / mL. In addition, the dosage or transplantation amount and the number of administrations or transplantations of the cell population or pharmaceutical composition of the present invention can be appropriately determined according to the age, body weight, symptoms, etc. of the mammal to which it is administered.

[0074] The cell transplantation therapeutic agent of the present invention is provided in a cryopreserved state under conditions normally used for cryopreserving cells, and can also be thawed and used at the time of use. In that case, it may further contain serum or its substitute, an organic solvent (e.g., DMSO), etc. In this case, the concentration of serum or its substitute is not particularly limited, but can 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 can be 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0075] The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto in any way.

Examples

[0076] Example 1: Investigation of the expression of fructose metabolism-related genes during the process of inducing differentiation from human iPS cells into hepatocytes The gene expressions of ketohexokinase (KHK: there are two isoforms, KHK-A and KHK-C, and it is KHK-C that is related to fructose metabolism), a fructose-metabolizing enzyme specifically expressed in hepatocytes, and Glut5, a fructose transporter, were investigated as to when they start to be expressed during the process of inducing differentiation of human iPS cells into hepatocytes. In addition, comparisons were also made with the expressions of human iPS cells and cardiomyocytes derived from human iPS cells.

[0077] Human iPS cells (KMUR001 strain established at this facility, already deposited with RIKEN RBC) were cultured feeder-free on iMatrix-511 (nippi) using the following medium to maintain undifferentiated state. <Medium for human iPS cells> StemFit (registered trademark) AK02N medium (REPROCELL)

[0078] The induction of differentiation of human iPS cells into hepatocytes was performed with a partial modification of the method described in the literature Siller R. et al., Stem Cell Reports. 4(5): 939-952 (2015). The hepatic differentiation induction schedule is shown in Figure 2 above.

[0079] To induce the differentiation of human iPS cells into hepatocytes, 25,000 - 30,000 cells / cm 2 were seeded at a density on dishes coated with Matrigel® Growth Factor Reduced (GFR) (Corning). Three days later, to differentiate human iPS cells into endoderm, after washing the cells with RPMI1640 medium, the cells were replaced with the following medium (day 0 of differentiation). <Endoderm Differentiation Inducing Medium (1)> RPMI1640 medium (FUJIFILM Wako) 2% B-27 TM Supplement (Thermo Fisher Scientific) 1% GlutaMAX (Thermo Fisher Scientific) 50 ng / ml Activin A (Nacalai Tesque) 3 μM - 6 μM CHIR-99021 (MCE cat. NO.HY-10182)

[0080] After replacing with the endoderm differentiation inducing medium (1) and 24 hours later, the medium was replaced with the following medium and CHIR-99021 was removed (day 1 of differentiation). <Endoderm Differentiation Inducing Medium (2)> RPMI1640 medium (FUJIFILM Wako) 2% B-27 TM Supplement (Thermo Fisher Scientific) 1% GlutaMAX (Thermo Fisher Scientific) 50 ng / ml Activin A (Nacalai Tesque)

[0081] After changing to the medium for inducing endoderm differentiation (2) and after 1 day, in order to induce the differentiation of hepatic progenitor cells, the medium was changed to the following medium and cultured for 5 days (day 2 of differentiation). <Medium for inducing hepatic progenitor cell differentiation> RPMI1640 medium (FUJIFILM Wako) 2% B-27 TM Supplement (Thermo Fisher Scientific) 1% GlutaMAX (Thermo Fisher Scientific) 1% Dimetyl Sulfoxide (DMSO; FUJIFILM Wako)

[0082] Subsequently, in order to differentiate into mature hepatocytes, the medium was changed to the following medium (day 7 of differentiation). <Medium for hepatocyte maturation> Leibovitz L-15 medium (FUJIFILM Wako) (containing galactose as sugar) 8.3% tryptose phosphate broth solution (Sigma) 0.58% Insulin-Transferrin-Selenium (ITS; Thermo Fisher Scientific) 1% GlutaMAX (Thermo Fisher Scientific) 5% fetal bovine serum (Biowest) 10 μM hydrocortisone 21-hemisuccinate (Sigma) 50 μg / ml sodium-L-ascorbate (Sigma) 100 nM dexamethasone (FUJIFILM Wako) 100 nM N-hexanoic-Tyr, Ile-(6) aminohexanoic amide (dihexa; Toronto Research Chemicals) or 40 ng / ml HGF (Peprotech) The culture medium for hepatocyte maturation was replaced every other day and cultured for up to 11 days (until day 18 of differentiation).

[0083] Cells at the stages of day 0 (human iPS cells), day 4 (endoderm), day 7 (hepatic progenitor cells), day 12 (hepatocytes), and day 18 (mature hepatocytes) from the start of differentiation were observed and photographed. Cells cultured as such in the medium at each stage and cells cultured in the following fructose medium for 3 hours after replacement were prepared. Each was washed with PBS(-) and subjected to RNA extraction. In addition, the induction of cardiomyocyte differentiation from human iPS cells was performed with reference to the method of Burridge P. W. et al., Nature Methods 11(8), 855 - 60 (2014). Cardiomyocytes approximately 1 month after the start of differentiation were prepared from cells cultured in the CDM3 medium described in the literature and cells cultured in the following fructose medium for 3 hours after replacement. Each was washed with PBS(-) and subjected to RNA extraction. <Fructose Medium> DMEM No Glucose (with L - Gln, without Sodium Pyruvate; Nacalai Tesque) 10 mM Fructose (FUJIFILM Wako)

[0084] RNA extraction was performed using ISOGEN (Nippon Gene) according to the manual. Subsequently, cDNA synthesis was performed using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific). The real - time PCR reaction was carried out using the Power SYBR TM Green PCR Master Mix (Thermo Fisher Scientific) on a Rotor - Gene Q 2plex HRM System (Qiagen). The primers for each gene were the same as the sequences described in the literature (Mirtschink P et al., Nature 552, 444 - 449 (2015)) and are shown in Table 1.

[0085]

Table 1

[0086] During the process of inducing differentiation from human iPS cells into hepatocytes, polygonal cells arranged in a paving stone-like pattern, which are characteristic of hepatocytes, were observed around 7 to 12 days after the start of differentiation (in the middle of Fig. 2). As a result of real-time PCR, strong expression of genes related to fructose metabolism was observed on the 18th day after the start of hepatocyte differentiation (at the bottom of Fig. 2). In addition, it was shown that these genes were induced regardless of the presence or absence of fructose in the medium. On the other hand, in human iPS cells (day 0 of differentiation start) and human iPS cell-derived cardiomyocytes, almost no expression of these genes was observed, indicating that fructose metabolism by KHK was not possible (at the bottom of Fig. 2).

[0087] Example 2: Induction of hepatocyte differentiation from human iPS cells and purification of hepatocytes using fructose medium and hexokinase (HK) inhibitor Human iPS cells (HiPS-RIKEN-2F strain (2F strain) purchased from the RIKEN Center for Developmental Biology) were cultured in feeder-free conditions on iMatrix-511 (nippi) using the following medium to maintain undifferentiated state. <Medium for human iPS cells> StemFit (registered trademark) AK02N medium (REPROCELL)

[0088] Induction of differentiation of human iPS cells into hepatocytes was carried out in the same manner as in Example 1. On the 14th day after the start of differentiation, the cells were washed twice with DMEM No Glucose (with L-Gln, without Sodium Pyruvate; Nacalai Tesque) medium and then replaced with the following medium for culture to purify hepatocytes. <Medium for hepatocyte purification> DMEM No Glucose (with L-Gln, without Sodium Pyruvate; Nacalai Tesque) 8 mM Fructose (FUJIFILM Wako) 80 μM 3-Bromopyruvic acid ( 3-Bromopyruvic Acid) (Tokyo Chemical Industry) (HK2 inhibitor)

[0089] After replacing with the hepatocyte purification medium and observing the cells 24 hours later, dead non-hepatocytes were detached from the dish, and it was confirmed that the hepatocytes were viable (lower part of Figure 3). Half of the hepatocyte purification medium was removed, and half of the hepatocyte maturation medium was added and cultured for 1 day. Then, it was completely replaced with the hepatocyte maturation medium. The next day, to confirm the purity of the hepatocytes before and after purification, FACS analysis was performed using antibodies against HNF4α and α-Fetoprotein (AFP) expressed in hepatocytes according to the following procedure. After washing the cells with PBS(-), to minimize cell damage, a solution containing the following enzymes was added, and the cells were shaken on a shaker at room temperature until they detached from the plate and became round. After detachment, they were made into single cells by pipetting and collected into a tube. <Enzyme solution> Accutase(Innovative Cell Technologies) 0.1% Collagenase(FUJIFILM Wako) 20 nM Cyclosporin A(FUJIFILM Wako) 10 μM Y-27632(FUJIFILM Wako) 50 μg / ml sodium-L-ascorbate(sigma)

[0090] An equal volume of hepatocyte maturation medium was added to the cells collected in the tube to the enzyme solution, followed by centrifugation at 200 g for 5 minutes, and the supernatant was removed. After washing once with PBS(-), it was fixed with 4% paraformaldehyde-phosphate buffer (FUJIFILM Wako) for 5 minutes. After washing with Tris Buffered Saline with 0.1% Tween (registered trademark) 20 (Nacalai Tesque) (TBS-T), it was treated with 0.1% Triton X-100 (Nacalai Tesque) for 5 minutes. After washing twice with TBS-T, blocking was performed with ImmunoBlock (KAC) for 30 minutes. After removing the ImmunoBlock, the primary antibody in Table 2 was reacted at 4°C for 1 hour. Then, after washing twice with TBS-T, the secondary antibody in Table 2 was reacted at room temperature for 30 minutes. Then, as a result of performing FACS analysis, it was confirmed that the proportion of cells expressing HNF4α or AFP increased before and after purification, and that hepatocytes could be concentrated in this purification medium (Figure 4).

[0091]

Table 2

[0092] Example 3: Verification of the hypoxia tolerance of non-hepatocytes derived from human iPS cells Initially, it was thought that non-hepatocytes without KHK would have their metabolism suppressed and die under a glucose-free fructose-containing medium and hypoxic conditions. However, contrary to expectations, they survived. The reason was considered to be that phosphofructokinase (PFK), the rate-limiting enzyme of the glycolytic pathway also possessed by non-hepatocytes, was enhanced by hypoxia. To prove this, it was examined whether the gene expression of PFKFB3, an enzyme related to the enhancement of PFK, increased by subjecting non-hepatocytes to hypoxic conditions.

[0093] The induction of differentiation from human iPS cells into non-hepatocytes was carried out in the same medium and schedule as the hepatocyte differentiation induction in Example 1, but without adding CHIR-99021 that activates the Wnt signal, and by changing Activin A to a low concentration (20 ng / ml), to intentionally differentiate into cells that are not hepatocytes. 32 days after the induction of differentiation, the cells were washed twice with DMEM No Glucose medium, and then the medium was exchanged with a fructose medium (DMEM No Glucose containing 10 mM Fructose), and cultured for 24 hours under normal oxygen concentration (20%) and low oxygen (1%) as it was. The low oxygen environment was obtained using the AnaeroPack system of Sugiyama Gen. That is, the cell culture plate and the oxygen concentration meter were placed at the back of the pouch bag, and after putting AnaeroPack (registered trademark) Kenki 5% (oxygen absorber and carbon dioxide generator) in front, the mouth was closed with a sealing clip. When the oxygen concentration reached 1%, the space between the dish and the AnaeroPack was closed with a sealing clip to maintain an oxygen concentration of 1%, and cultured in a 37°C 5% CO2 incubator. Then, RNA extraction was performed after 24 hours. RNA extraction, cDNA synthesis, and Real-time PCR were performed in the same manner as in Example 1. The primers were the same as the sequences described in the literature (Pobuda A. C. et al., Oncotarget 6(30), 29753-29770. (2015)) and are shown in Table 3.

[0094]

Table 3

[0095] As a result of Real-time PCR, it was found that in non-hepatocytes derived from human iPS cells, the expression of the PFKFB3 gene was increased by low oxygen culture, suggesting that the glycolytic system of anaerobic metabolism may be enhanced (Figure 5). Therefore, it was suggested that the addition of low oxygen and a PFKFB3 inhibitor may promote non-hepatocyte death by suppressing both aerobic and anaerobic metabolism of non-hepatocytes.

[0096] Example 4: Purification of hepatocytes derived from human iPS cells by fructose medium, Phosphofructokinase (PFK) inhibitor, and hypoxic culture Undifferentiated culture of human iPS cells (using the 2F strain) and induction of differentiation into hepatocytes from human iPS cells were performed in the same manner as in Example 1. Hepatocyte differentiation induction was carried out in one 6-well plate. After 18 days of differentiation, the cells were washed twice with DMEM No Glucose (with L-Gln, without Sodium Pyruvate; Nacalai Tesque) medium and then replaced with the following medium. One well was left with the hepatocyte maturation medium. <Hepatocyte purification medium> DMEM No Glucose (with L-Gln, without Sodium Pyruvate; Nacalai Tesque) 10 mM Fructose (FUJIFILM Wako) PFK15 (Selleck) (PFK inhibitor, added concentrations were 0 μM, 20 μM, 30 μM, 35 μM, 40 μM, diluted in the medium in the tube in advance and then added to each well of the cell culture plate.)

[0097] Also, the low oxygen environment was obtained using Sugiyama Gen's AnaeroPack system. That is, the cell culture plate and the oxygen concentration meter were placed at the back of the pouch bag, and AnaeroPack (registered trademark) Kenki 5% (oxygen absorber and carbon dioxide generator) was placed in front, and then the mouth was closed with a sealing clip. When the oxygen concentration reached 1%, the space between the dish and the AnaeroPack was closed with a sealing clip to maintain an oxygen concentration of 1%, and the cells were cultured overnight in a 37°C 5% CO2 incubator. The next morning, the clip was removed, and when the oxygen concentration reached 4.5%, the clip was closed again. After culturing for 2 days, the dish was taken out from the pouch bag and cultured in a 37°C 5% CO2 incubator. After about 5 hours, it was washed with Leibovitz L-15 medium and then replaced with the hepatocyte maturation medium.

[0098] The next day, to confirm the purity of the purified hepatocytes, FACS analysis was performed using an antibody against Asialoglycoprotein receptor 1 (ASGPR1), a hepatocyte-specific receptor, according to the following procedure. After washing the cells with PBS(-), to minimize cell damage, a solution containing the following enzymes was added, and the cells were shaken on a shaker at room temperature until they detached from the plate and became round. After detachment, the cells were made into single cells by pipetting and collected into a tube. <Enzyme solution> Accutase (Innovative Cell Technologies) 0.1% Collagenase (FUJIFILM Wako) 20 nM Cyclosporin A (FUJIFILM Wako) 10 μM Y-27632 (FUJIFILM Wako) 50 μg / ml sodium-L-ascorbate (sigma)

[0099] To the cells collected in the tube, an equal volume of hepatocyte maturation medium was added to the enzyme solution, and centrifugation was performed at 200 g for 5 minutes to remove the supernatant. To remove dead cells by gating during FACS analysis, dead cell staining was performed using LIVE / DEAD® Fixable Dead Cell Stains (Thermo Fisher Scientific). Then, the cells were fixed with 4% paraformaldehyde-phosphate buffer (FUJIFILM Wako), washed with TBS-T, and blocked with ImmunoBlock (KAC) for 30 minutes. After removing the ImmunoBlock, the primary antibody in Table 4 was reacted at 4°C for 1 hour. Then, after washing twice with TBS-T, the secondary antibody in Table 4 was reacted at room temperature for 30 minutes. After washing twice with TBS-T and performing FACS analysis, it was found that hepatocytes were more concentrated in the cells treated with 35 μM PFK15 (Figure 7). However, ASGPR1 is expressed in only some cells in hepatocytes derived from human iPS cells and may not represent the exact purity of hepatocytes.

[0100]

Table 4

[0101] Example 5: Confirmation of glycogen by Periodic Acid Schiff reaction (PAS) staining of hepatocytes derived from human iPS cells Undifferentiated culture of human iPS cells (using the 2F strain) and induction of hepatocyte differentiation from human iPS cells were performed in the same manner as in Example 1. On the 22nd day after the start of differentiation, the cells were washed with PBS(-) and then fixed with 4% paraformaldehyde·phosphate buffer (FUJIFILM Wako) for 5 minutes. Thereafter, glycogen staining was performed manually using a PAS kit (sigma). As a result, it was found that the hepatocyte portion was stained darker than non-hepatocytes and accumulated more glycogen (right in Figure 8).

[0102] Example 6: Purification of hepatocytes derived from human iPS cells using sugar-free medium Undifferentiated culture of human iPS cells (HiPS-RIKEN-2F strain (2F strain), 253G1 strain (G1 strain) purchased from the RIKEN Center for Developmental Biology) and induction of hepatocyte differentiation from human iPS cells were performed in the same manner as in Example 1. Thirty days after differentiation, the cells were washed twice with DMEM No Glucose (with L-Gln, without Sodium Pyruvate; Nacalai Tesque) medium and then replaced with DMEM No Glucose (with L-Gln, without Sodium Pyruvate; Nacalai Tesque) medium, and cultured until non-hepatocytes died. Five days later, it was observed under a microscope that non-hepatocytes had died and hepatocytes were surviving (Figure 9). As a control, cells cultured in the hepatocyte maturation medium without purification were prepared.

[0103] After completion of purification, in order to confirm the approximate ratio of hepatocytes, immunostaining of HNF4α, a protein expressed in the nuclei of hepatocytes, was performed according to the following procedure (for the 2F strain only). After the death of non-hepatocytes, half of the purification medium was removed and half of the hepatocyte maturation medium was added. The next day, after washing several times with Leibovitz L-15 medium (FUJIFILM Wako) to remove dead cells, washing once with PBS(-), and fixing with 4% paraformaldehyde phosphate buffer (FUJIFILM Wako) for 5 minutes. Then, washing twice with TBS-T and treating with 0.1% Triton X-100 (Nacalai Tesque) for 5 minutes. Then, washing twice with TBS-T and blocking with immunoblock for 30 minutes. The primary antibody in Table 5 was added and left overnight at 4°C. Then, after washing twice with TBS-T, immunoblock was added and left for 15 minutes. The secondary antibody in Table 5 was added and left for 30 minutes. Then, washing twice with TBS-T, adding DAPI (Thermo Fisher Scientific) diluted with TBS-T, and staining the nuclei of all cells for 30 minutes. Washing twice with TBS-T and observing with a fluorescence microscope. As a result, in the unpurified state, only DAPI was stained and there were many HNF4α-negative cells, but after purification, it was almost occupied by cells where DAPI overlapped with HNF4α-positive cells, confirming that hepatocytes were concentrated (Figure 10).

[0104]

Table 5

[0105] Example 7: Purification of hepatocytes derived from human iPS cells using sugar-free medium and hexokinase (HK) inhibitor Undifferentiated culture of human iPS cells (2F strain) and induction of hepatocyte differentiation from human iPS cells were performed in the same manner as in Example 1. 15 days after differentiation, after washing the cells twice with DMEM No Glucose (with L-Gln, without Sodium Pyruvate; Nacalai Tesque) medium, the cells were replaced with the following medium and cultured. As a control, cells were prepared without purification and remained in the hepatocyte maturation medium. <Hepatocyte Purification Medium> DMEM without Glucose (with L-Gln, without Sodium Pyruvate; Nacalai Tesque) 50 μM 3-Bromopyruvic Acid (Tokyo Chemical Industry)

[0106] After 21 hours, it was confirmed that non-hepatocytes were in a state of death (Figure 13). Therefore, half of the medium was removed, and half of the hepatocyte maturation medium was added, followed by culturing for 2 days. Thereafter, the medium was completely replaced with the hepatocyte maturation medium, and culturing was continued for 4 days. In the case of unpurified cells, all cells survived (Figure 12). To confirm the purity of unpurified and purified hepatocytes, FACS analysis was performed using an antibody against HNF4α expressed in the nuclei of hepatocytes according to the following procedure. After washing the cells with PBS(-), to minimize cell damage, a solution containing the following enzymes was added, and the cells were shaken on a shaker until they detached in a 37°C incubator. After detachment, the cells were made into single cells by pipetting and collected into a tube. <Enzyme solution> TrypLE Express (Thermo Fisher Scientific) 0.1% Collagenase (FUJIFILM Wako) 20 nM Cyclosporin A (FUJIFILM Wako) 10 μM Y-27632 (FUJIFILM Wako) 50 μg / ml sodium-L-ascorbate (sigma) 0.1 mg / ml DNase I (sigma)

[0107] An equal volume of hepatocyte maturation medium was added to the cells collected in the tube with the enzyme solution, and centrifugation was performed at 200 g for 5 minutes to remove the supernatant. To remove dead cells by gating during FACS analysis, dead cell staining was performed using LIVE / DEAD (registered trademark) Fixable Dead Cell Stains (Thermo Fisher Scientific). Then, it was fixed with 4% paraformaldehyde phosphate buffer (FUJIFILM Wako), washed twice with TBS-T, and then treated with 0.1% Triton X-100 (Nacalai Tesque) for 5 minutes. After washing twice with TBS-T, blocking was carried out overnight with immunoblock. Then, the primary antibodies in the following table were reacted overnight at 4°C. The samples were washed twice with TBS-T, and after blocking for 15 minutes, the secondary antibodies in the following table were reacted at room temperature for 30 minutes. After washing twice with TBS-T and passing through the filter, FACS analysis was performed. As a result, the proportion of cells expressing HNF4α increased after purification compared to that before purification, and it was confirmed that hepatocytes could be purified with this purification medium (Figure 14).

[0108]

Table 6

[0109] Example 8: Comprehensive metabolome analysis of hepatocytes derived from human iPS cells in hepatocyte maturation medium and sugar-free medium To investigate the mechanism by which human iPS cell-derived hepatocytes can survive for several days under sugar-free conditions, and to search for substances other than sugar that are beneficial for the survival of hepatocytes, the metabolites in human iPS cell-derived hepatocytes in normal hepatocyte maturation medium (containing galactose and glucose as sugars) and sugar-free medium were comprehensively analyzed.

[0110] Undifferentiated culture of human iPS cells (2F strain) was performed in the same manner as in Example 1. For the induction of hepatocyte differentiation from human iPS cells, in order to exclude non-hepatocytes as much as possible for the analysis, the following method with higher hepatocyte differentiation efficiency was used. Metabolome analysis was outsourced to Toray Research Center. To induce hepatocyte differentiation from human iPS cells, 22,000 cells / cm 2 were seeded at a density of on a 10-cm dish coated with Matrigel (registered trademark) Growth Factor Reduced (GFR) (Corning). Three days later, to differentiate the human iPS cells into endoderm, after washing the cells with RPMI1640 medium, the cells were replaced with the following medium (day 0 of differentiation). <Endoderm differentiation induction medium (1)> RPMI1640 medium (FUJIFILM Wako) 1% B-27 TM Supplement (Thermo Fisher Scientific) 1% GlutaMAX (Thermo Fisher Scientific) 50 ng / ml Activin A (Nacalai Tesque) 6 μM CHIR-99021 (MCE cat. NO.HY-10182)

[0111] After replacing with the endoderm differentiation induction medium (1) and 24 hours later (day 1 of differentiation), the medium was replaced with the following medium, and CHIR-99021 was removed. Also, the medium was replaced with the same medium on day 3 of differentiation. <Endoderm differentiation induction medium (2)> RPMI1640 medium (FUJIFILM Wako) 1% B-27 TM Supplement (Thermo Fisher Scientific) 1% GlutaMAX (Thermo Fisher Scientific) 50 ng / ml Activin A (Nacalai Tesque)

[0112] On the next day (day 4 of differentiation), the cells were transferred to the following culture medium for inducing hepatic progenitor cell differentiation. The medium was changed again on day 6 of differentiation using the same medium. <Culture medium for inducing hepatic progenitor cell differentiation> RPMI1640 medium (FUJIFILM Wako) 1% B-27 TM Supplement (Thermo Fisher Scientific) 1% GlutaMAX (Thermo Fisher Scientific) 10 ng / ml BMP4 (Nacalai Tesque)

[0113] Two days later (day 8 of differentiation), to induce differentiation into mature hepatocytes, the medium was changed to the following medium. <Culture medium for hepatocyte maturation> Leibovitz L-15 medium (FUJIFILM Wako) 8.3% tryptose phosphate broth solution (Sigma) 0.58% Insulin-Transferrin-Selenium (ITS; Thermo Fisher Scientific) 1% GlutaMAX (Thermo Fisher Scientific) 5% fetal bovine serum (Biowest) 10 μM hydrocortisone 21-hemisuccinate (Sigma) 50 μg / ml sodium-L-ascorbate (Sigma) 100 nM dexamethasone (FUJIFILM Wako) 100 nM N-hexanoic-Tyr, Ile-(6) aminohexanoic amide (dihexa; Toronto Research Chemicals)

[0114] The hepatocyte maturation medium was changed every other day and cultured until day 14 of differentiation. On day 14 of differentiation, the cells were washed twice with DMEM No Glucose (with L-Gln, without Sodium Pyruvate; Nacalai Tesque) medium, and then four dishes of cells cultured in DMEM No Glucose for about 15 hours and four dishes of cells cultured in the hepatocyte maturation medium as they were were prepared and subjected to metabolome analysis.

[0115] As a result of the analysis, in the DMEM No Glucose medium (NG), compared with the hepatocyte maturation medium (MM), metabolites were generally decreased, but the amount of ATP was kept without difference. Also, in the NG medium, creatine phosphate was significantly increased (Figure 16). Creatine phosphate is an energy storage substance that rapidly resynthesizes ATP by transferring a phosphate group to ADP. It is mainly abundant in skeletal muscle, but it was found to increase also in hepatocytes as a preparation for starvation. Also, ornithine was significantly increased (Figure 16). Ornithine is an intermediate metabolite of the urea cycle that detoxifies ammonia in hepatocytes and activates this cycle. Since an increase in ammonia due to amino acid metabolism is predicted under sugar-free conditions, ornithine is important. Also, in the NG medium, reduced glutathione (GSH) was significantly decreased (Figure 16), which is considered to be due to the generation of reactive oxygen species under sugar-free conditions. Therefore, it was suggested that by adding these substances with significant differences and substances related to synthesis to the medium, it might be possible to improve the medium to be favorable for the survival of hepatocytes.

[0116] Example 9: Improvement of hepatocyte purification medium by amino acid addition (extension of the survival period of hepatocytes derived from human iPS cells under sugar-free conditions) In a sugar-free starvation state, amino acids are catabolized and used for gluconeogenesis, and at that time, toxic ammonia is generated. Since hepatocytes have an ornithine circuit for converting ammonia to urea, adding ornithine activates this circuit. Also, since the main glucogenic amino acid in hepatocytes is alanine, this was added. Thereby, without affecting the death of non-hepatocytes, it was observed whether the survival period of hepatocytes was extended.

[0117] A stock solution of amino acids to be added to the medium was prepared. 0.4M L-Ornithine-hydrochloride 0.674 g of L-ornithine hydrochloride (Nacalai Tesque) was dissolved in 10 ml of PBS(-). It was passed through a 0.22 μm filter and stored at 4°C. 22.5 g / l L-Alanine 0.225 g of L-alanine (FUJIFILM Wako) was dissolved in 10 ml of PBS(-). It was passed through a 0.22 μm filter and stored at 4°C.

[0118] Undifferentiated culture of human iPS cells (2F strain) and induction of hepatocyte differentiation from human iPS cells were carried out in a 6-well plate in the same manner as in Example 1. Fourteen days after differentiation, the cells were washed twice with DMEM No Glucose (with L-Gln, without Sodium Pyruvate; Nacalai Tesque) medium, and then cultured under the following conditions. The day when purification was completed (the day when non-hepatocytes died) and the day when hepatocytes completely died were compared for each condition. [Hepatocyte purification medium] Each condition was performed in two wells (1) DMEM No Glucose only (2) DMEM No Glucose + 1 mM L-ornithine hydrochloride (3) DMEM No Glucose + 225 mg / l L-alanine (4) DMEM No Glucose + 50 μM 3-Bromopyruvic Acid (3BP) (5) DMEM No Glucose + 50 μM 3BP + 1 mM L-ornithine hydrochloride (6) DMEM No Glucose + 50 μM 3BP + 225 mg / l L-alanine

[0119] Under all conditions, the day when non-hepatocytes died was the third day after purification (Figure 17). It was found that the addition of alanine or ornithine did not protect non-hepatocytes and had no effect on their death. After the completion of purification, the culture was continued in the hepatocyte purification medium. At the fourth day, the proportion of surviving hepatocytes seemed to be higher in conditions (2), (3), (5), and (6) with added amino acids than in conditions (1) and (4) (Figure 18). At the fifth day, hepatocytes had almost died in conditions (1), (4), and (5), but more hepatocytes survived in (2), (3), and (6), especially in the wells with added alanine (Figure 19), and they died the next day. Ornithine and alanine improved the nutritional status of hepatocytes and extended the survival period by one day even under starvation conditions without sugar (Figure 20).

[0120] It is expected that similar effects to alanine can also be obtained by adding glycogenic amino acids other than alanine, such as glycine, serine, threonine, cysteine, tryptophan, glutamic acid, glutamine, arginine, histidine, proline, isoleucine, methionine, valine, phenylalanine, tyrosine, aspartic acid, and asparagine. Also, it is expected that similar effects to ornithine can be obtained by adding citrulline, aspartic acid, argininosuccinic acid, and arginine, which are intermediate metabolites of the ornithine cycle other than ornithine.

[0121] Example 10: Effect of adding 3-Bromopyruvic Acid to sugar-free medium on non-hepatocyte death Undifferentiated culture of human iPS cells (253G4 strain (G4 strain)) and induction of hepatocyte differentiation from human iPS cells were performed in the same manner as in Example 1. Fourteen days after differentiation, the cells were washed twice with DMEM No Glucose (with L-Gln, without Sodium Pyruvate; Nacalai Tesque) medium, and then replaced with DMEM No Glucose or a medium obtained by adding 60 μM 3-Bromopyruvic Acid to DMEM No Glucose for culture. After 21 hours of culture, the cells were observed under a phase-contrast microscope. It was confirmed that non-hepatocytes survived in the DMEM No Glucose medium, while non-hepatocytes died in the medium supplemented with 3-Bromopyruvic Acid (Figure 21). From these results, it was confirmed that hepatocytes can be purified using only a sugar-free medium, but the addition of 3-Bromopyruvic Acid accelerates non-hepatocyte death. Under sugar-free conditions, hepatocytes release glucose, protecting non-hepatocytes. However, it was considered that the addition of 3-Bromopyruvic Acid, a glycolysis inhibitor for non-hepatocytes, inhibits non-hepatocyte protection and accelerates cell death.

[0122] Example 11: Effect of adding 3-Bromopyruvic Acid under sugar-free conditions on suppressing reactive oxygen species production in hepatocytes Cells produce reactive oxygen species (ROS) when they are in a nutrient-starved state such as serum-free. The effects of sugar-free conditions and the addition of 3-Bromopyruvic Acid (3BP) on ROS production were investigated using the cell-permeable ROS indicator CM-H2DCFDA (Thermo Fisher Scientific).

[0123] Undifferentiated culture of human iPS cells (G4 strain) and induction of hepatocyte differentiation from human iPS cells were performed in the same manner as in Example 1, and cells in which almost all cells had differentiated into hepatocytes were used. On the 16th day of differentiation, the cells were detached from the plate by enzymatic treatment and evenly seeded onto a 96-well black clear-bottom plate (Falcon) coated with Matrigel (registered trademark) Growth Factor Reduced (GFR) (Corning), and cultured with 100 μl / well of hepatocyte maturation medium (HMM). On the 4th day of culture, 100 μl / well of HMM was added. The next day, after washing with 300 μl of DMEM No Glucose medium (NG), the medium was replaced with HMM, NG, NG + 3BP (the added concentrations of 3BP were 20 μM, 40 μM, 60 μM, 80 μM, and 100 μM) (n = 4). At the time of medium replacement and 2 days after replacement, the cells were stained with 1 μM CM-H2DCFDA, and the fluorescence of each well was measured with a plate reader. Thereafter, to normalize the RFU of CM-H2DCFDA by the number of viable cells, Hoechst staining was performed and the fluorescence was measured with a plate reader (measurements were taken 1 hour later and 48 hours later). The results are shown in Figure 22. It was found that the production of reactive oxygen species was suppressed in cells with 3BP added compared to NG both 1 hour later and 48 hours later. Also, at 48 hours, some hepatocyte death was observed in wells with 80 μM and 100 μM of 3BP added, but the reactive oxygen species had a high value. At 48 hours, the reactive oxygen species had decreased in the well with 60 μM 3BP. It was found that reactive oxygen species were produced by making the human iPS cell-derived hepatocytes sugar-free, but it was suppressed to some extent by the addition of 3BP, and the optimal 3BP concentration was found to be 60 μM.

[0124] Example 12: Effect of adding alanine and ornithine on maintaining mitochondrial membrane potential of hepatocytes derived from human iPS cells under sugar-free conditions In Example 9, in hepatocytes derived from human iPS cells, an effect of extending the survival days was confirmed by adding alanine or ornithine under glucose-free conditions. Whether the metabolism of hepatocytes was improved by the addition of alanine or ornithine was examined using Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM) (Thermo Fisher Scientific), a fluorescent dye incorporated into the mitochondrial membrane potential.

[0125] Undifferentiated culture of human iPS cells (G4 strain) and induction of differentiation into hepatocytes from human iPS cells were performed in the same manner as in Example 1. On the 14th day of differentiation, the cells were washed twice with DMEM No Glucose (with L-Gln, without Sodium Pyruvate; Nacalai Tesque) medium, and then cultured under the following conditions: (I) DMEM No Glucose (NG), (II) NG + 60 μM 3-Bromopyruvic Acid (3BP), (III) NG + 60 μM 3BP + 225 mg / l L-alanine (A), (IV) NG + 60 μM 3BP + 4 mM L-ornithine hydrochloride (O), (V) NG + 60 μM 3BP + 225 mg / l A + 4 mM O

[0126] On the day after medium exchange, 50 nM TMRM was added to the medium. After staining at 37°C for 40 minutes, the medium was exchanged to the medium under the above conditions to remove the dye. Then, observation was performed with a fluorescence microscope (Nikon ECLIPSE Ti2), and all conditions were photographed with the same laser intensity and exposure time. Incubation was continued as it was, and photographs were also taken 2 days, 3 days, and 4 days after the purification conditions were established. ROIs were set from 1 to 6 on the photographed images (excluding locations with few cells and out-of-focus areas), and the average fluorescence intensity within each ROI was calculated using the ROI statics function. Furthermore, the average values of the ROIs from 1 to 6 were calculated, and the daily changes in the fluorescence intensity of TMRM under each purification condition were graphed (Figure 23). As a result, in (V) of NG + 60 μM 3BP + A + O, the fluorescence intensity was the highest, and it was considered that the mitochondrial membrane potential was maintained. From this, it was shown that alanine and ornithine are advantageous factors for hepatocytes. On the 5th day of purification, hepatocytes survived under the conditions (III) and (V) containing alanine, but were almost dead under other conditions (Figure 24). As a result of showing that hepatocytes derived from human iPS cells perform gluconeogenesis from alanine, metabolize it as an energy source, and are efficiently detoxified of ammonia by ornithine, it was considered that they maintained a sound mitochondrial activity (membrane potential) for a longer period compared to other conditions.

[0127] Example 13: Effect of removing undifferentiated cells by hepatocyte purification When undifferentiated iPS cells were mixed into hepatocytes and cultured by the liver purification method of condition (V) in Example 12, it was confirmed by Real-time PCR of undifferentiated markers whether the undifferentiated iPS cells died and were removed.

[0128] First, using a cell culture insert flexiPERM disc (BMS), human iPS cells (2F strain), hepatocytes and non-hepatocytes differentiated from human iPS cells were seeded in the wells of the flexiPERM disc, respectively, to culture the three types of cells in the same dish. The induction of hepatocyte differentiation was carried out by the method of Example 1, and non-hepatocytes were obtained by differentiating in a medium obtained by removing Activin A and CHIR-99021 from the method of Example 1. Two days after culturing, the flexiPERM disc was removed, washed twice with DMEM No Glucose medium (NG), and cultured in a purified medium (NG + 50 μM 3BP + 225 mg / l L-alanine + 4 mM L-ornithine-hydrochloride). A dish of unpurified control cells was also prepared. From two days after purification, the medium was replaced little by little with the hepatocyte maturation medium, and four days later, the cells were lysed in ISOGEN (Nippon Gene) for RNA extraction. RNA extraction was carried out according to the manual of ISOGEN. Then, it was treated with DNase I (Nippon Gene), and cDNA synthesis was carried out using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific). The undifferentiated marker genes investigated by real-time PCR were referred to the literature (Lemmens M et al., Cytotherapy 25(1), 59-67 (2023)). The PCR reaction was carried out using the TaqMan TM Gene Expression Assay and TaqMan TM Non-coding RNA Assay on a Rotor-Gene Q 2plex HRM System (Qiagen).

[0129]

Table 7

[0130] The results of investigating the gene expression of undifferentiated markers in unpurified and purified hepatocytes by real-time PCR are shown in Fig. 25 (n = 3). The expression levels of each undifferentiated marker after purification decreased (decreased at a ratio of POU5F1: 1 / 31, ESRG: 1 / 4,046, CAMKV: 1 / 2,040, ZSCAN10: 1 / 5,735), and among them, LINC00678 was below the detection limit. POU5F1 is thought to be expressed in human fetal liver as well as in hepatocytes derived from human iPS cells. The removal effect of human iPS cells by this purification method was almost shown.

Industrial Applicability

[0131] According to the present invention, a large amount of hepatic parenchymal cells can be purified at a low cost by simply changing the medium without generating residues such as antibodies and mRNAs caused by the cell sorting method or the mRNA switch method. In addition, since the present invention can remove the remaining undifferentiated cells, it is particularly useful in the medical field.

Claims

**Claim 1** A method for producing a cell population enriched in hepatocytes, comprising the step of culturing a cell population containing hepatocytes in the absence of glucose, fructose, and galactose. **Claim 2** The method according to claim 1, wherein the culturing is performed in the presence of a hexokinase inhibitor. **Claim 3** A method for producing a cell population enriched in hepatocytes, comprising the step of culturing a cell population containing hepatocytes in the absence of glucose and in the presence of a hexokinase inhibitor. **Claim 4** The method according to claim 3, wherein the culturing is performed in the presence of fructose. **Claim 5** The method according to claim 2, wherein at least one of the hexokinase inhibitors is 3-bromopyruvate. **Claim 6** The method according to claim 3, wherein at least one of the hexokinase inhibitors is 3-bromopyruvate. **Claim 7** A method for producing a cell population enriched in hepatocytes, comprising the step of culturing a cell population containing hepatocytes in the absence of glucose, in the presence of fructose and a phosphofructokinase inhibitor, and under conditions where the oxygen concentration is 5% or less. **Claim 8** The method according to claim 3, wherein the culturing is performed in the absence of galactose. **Claim 9** The method according to claim 1, wherein the culturing is performed in the presence of a glycogenic amino acid. **Claim 10** The method according to claim 1, wherein the culturing is performed in the presence of an intermediate metabolite of the urea cycle. **Claim 11** The method according to claim 1, wherein the culturing is performed in the presence of a glycogenic amino acid and an intermediate metabolite of the urea cycle. **Claim 12** The method according to claim 9, wherein at least one of the glycogenic amino acids is alanine. **Claim 13** The method according to claim 10, wherein at least one of the intermediate metabolites of the urea cycle is ornithine. **Claim 14** The method according to any one of claims 1 to 13, wherein the culturing is performed in the presence of alanine and ornithine. **Claim 15** The method according to claim 14, wherein the culturing is performed in the presence of 3-bromopyruvate. **Claim 16** The method according to any one of claims 1 to 13, wherein the culturing is performed in the presence of one or more compounds selected from the group consisting of creatine, arginine, glycine, guanidinoacetic acid, methionine, reduced glutathione, and N-acetylcysteine. **Claim 17** The method according to any one of claims 1 to 13, wherein the cell population containing the hepatocytes is derived from pluripotent stem cells.

18. The method according to any one of claims 1 to 13, wherein the culture period is 6 hours to 7 days.

19. A method for culturing hepatocytes, comprising the step of culturing a cell population produced by the method according to any one of claims 1 to 13 in the presence of glucose.

20. A cell population obtained by the method according to any one of claims 1 to 13.

21. A cell population in which 80% or more of all cells are hepatocytes, which does not contain cells labeled with an antibody, and which does not contain cells having exogenous mRNA that expresses a protein in response to miRNA.

22. A cell transplantation therapeutic agent comprising the cell population according to claim 20.

23. A cell transplantation therapeutic agent comprising the cell population according to claim 21.

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