JP2018011538A - Method for producing hepatic precursor cells - Google Patents

Abstract

The present invention relates to a method for producing hepatic progenitor cells from pluripotent stem cells in comparison with a conventional medium (particularly HDI medium or L15 medium) that can induce hepatic progenitor cells from pluripotent stem cells. Liver progenitor cells can be cultured for a long period of time, or more hepatic progenitor cells can be obtained by suppressing the decrease of hepatic progenitor cells. The object is to provide a medium additive to be added to a differentiation-inducing medium from stem cells to hepatic progenitor cells and a medium for inducing differentiation from pluripotent stem cells to hepatic progenitor cells. A pluripotent stem cell is cultured in a hepatic progenitor cell induction medium containing a glycolytic inhibitor and glucose. Examples of such glycolytic inhibitors include analog substances that constitute glycolysis and inhibitors of enzymes that constitute glycolysis, among which 2-deoxy-D-glucose and 3-bromopyruvic acid. Are preferred. As the hepatic progenitor cell induction medium containing the aforementioned glycolytic inhibitor and glucose, William E medium containing a glycolytic inhibitor is preferably exemplified. [Selection figure] None

Description

  The present invention relates to a method for producing hepatic progenitor cells, a medium additive added to a medium for inducing differentiation from pluripotent stem cells to hepatic progenitor cells, and a medium for inducing differentiation from pluripotent stem cells to hepatic progenitor cells.

  A pluripotent stem cell is a cell that has both self-replicating ability and pluripotency to replicate itself. In recent years, transplantation therapy using cells differentiated from pluripotent stem cells has attracted attention in regenerative medicine for the purpose of regenerating or recovering the function, function, or failure of cells, tissues, or organs. In addition, it is expected that cells differentiated from pluripotent stem cells will be applied to drug toxicity tests. Thus, inducing differentiation and amplification of a target cell from pluripotent stem cells is a major problem in the pharmaceutical field.

  Examples of pluripotent stem cells include induced pluripotent stem cells (hereinafter sometimes abbreviated as iPS cells) and embryonic stem cells (hereinafter abbreviated as ES cells). Etc. are known. These pluripotent stem cells are expected to be applied to regenerative medicine such as transplantation therapy used by inducing differentiation into the target cells and to drug toxicity tests. In particular, iPS cells are an ideal cell source for transplantation therapy because they have no ethical problems like ES cells, and iPS cells prepared from the patient's own cells have no immune rejection problems.

  For example, if differentiation of hepatocytes can be induced from human iPS cells, development to transplantation therapy for liver failure cases is expected. The liver is mainly composed of hepatocytes that are hepatocytes, non-hepatocytes such as biliary epithelial cells, and the like. In addition to the secretion of bile, filtration and detoxification of absorbed nutrients, drug metabolism, storage of sugar and regulation of blood glucose, the liver is a vital organ that is a vital organ, such as fibrinogen, heparin, and anemia inhibitor. Therefore, liver failure in which functioning hepatocytes are extremely reduced is a fatal disease state, and in cases of liver failure, a very serious state such as bleeding tendency due to insufficient coagulation factor and hepatic coma. Hepatocyte transplantation can be a fundamental treatment for such pathologies.

  As a technique for inducing the differentiation of hepatocytes from human iPS cells, a method has been reported in which growth factors and / or transcription factors that promote differentiation into hepatocytes are added to the culture of human iPS cells, respectively, and introduced (Non-Non-Patent Document) Patent Documents 1 to 3). In addition, a method for forming a liver from human iPS cells has been reported (Non-patent Document 4). In this method, differentiation from iPS cells to hepatocytes is first promoted in vitro, and then mixed with vascular endothelial cells and mesenchymal stem cells and transplanted into the mouse brain to produce the liver. is there. When applied to humans, the condition is that there is no exposure to foreign proteins. In addition, iPS cells that have been promoted to differentiate into hepatocytes are killed if maintained in vitro without being transplanted to the mouse brain. Furthermore, induction of differentiation into hepatocytes in vitro by such a method takes several weeks, and mature hepatocytes cannot be obtained (Non-patent Document 5). Therefore, in order to apply to humans, it is essential to develop a method for producing hepatocytes in a short time from iPS cells that are not exposed to heterologous proteins.

  Thus, iPS cells are expected to be applied to regenerative medicine. However, when differentiation of target cells from iPS cells is performed in vitro, the resulting cell group includes undifferentiated iPS cells in addition to target cells. Therefore, when transplanted, remaining undifferentiated iPS cells may form a tumor. Therefore, in order to use cells induced to differentiate from iPS cells for regenerative medicine such as transplantation, it is necessary to separate the cells induced to differentiate from the cell group including the cells induced to differentiate and undifferentiated iPS cells. is there.

  The present inventors have disclosed a method of killing human iPS cells and recovering only human primary cultured hepatocytes from a co-culture of human iPS cells and human primary cultured hepatocytes, and a hepatocyte selection medium used in the method ( hepatocyte selection medium (HSM) has been developed and reported (Patent Document 1 and Non-Patent Document 6). This HSM medium is prepared by adding galactose and ornithine except for glucose and arginine which are essential for cell survival. Hepatocytes have a series of enzymes related to the gluconeogenesis pathway and the urea cycle, and can synthesize glucose from galactose by the gluconeogenesis pathway and arginine from ornithine by the urea cycle. Can survive. However, since human iPS cells do not have a gluconeogenic pathway or urea cycle, they die in 3 days when cultured in HSM medium. Therefore, when a method for producing hepatocytes from human iPS cells has been established, the remaining undifferentiated human iPS cells can be removed from the cell group by culturing the produced cell group in an HSM medium for 3 days. And a cell culture substantially consisting of hepatocytes can be obtained. Since the HSM medium is composed of only components contained in a normal medium, the possibility of damage to the obtained hepatocytes is extremely low.

  When the present inventors cultured human iPS cells in HSM medium for 2 days, α-fetoprotein (α, which is a marker for hepatic progenitor cells such as embryonic hepatocytes (hepatoblasts) and hepatocyte cells such as hepatocytes. -feto protein: AFP) was found to be significantly enhanced (Patent Document 2). The inventor further added “oncostatin M”, “hepatocyte functional proliferation inducer 1 (FPH1)”, “M50054 which is an apoptosis inhibitor”, “ Hepatocyte differentiation induction prepared by adding “non-essential amino acid (NEAA)”, “sodium pyruvate”, “nicotinamide”, and “L-glutamine” effective for survival of mouse ES cells A substance (hepatocyte differentiation inducer; HDI) medium has been developed and reported (Patent Document 2). When the present inventors cultured human iPS cells in HDI medium for 2 days, both AFP, a hepatocyte marker, and γ-glutamyl trans peptidase (G-GTP), a bile duct epithelial cell marker, were obtained. It has been found that increased expression is observed, and that increased expression of delta like-1 homolog (DLK-1), which is a marker of hepatic progenitor cells such as hepatoblasts, has been found. (Patent Document 2). Thus, when human iPS cells are cultured in HSM medium or HDI medium, hepatic progenitor cells such as hepatoblasts (undifferentiated hepatocytes and cells capable of differentiating into hepatocytes and bile duct epithelial cells) are obtained. It was known to be obtained.

  However, even if human iPS cells are cultured in HSM medium, hepatic progenitor cells are almost killed in 3 days. Even if human iPS cells are cultured in HDI medium, hepatic progenitor cells are almost killed in 7 days. have done. Therefore, when producing hepatic progenitor cells from human iPS cells, a hepatic progenitor cell induction medium in which such cells can survive for more than 7 days and a method for producing hepatic progenitor cells have been demanded.

The glycolytic system is a metabolic pathway of sugar in the living body, and the glycolytic system in eukaryotes and anaerobic eubacteria is particularly called the Emden-Meyerhof pathway (EM pathway). This EM pathway is also called anaerobic glycolysis because it does not require oxygen. The EM pathway is a metabolic pathway in which glucose is anaerobically degraded to pyruvate or lactic acid, and throughout this pathway, 2 mol of ATP is produced per mol of glucose and 2 mol of NAD ⁺ is reduced to produce NADH. Pyruvate, the end product of the glycolysis, then enters the citrate cycle to generate more energy necessary for cell survival.

  For various substances, analogs of the substances are known. 2-Deoxy-d-glucose (2-DG) is known as an analog of glucose, which is a glycolytic starting material. Such 2DG is taken up into cells like glucose, but has the property of accumulating in cells without being metabolized through the EM pathway or the like. 2DG labeled with fluorine 19 is applied to positron emission tomography (PET) (Non-patent Document 7). Moreover, 3-bromopyruvate (3-BP) is known to suppress the glycolysis as an analog of pyruvate, which is the final product of glycolysis (Non-patent Document 8).

  However, it has not been known that when pluripotent stem cells are cultured in a hepatic progenitor cell induction medium containing a glycolytic inhibitor and glucose, such hepatic progenitor cells can survive for more than 7 days.

JP 2014-128210 A Japanese Patent Laying-Open No. 2006-026490

Tomizawa M et al., “Liver”, 2011, Volume 52, (Extra Number 2): A 680. Inamura et al., “Efficient Generation of Hepatoblasts Human ES Cells and iPS Cells by Transient Overexpression of Homeobox Gene HEX”, Molecular Therapy, 2011, Vol. 19, No. 2, p. 400-407. Tomizawa M et al., “Single-step protocol for the differing ntiation of human-induced pluripotent stem cells into hepatic progenitor-like cells”, Biomedical Reports, 2013, Volume 1, p.18-22 (Published online on Monday, August 13, 2012 as Doi: 10.3892 / br.2012.2). Takebe T et al., “Vascularized and functional human liver from a iPSC-derived organ bud transplantation”, Nature, 2013, 499, p.481-489. Si-Tayeb K et al., “Highly efficient generation of human hepaotcyte-like cells from induced pluripotent stem cells”, Hepatology, 2010, 51, p.297-305. Tomizawa et al. Survival of primary human hepatocytes and death of induced pluripotent stem cells in media lacking glucose and arginine. PLoS One 8, e71897, 2013) Zhang D, Li J, Wang F, Hu J, Wang S and Sun Y: 2-Deoxy-D-glucose targeting of glucose metabolism in cancer cells as a potential therapy.Cancer Lett 355, 176-183, 2014. Tomizawa et al. Suppressive effects of 3-bromopyruvate on the proliferation and the motiligy of hepatocellular carcinoma cells. Oncol Rep 35, 59-63, 2016.

  The object of the present invention is to compare hepatic progenitor cells such as hepatoblasts from pluripotent stem cells when producing hepatic progenitor cells from pluripotent stem cells (particularly HDI medium or L15 medium). Thus, hepatic progenitor cells can be cultured for a longer period of time, or more hepatic progenitor cells can be obtained by suppressing the decrease in hepatic progenitor cells, The object is to provide a “medium additive to be added to a differentiation-inducing medium from pluripotent stem cells to hepatic progenitor cells” and “a medium for inducing differentiation from pluripotent stem cells to hepatic progenitor cells”.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive research. As a result, hepatic progenitor cell-inducing medium containing glucose (for example, William E medium) has a “glycolytic inhibitor 2-deoxy-D. -When pluripotent stem cells are cultured in a medium supplemented with “glucose (2DG) and / or 3-bromopyruvic acid (3BP)”, hepatic progenitor cell-inducing medium containing no glycolytic inhibitor and glucose (particularly HDI) Compared with the case of culturing in medium or L15 medium), hepatic progenitor cells can be cultured for a longer period of time, and more hepatic progenitor cells can be obtained by suppressing the decrease in hepatic progenitor cells. As a result, the present invention has been completed.

That is, the present invention includes (1) a method for producing hepatic progenitor cells, comprising a step of culturing pluripotent stem cells in a hepatic progenitor cell induction medium containing a glycolytic inhibitor and glucose,
(2) The hepatic progenitor cell according to (1) above, wherein the glycolytic inhibitor is an analog substance of a substance constituting a glycolytic system or an inhibitor of an enzyme constituting a glycolytic system Manufacturing method,
(3) The hepatic progenitor cell according to (1) or (2) above, wherein the glycolytic inhibitor includes any one or both of 2-deoxy-D-glucose and 3-bromopyruvic acid Manufacturing method,
(4) The method for producing hepatic progenitor cells according to (3) above, wherein the concentration of 2-deoxy-D-glucose in the hepatic progenitor cell induction medium is 0.05 to 30 μM,
(5) The method for producing hepatic progenitor cells according to (3) or (4) above, wherein the concentration of 3-bromopyruvic acid in the hepatic progenitor cell induction medium is 0.05 to 30 μM,
(6) The method for producing hepatic progenitor cells according to any one of (1) to (5) above, wherein the hepatic progenitor cell induction medium is William E medium containing a glycolytic inhibitor;
(7) The present invention relates to the method for producing hepatic progenitor cells according to any one of (1) to (6) above, wherein the cells are cultured for at least 7 days.

The present invention also provides:
(8) A medium additive for adding to a differentiation-inducing medium from pluripotent stem cells to hepatic progenitor cells, a medium additive containing a glycolytic inhibitor,
(9) The culture medium additive according to (8) above, wherein the glycolytic inhibitor is an analog substance of a substance constituting a glycolytic system or an inhibitor of an enzyme constituting a glycolytic system Or
(10) The medium additive according to (8) or (9), further comprising glucose.

Furthermore, the present invention provides
(11) A medium for inducing differentiation from pluripotent stem cells to hepatic progenitor cells, comprising a glycolytic inhibitor and glucose.

  According to the present invention, when producing hepatic progenitor cells from pluripotent stem cells, hepatic progenitor compared to conventional media (particularly HDI medium or L15 medium) that can induce hepatic progenitor cells from pluripotent stem cells. Cells can be cultured for a longer period and / or more hepatic progenitor cells can be obtained by suppressing the decrease in hepatic progenitor cells.

It is a figure which shows the influence which the kind of culture medium which culture | cultivates an iPS cell has on the expression of a liver specific gene. The vertical axis represents the expression level of each gene. A: The results for cytosolic aspartate aminotransferase (Cyto AST) gene are shown. B: The results for the mitochondrial aspartate aminotransferase (Mito AST) gene are shown. C: The results for the alanine aminotransferase (ALT) gene are shown. D: The result about a glycogen synthase (GS) gene is shown. “Fetal” represents the result of the fetal liver, and “adult” represents the result of the adult liver. The error bar represents the standard deviation, and “*” indicates that the P value <0.05 compared to the case of FF. n = 3 It is a figure which shows the influence on the expression of the alpha-fetoprotein gene by the presence or absence of galactose in a culture medium. The vertical axis represents the expression level of the α-fetoprotein gene. “Medium” represents the type of medium, “−” in “Galactose” represents a medium without galactose, and (+) represents a medium with galactose added. Error bars represent standard deviation, and “*” indicates P value <0.05 compared to FF. n = 3 It is a figure which shows the influence on GALK1 promoter activity and GALK2 promoter activity of not containing glucose in a culture medium but containing galactose. The vertical axis in the figure represents the relative light emission amount (%) relative to the light emission amount (CMV) of Metridia luciferase when pMetLuc2-control is used. The black bar graph represents the result (FF) using ReproFF medium, the white bar graph represents the result using WE medium (WE), and the gray bar graph represents the result using HSM medium ( HSM). Error bars represent standard deviation, and “*” indicates P value <0.05 compared to FF. n = 4 It is a figure showing the influence on the expression of the alpha-fetoprotein gene by the presence or absence of 2DG in a culture medium. The vertical axis of the figure represents the expression level of the AFP gene with a display of x10. Error bars represent standard deviations, “*” indicates a P value <0.05 compared to FF with no 2DG added and galactose added, “***” is FF, P value <0.05 compared to the case of adding 0.1 μM 2DG and no addition of galactose. n = 3 It is a figure showing the influence on the morphological characteristic of a cell with the presence or absence of 2DG in a culture medium, and the presence or absence of 3BP. The result of having observed the cell culture | cultivated for 7 days in each culture medium with the microscope is represented. The magnification at the time of microscopic observation is 400 times, and the scale bar represents 100 μm. It is a figure showing the influence on the expression of the alpha-fetoprotein gene by the presence or absence of 2DG in a culture medium, and the presence or absence of 3BP. The vertical axis in the figure represents the expression level of the AFP gene. Error bars represent standard deviation, and “*” indicates P value <0.05 compared to FF. n = 3

  The present invention is directed to “a method for producing hepatic progenitor cells, comprising a step of culturing pluripotent stem cells in a hepatic progenitor cell induction medium containing a glycolytic inhibitor and glucose” (hereinafter referred to as “the present invention”). And "a medium additive to be added to a medium for inducing differentiation from pluripotent stem cells to hepatic progenitor cells, comprising a glycolytic inhibitor and glucose." "Additives" (hereinafter also referred to as "medium additive of the present invention") and "differentiation induction medium from pluripotent stem cells to hepatic progenitor cells containing a glycolytic inhibitor and glucose" (hereinafter, It is also indicated as “differentiation induction medium of the present invention”).

  The “pluripotent stem cell” in the present invention is an embryonic stem cell (embryonic stem cells: ES cell) isolated from an early embryo or an embryonic germ cell (embryonic germ cells: EG cells) (for example, see Proc Natl Acad Sci US A. 1998, 95: 13726-31) and germline stem cells (GS cells) isolated from testis immediately after birth (for example, Nature. 2008, 456: 344-9), mesenchymal stem cells such as iliac bone marrow and jaw bone marrow, stem cells derived from adipose tissue, and mesenchymal tissues such as skin and bone marrow. Introducing multiple genes into somatic cells such as muscular cells (Muse cells [Multi-lineage differentiating Stress Enduring cells]) and skin cells, etc., induces dedifferentiation of the subject's own somatic cells Induced pluripotency derived from somatic cells having pluripotency similar to ES cells Sexual stem cells (iPS; induced pluripotent stemcell) can be mentioned, and among these, iPS cells are preferred. In addition, when hepatic progenitor cells produced by the method for producing hepatic progenitor cells of the present invention are transplanted into mammalian cells and used for the treatment of liver disease, considering the risk of rejection due to transplantation, patients suffering from liver disease Obtained from iPS cells obtained from somatic cells derived from the subject, or from somatic cells derived from a subject (human) having the same or substantially the same human leukocyte antigen (HLA) genotype as the patient suffering from liver disease The obtained iPS cells can be preferably exemplified. Here, “the HLA genotype is substantially the same” means that the HLA genotype matches to such an extent that the immune response can be suppressed by the immunosuppressive agent for the transplanted cells. Therefore, as a somatic cell derived from a subject (human) having substantially the same HLA genotype, specifically, three HLA genotypes of HLA-A, HLA-B and HLA-DR, or such HLA Examples include somatic cells derived from a subject (human) having the same HLA genotype in which four HLA genotypes obtained by adding HLA-C to three genotypes are identical.

  The biological species from which the “pluripotent stem cell” in the present invention is derived is not particularly limited as long as it is a mammal. Among them, humans, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, monkeys, Preferred examples include mammals such as sheep, goats, and pigs, and particularly preferred examples include humans.

  As the “glycolytic inhibitor” in the present invention, any one or two or more glycolytic systems of mammalian cells (hereinafter, unless otherwise specified, the glycolytic system represents the Emden-Meyerhof pathway). The agent is not particularly limited as long as it is an agent having a property of suppressing the reaction of, for example, an analog substance of a substance constituting a glycolytic system (hereinafter also referred to as “glycolytic constituent substance”) or a glycolytic system Preferred examples include inhibitors of constituent enzymes (hereinafter also referred to as “glycolytic constituent enzymes”), and among them, analog substances of glycolytic constituents are more preferred. Examples of glycolytic constituent substances and analog substances thereof are shown in Table 1, and glycolytic constituent enzymes are shown in Table 2. Examples of glycolytic constituents other than those listed in Table 1 include, for example, fructose-6-phosphate, fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate Examples include acids, 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate.

  In the present invention, “analogue substance of glycolytic constituent substance A” is a substance having a structure similar to glycolytic constituent substance A, and when it is incorporated into mammalian cells, the glycolysis of the mammalian cells It means a substance having an action of suppressing the reaction of the system. As a substance having a structure similar to the above-mentioned glycolytic constituent substance A, the functional group of substance A is substituted with a hydrogen atom, a halogen atom, or a hydrocarbon group having 1 to 5 carbon atoms which may be halogenated. Substances.

  Whether or not a test substance having a structure similar to the glycolytic constituent A is an analog substance of the glycolytic constituent A can be confirmed by, for example, a general evaluation method known to those skilled in the art. Is possible. For example, the amount of glycolytic reaction in mammalian cells is compared in the presence and absence of the test substance. When the amount of glycolytic reaction in the presence of the test substance is reduced or deleted compared to the amount of glycolytic reaction in the absence of the test substance, the test substance is treated as a glycolytic constituent. It can be identified as an analog substance of A. Moreover, the analog substance of glycolytic constituent substance A can be obtained by, for example, a general screening method known to those skilled in the art. For example, the amount of glycolytic reaction in mammalian cells is compared in the presence and absence of the test substance. When the amount of glycolytic reaction in the presence of the test substance is reduced or deleted compared to the amount of glycolytic reaction in the absence of the test substance, the test substance is treated as a glycolytic constituent. It can be obtained as an analog substance of A.

  In the present invention, the “inhibitor of glycolytic constituent enzymes” means a substance capable of inhibiting the enzymatic reaction by the glycolytic constituent enzymes shown in Table 2 in the glycolytic system. Examples of such substances include sodium fluoride (NaF), which suppresses the enzyme reaction by enolase, iodoacetic acid, sesquiterpene lactone, BZL101 (Scutellaria barbata, which suppresses the enzyme reaction by glyceraldehyde-3-phosphate dehydrogenase. ) Water extract), coningic acid and the like.

  Whether or not a certain test substance is an inhibitor of glycolytic constituent enzymes can be confirmed by, for example, a general evaluation method known to those skilled in the art. For example, the activity of glycolytic constitutive enzymes is compared in the presence and absence of the test substance. If the enzyme activity in the presence of the test substance is reduced or deleted compared to the enzyme activity in the absence of the test substance, the test substance may be identified as an inhibitor of glycolytic constituent enzymes. it can. Moreover, the glycolytic constituent enzyme inhibitory substance can be obtained by, for example, a general screening method known to those skilled in the art. For example, the activity of glycolytic constitutive enzymes is compared in the presence and absence of the test substance. If the enzyme activity in the presence of the test substance is reduced or deleted compared to the enzyme activity in the absence of the test substance, the test substance may be obtained as an inhibitor of glycolytic constituent enzymes. it can.

  Suitable “glycolytic inhibitors” in the present invention include 2-deoxy-D-glucose (2DG), 3-bromopyruvic acid (3BP), 2-deoxy-D-glucose-6-phosphate, and fluoride. One type or two or more types selected from the group consisting of sodium, iodoacetic acid, sesquiterpene lactone, BZL101 and coningic acid can be mentioned, and among them, any one or both of 2DG and 3BP can be preferably mentioned.

  “Glucose” in the present invention is not particularly limited as long as it is glucose that can be assimilated by pluripotent stem cells, and for example, D-glucose is preferable.

  The “hepatic progenitor cell” in the present invention means “a cell in which the expression of delta like-1 homolog (DLK-1), which is a marker of hepatic progenitor cell, is enhanced”, or “hepatocyte lineage” Cells in which expression of both α-feto protein (AFP), which is a cell marker, and γ-glutamyl trans peptidase (G-GTP), which is a bile duct epithelial cell marker, are enhanced And preferably includes cells in which increased expression of DLK-1, AFP and G-GTP is observed. The expression level of a gene is enhanced in comparison with the expression level of the gene in cells cultured under the same conditions except that ReproFF (product number: RCHEMD004, manufactured by Reprocell) is used as a medium. Means increased. Increased expression of these genes can be measured by a known technique such as real-time quantitative PCR. As a method of such real-time quantitative PCR, for example, 5. Preferably, the method described in (1) can be mentioned.

  Hepatoblasts are tissue stem cells derived from the foregut endoderm and are essential for liver tissue development. Hepatoblasts are positioned as tissue stem cells in fetal liver. The abundance of tissue hepatocytes in mature liver is extremely small and is called liver progenitor cells. The definition of “hepatic progenitor cell” in the present specification is as described above, and is a concept including hepatoblasts. Hepatic progenitor cells present in the mature liver are activated with liver injury and are thought to play an important role in liver repair. Differentiation of hepatic progenitor cells such as hepatoblasts into hepatocytes involves various transcription factors and various extracellular substrates produced by non-parenchymal cells such as hepatocyte growth factor (HGF) and transforming growth. The involvement of factor β (Transforming growth factor β; TGFβ) has been reported. Hepatic progenitor cells such as hepatoblasts can be cultured in vitro, and can be differentiated into hepatocytes or bile duct epithelial cells by culturing under appropriate culture conditions.

  In the present specification, the term “hepatocyte” is used to mean cells including all differentiation stages in which differentiation into hepatocytes has been determined, such as hepatic progenitor cells and mature hepatocytes. Mature hepatocytes, also called mature liver parenchymal cells, are terminally differentiated to express a wide variety of liver-specific functions such as cholesterol synthesis ability, amino acid transport activity, and glucose-6-phosphatase activity. Although it is a cell, it has an active proliferation ability as is well known in the liver regeneration phenomenon. Hepatic progenitor cells are more proliferative than mature hepatocytes and also form bile duct epithelium, so when transplanted into the liver, they quickly form an existing liver structure and are lost more effectively than transplanting only hepatocytes. (Sangan CB et. Al., “Hepatic progenitor cells”, Cell and Tissue Research, 2010, Vol.342, No.2, p.131-137).

  In the present specification, a cell for which differentiation from a pluripotent stem cell to a hepatocyte is determined may be referred to as a hepatocyte cell. Hepatocyte lineage cells include cells in various differentiation processes from cells in the early stages of differentiation from pluripotent stem cells to hepatocytes to mature hepatocytes, such as hepatic progenitor cells such as hepatoblasts, Hepatocytes are preferably included.

  The “hepatic progenitor cell induction medium” in the present invention is a medium containing the above glycolytic inhibitor and glucose, and can be induced into hepatic progenitor cells when pluripotent stem cells are cultured in the medium. Means medium. As the concentration of the glycolytic inhibitor and glucose contained in the “hepatic progenitor cell induction medium” in the present invention, hepatic progenitor cells can be cultured for a longer period of time compared to the HDI medium or L15 medium, and / or Or as long as it can suppress the reduction | decrease of a hepatic progenitor cell and can obtain more hepatic progenitor cells, it will not restrict | limit in particular. The total concentration of the glycolytic inhibitor is, for example, in the range of 0.05 to 60 μM, preferably in the range of 0.12 to 50 μM. These concentration ranges can be used particularly preferably when the glycolytic inhibitor is 2-deoxy-D-glucose, 3-bromopyruvic acid, or both.

  When 2DG is used as a glycolytic inhibitor, the concentration of 2DG in the hepatic progenitor cell induction medium is in the range of 0.05 to 30 μM, and preferably in the range of 0.06 to 25 μM. In addition, the concentration of 3BP in the hepatic progenitor cell induction medium when 3BP is used as a glycolytic inhibitor includes a range of 0.05 to 30 μM, preferably a range of 0.06 to 25 μM. .

  In the “hepatic progenitor cell induction medium” of the present invention, as components other than the glycolytic inhibitor and glucose, one or more types of inorganic salt (s), one or more types of amino acids (s), and 1 Or 2 or more types of vitamin (s) and 1 or 2 or more types of other components are mentioned. The hepatic progenitor cell induction medium may further contain one or two or more kinds of sugar (s) in addition to glucose, but may not contain.

  Specific examples of the saccharides other than glucose include monosaccharides such as galactose, mannose, and fructose, and disaccharides such as sucrose, maltose, and lactose. Among them, galactose can be mentioned. These saccharides can be added alone or in combination.

  Specific examples of the inorganic salts include calcium chloride, calcium nitrate, copper sulfate pentahydrate, iron (III) nitrate nonahydrate, iron (II) sulfate heptahydrate, magnesium chloride hexahydrate. , Manganese chloride tetrahydrate, magnesium sulfate, potassium chloride, sodium chloride, sodium bicarbonate, disodium hydrogen phosphate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate, sodium dihydrogen phosphate monohydrate One or two or more inorganic salts (s) selected from Japanese, sodium dihydrogen phosphate dihydrate, sodium selenite pentahydrate, zinc sulfate heptahydrate, Any inorganic salt or a combination thereof can be used as long as it is a component that advantageously acts on the production of hepatic progenitor cells from pluripotent stem cells. As the inorganic salt, one or more selected from the group consisting of copper sulfate pentahydrate, iron (III) nitrate nonahydrate, manganese chloride tetrahydrate, and zinc sulfate heptahydrate are inorganic salts. It is preferable to include at least, and it is more preferable to include all inorganic salts of such a group. This is because these groups of inorganic salts are relatively characteristic of the WE medium, which is a suitable medium. These inorganic salts are not particularly limited in the degree of hydration, and anhydrides may be used.

  Specific examples of the amino acids include alanine, arginine, asparagine, aspartic acid, cystine, cysteine, glutamine, glycine, histidine, glutamic acid, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine. Or one or more amino acid (s) selected from, tryptophan, tyrosine, valine, etc., preferably L-amino acids and their derivatives and salts, and derivatives thereof such as hydrates thereof Can do. For example, examples of the arginine include arginine derivatives such as L-arginine hydrochloride and L-arginine monohydrochloride. Examples of the aspartic acid include L-aspartic acid sodium salt monohydrate and L-asparagine. Derivatives of aspartic acid such as acid monohydrate, potassium L-aspartate, magnesium L-aspartate, etc. can be mentioned, and examples of the cysteine include L-cysteine dihydrochloride and L-cysteine hydrochloride monohydrate Derivatives of cysteine such as L-lysine hydrochloride and derivatives of lysine such as L-lysine hydrochloride, and the glutamic acid may include derivatives of glutamine such as L-glutamic acid monosodium salt, Asparagine may include derivatives of asparagine such as L-asparagine monohydrate, Examples of tyrosine include derivatives of tyrosine such as L-tyrosine disodium dihydrate, and examples of histidine include derivatives of histidine such as histidine hydrochloride and histidine hydrochloride monohydrate. Examples of the lysine include lysine derivatives such as L-lysine hydrochloride. Further, NEAA (a non-essential amino acid effective for survival of mouse ES cells) can be preferably mentioned. The amino acids preferably include at least one or two or more amino acids selected from the group consisting of alanine, asparagine and glutamic acid, and more preferably include all amino acids of such group. This is because these groups of amino acids are relatively characteristic amino acids in the WE medium, which is a preferred medium.

  Specific examples of the vitamins include biotin, choline, folic acid, inositol, niacin, pantothenic acid, pyridoxine, riboflavin, thiamine, vitamin B12, paraaminobenzoic acid (PABA), ascorbic acid, ergocalciferol (vitamin D2). And one or more vitamin (s) selected from vitamin A and α-tocopherol, and derivatives of each of these components, their salts, and their hydrates. For example, examples of the choline include choline derivatives such as choline chloride, and examples of niacin include niacin derivatives such as nicotinic acid, nicotinic acid amide (nicotinamide), and nicotinic alcohol. As pantothenic acid, derivatives of pantothenic acid such as calcium pantothenate, sodium pantothenate, panthenol and the like can be mentioned. As pyridoxine, pyridoxine such as pyridoxine hydrochloride, pyridoxal hydrochloride, pyridoxal phosphate and pyridoxamine Examples of thiamine include thiamine hydrochloride, thiamine nitrate, bisthiamine nitrate, thiamine dicetyl sulfate ester salt, thulamine derivative such as fursultiamine hydrochloride, octothiamine, benfotiamine and the like. so Ascorbic acid is derived from ascorbic acid such as ascorbic acid 2-phosphate, magnesium ascorbate phosphate, sodium ascorbate sulfate, ascorbylaminopropyl phosphate, sodium ascorbate phosphate, etc. Examples of α-tocopherol include α-tocopherol derivatives such as sodium α-tocopheryl phosphate. Examples of the vitamins include ascorbic acid, ergocalciferol (vitamin D2), menadione sodium bisulfite (vitamin K3), vitamin A (preferably vitamin A acetate) and α-tocopherol (preferably sodium α-tocopheryl phosphate). It is preferable to include at least one or two or more selected from the group consisting of) as vitamins, and more preferably to include all vitamins of such group. This is because these groups of vitamins are relatively characteristic vitamins in the WE medium, which is the preferred medium.

  Examples of other components include serum or serum substitutes, buffers such as HEPES, nucleic acids such as nucleotides, pyruvate, glutathione, linoleic acid and derivatives thereof, and derivatives thereof such as phenol red, phenol red And so on.

  As the above-mentioned serum, any hepatic progenitor cells such as pluripotent stem cells and hepatoblasts, and those generally used for culturing hepatocytes can be used. When using serum, it is preferable to use serum derived from the same species as the cell species to be cultured. For example, when the cells to be cultured are human cells, it is preferable to use human-derived serum. The serum substitute substance is a substance used for maintenance and growth of cells instead of serum, and means a composition having a clear chemical composition. Serum replacement substances may be any of hepatic progenitor cells such as pluripotent stem cells and hepatoblasts, and those generally used for culturing hepatocytes, such as Knockout (registered trademark) Serum Replacement ( Life Tec hnologies), CDM-HD serum substitute (FiberCell Systems), StemSure serum substitute (Wako Pure Chemical Industries, Ltd.), Nu-Serum trademark (Becton Dickinson). The dose of serum or serum replacement can be determined by simple repeated experiments.

  As the nucleotide, ATP, UTP, GTP, CTP, preferably an equimolar mixture of these 4 types) can be preferably mentioned, and as a derivative of pyruvic acid, sodium pyruvate can be preferably mentioned.

  In addition, as other components, growth promoters such as Oncostatin M, epidermal growth factor, retinoic acid, dexamethasone, insulin, transferrin, FPH1 (hepatocyte functional proliferation inducer 1), M50054; FOXA2, GATA4, HEX , Transcription factors such as C / EBPα, and the like.

  Specific examples of preferable “hepatic progenitor cell induction medium” in the present invention include the following medium containing a glycolytic inhibitor in “basic medium for mammalian cell culture containing glucose”. "Basic medium for culturing mammalian cells containing glucose" includes William E's medium (William's E medium, Life Technologies), ReproFF (product number: RCHEMD004, manufactured by Reprocell), Eagle's minimum essential medium (product number) : 11090-081, Life Technologies, Inc.) Improved Minimum Essential Medium (Product Number: 10373-017, Life Technologies), Iscove's Modified Dulbecco's Medium, Product Number: 12440-053, Life Technologies), Connaught Medical Research Laboratories Medium 1066 (Connaught Medical Research Laboratories Medium 1066, Product No. 11530-037, Life Technologies), Eagle Basal Medium (Basal Medium Eagle, Product No. 21010-046, Life Technologies) Name of basic medium for mammalian cell culture containing glucose, such as McCoy's 5A Medium (product number: 16600-082, manufactured by Life Technologies). Can, among others, William E medium, can be preferably exemplified a ReproFF, among others, can be cited more preferably William E medium. In addition, since these basic culture media for mammalian cell culture contain glucose but do not contain a glycolytic inhibitor, a glycolytic inhibitor is added for use in the present invention. The composition of the basic medium for mammalian cell culture listed above is known, but the composition of William E medium is shown in Table 3 for reference. When the numerical value of “weight part of solid content” of each component in Table 3 is expressed in mg / L, it becomes equal to the “concentration (mM)” of the component. For example, when glycine is 50 mg / L, the concentration is 0.667 mM. The “hepatic progenitor cell-inducing medium containing glucose” in the present invention is independent for each component with respect to the concentration of each component in the basic medium for mammalian cell culture (preferably William E medium) listed above. And a medium containing each component at a concentration in the range of 70% to 130%.

As described above, the method for producing hepatic progenitor cells of the present invention is particularly limited as long as it has a step of culturing pluripotent stem cells in a hepatic progenitor cell induction medium containing a glycolytic inhibitor and glucose. Not. The culture conditions for such culture are not particularly limited as long as hepatic progenitor cells can be produced by culturing pluripotent stem cells in the hepatic progenitor cell induction medium. The culture temperature is usually in the range of about 30 to 40 ° C, preferably 37 ° C. The CO ₂ concentration at the time of culture is usually in the range of about 1 to 10%, preferably about 5%. Moreover, the humidity at the time of culture | cultivation is in the range of about 70-100% normally, Preferably it exists in the range of about 95-100%. Further, the O ₂ concentration during the culture may be a normal oxygen concentration (18 to 22% O ₂ ) or a low oxygen concentration (0 to 10% O ₂ ). The culture period is usually within a range of 2 to 15 days, preferably within a range of 3 to 15 days, more preferably within a range of 7 to 15 days, still more preferably within a range of 7 to 14 days, more preferably 7 Within the range of ˜12 days. The culture period can be appropriately adjusted according to the purpose. When the culture is continued for a longer period, the number of cells obtained can be reduced, but hepatic progenitor cells that are more differentiated into hepatocytes can be obtained. In addition, culture medium replacement | exchange can be performed as needed, Preferably it is performed once every 1 to 2 days, More preferably, it is performed once every 2 days.

  Pluripotent stem cells are cultured in an extracellular matrix (for example, laminin-1-12, collagen, fibronectin, vitronectin, matrigel (manufactured by Becton Dickinson), Geltrex (manufactured by Invitrogen) Etc.) is preferable. Pluripotent stem cells may or may not be co-cultured with feeder cells. Any cells can be used as feeder cells as long as they contribute to the proliferation and maintenance of pluripotent stem cells. For example, C3H10T1 / 2 cell line, OP9 cells, NIH3T3 cells, ST2 cells, PA6 cells, Mouse embryonic fibroblasts (MEF cells), SL10 cells and the like can be used. When using feeder cells, it is preferable to inhibit cell growth by, for example, mitomycin C treatment or radiation irradiation.

  The method for producing hepatic progenitor cells of the present invention includes a step of culturing pluripotent stem cells (hereinafter also referred to as “step A”) in a hepatic progenitor cell induction medium containing a glycolytic inhibitor and glucose. , Any process may be included. Examples of the optional step include a step of separating hepatic progenitor cells from the hepatic progenitor cell induction medium after step A.

  The method for producing hepatic progenitor cells of the present invention may include a step P of culturing pluripotent stem cells in a medium not containing glucose (for example, an HSM medium or an HDI medium) before performing Step A. It is preferably not included. This is because before the step A is performed, the reduction in the number of hepatic progenitor cells can be suppressed more and a larger number of hepatic progenitor cells can be obtained if the step P is not included.

  Moreover, although the manufacturing method of the hepatic progenitor cell of this invention may include the process Q which culture | cultivates a hepatic progenitor cell in a HDI culture medium after the process A, it is preferable not to include. In addition, the method for producing hepatic progenitor cells of the present invention may not include Step R of culturing hepatic progenitor cells in an HSM medium after Step A. The hepatic progenitor cell induction medium of the present invention is a medium that induces differentiation from pluripotent stem cells to hepatic progenitor cells, and has the property of inducing differentiation into hepatic progenitor cells and slowly selecting hepatic progenitor cells. This is because the hepatic progenitor cell induction medium of the present invention has a glycolytic inhibitor and inhibits metabolism of glucose, so that cells other than hepatocyte cells tend not to survive. However, from the viewpoint of sorting or isolating hepatocyte cells (preferably hepatic progenitor cells) such as hepatic progenitor cells more selectively and with higher accuracy, the method for producing hepatic progenitor cells of the present invention comprises step A. After that, it is preferable to include Step R of culturing hepatic progenitor cells in HSM medium. As described in the background art, HSM medium allows hepatocyte cells such as hepatic progenitor cells to survive for several days, but cells other than hepatocyte cells die quickly, so It is excellent as a selective medium. The composition of the HSM medium is shown in Tables 4 and 5 below, the composition of the HDI medium is shown in Table 6 below, and the composition of the L15 medium is shown in Table 7 below. The composition of the HSM medium in Table 5 is the same as the composition of the HSM medium in Table 4 except that it does not contain insulin, dexamethasone, or aprotinin.

  The differentiation of hepatic progenitor cells and hepatocytes from pluripotent stem cells by the method for producing hepatic progenitor cells of the present invention can be confirmed by detecting expression of known markers of hepatic progenitor cells and hepatocytes. it can. Markers of hepatic progenitor cells such as hepatoblasts include AFP and DLK-1, and hepatocyte markers include CYP3A4 related to drug metabolism, ALDH2 related to alcohol metabolism, and albumin.

  Detection of hepatic progenitor cells and hepatocyte markers is performed by reverse transcription of the target protein mRNA in the cell, followed by polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), or real-time quantitative polymerase chain reaction. However, it is not limited to these methods, and a known method can be used, which can be confirmed by an enzyme immunoassay method (ELISA method) or an immunostaining method using an antibody against a test protein. Either can be used.

  The cell culture obtained by the method for producing hepatic progenitor cells of the present invention is a cell culture containing hepatic progenitor cells. “Cell culture” refers to a group of cells obtained after culturing cells. The cell culture in the present invention does not substantially contain cells having undifferentiated ability. “Substantially not contained” means that the ratio of hepatic progenitor cells to undifferentiated cells (hepatic progenitor cells: undifferentiated cells) is 1000: 1 or less, preferably 10,000: 1 or less, More preferably, it means 100000: 1 or less. In addition to the hepatic progenitor cells, the cell culture may contain cells further differentiated from the hepatic progenitor cells.

  The cultured cell product obtained by the method for producing hepatic progenitor cells of the present invention includes, for example, regenerative medicine including transplantation treatment for liver diseases such as fulminant hepatitis, partial hepatectomy, or liver failure that occurs during the natural course of cirrhosis. It can be used as a medicine. A medicament containing a cell culture obtained by the method for producing hepatic progenitor cells of the present invention as an active ingredient may contain a pharmacologically acceptable physiological saline, an additive, a medium, etc. Those free from impurities such as viruses are preferred.

  The cell culture obtained by the method for producing hepatic progenitor cells of the present invention can also be differentiated into hepatocytes or bile duct epithelial cells by culturing in vitro under appropriate culture conditions. For example, the differentiation of hepatic progenitor cells into hepatocytes has been reported to involve many transcription factors and various extracellular substrates such as HGF and TGFβ produced by nonparenchymal cells. Differentiation from hepatic progenitor cells to hepatocytes can be induced in vitro. Thus, the cell culture obtained by the method for producing hepatic progenitor cells of the present invention can be used to obtain hepatocytes or bile duct epithelial cells by further inducing differentiation by a known method.

  Furthermore, a cell culture containing hepatocytes can be used for in vitro tests on drug metabolism functions and alcohol metabolism functions possessed by hepatocytes, drug toxicity tests on hepatocytes, and the like.

  The medium additive of the present invention is a medium additive for adding to a differentiation induction medium from pluripotent stem cells to hepatic progenitor cells, and is not particularly limited as long as it contains a glycolytic inhibitor. The culture medium additive of the present invention contains a glycolytic inhibitor as an active ingredient. When the hepatic progenitor cells are induced from pluripotent stem cells, the culture medium additive of the present invention cultures hepatic progenitor cells for a longer period and / or suppresses the decrease in hepatic progenitor cells, thereby increasing more hepatic progenitors. It is a medium additive for obtaining cells.

  The culture medium additive of the present invention may be in a solid form such as powder, granule, granule or the like, or in a liquid form. The solid medium additive can be prepared using a solid glycolytic inhibitor. The liquid medium additive can be prepared by dissolving or suspending the glycolytic inhibitor in a solvent such as water.

  Since the usage mode of the culture medium additive of the present invention may include a mode in which the medium additive is diluted when added to the culture medium, the content of the glycolytic inhibitor in the culture medium additive is not particularly limited. For example, the ratio to the total amount of the additive is in the range of 0.01 to 99.99% by weight, preferably in the range of 0.1 to 99.9% by weight, more preferably in the range of 1 to 99% by weight, and still more preferably. Within the range of 3 to 80% by weight, more preferably within the range of 10 to 60% by weight.

  The culture medium additive of the present invention may contain only a glycolytic inhibitor or may contain other optional components. Examples of optional components include glucose, a carrier, and a solvent.

  When the culture medium additive of the present invention contains glucose, the glucose concentration is not particularly limited, but the ratio of the total molar concentration of glycolytic inhibitors to the molar concentration of glucose is, for example, 1: 185 to 1: 222000. Within the range, preferably within the range of 1: 222 to 1: 92500.

  The culture medium additive of the present invention can be used by adding it to the above-mentioned hepatic progenitor cell induction medium. The hepatic progenitor cell induction medium to which the medium additive of the present invention is added preferably contains a glycolytic inhibitor and glucose. The hepatic progenitor cell induction medium thus prepared can be suitably used for culture from pluripotent stem cells to hepatic progenitor cells.

  The aspect and concentration of the glycolytic inhibitor (preferably 2DG, 3BP), glucose, and other medium components in the hepatic progenitor cell induction medium to which the medium additive of the present invention is added are described in “Hepatic progenitor cell induction medium” in the present invention. Is the same as described above.

Further, the culture medium additive of the present invention and the HSM medium can be combined to form a culture kit. Such a culture kit includes at least the medium additive of the present invention and an HSM medium. Such a culture kit is:
Step A of culturing pluripotent stem cells in a hepatic progenitor cell induction medium containing a glycolytic inhibitor and glucose, and
Step R of culturing hepatic progenitor cells in HSM medium after step A
Can be suitably used in the method for producing hepatic progenitor cells of the present invention. That is, this culture kit is a culture kit for inducing differentiation from pluripotent stem cells to hepatic progenitor cells and selecting hepatocyte cells (preferably hepatic progenitor cells). The composition of the HSM medium is shown in Table 4 above.

  The differentiation induction medium of the present invention is a differentiation induction medium from pluripotent stem cells to hepatic progenitor cells, and is not particularly limited as long as it contains a glycolytic inhibitor and glucose. The differentiation-inducing medium of the present invention contains a glycolytic inhibitor and glucose as active ingredients. The differentiation induction medium of the present invention is a medium for inducing differentiation from pluripotent stem cells to hepatic progenitor cells. More specifically, when inducing hepatic progenitor cells from pluripotent stem cells, the hepatic progenitor cells are longer. It is a “culture medium for inducing differentiation from pluripotent stem cells to hepatic progenitor cells” for culturing for a period and / or obtaining more hepatic progenitor cells by suppressing the decrease in hepatic progenitor cells.

The differentiation-inducing medium of the present invention may be in a solid form such as powder, granule, granule or the like, or in a liquid form. Since the usage mode of the differentiation-inducing medium of the present invention may include a mode in which the differentiation-inducing medium is diluted with a solvent such as water, the content of the glycolytic inhibitor in the differentiation-inducing medium is not particularly limited. The ratio of the sugar-based inhibitor (dry weight) to the total solid content (dry weight) of the differentiation-inducing medium is, for example, in the range of 1.0 × 10 ^{−7 to} 1.0 wt%, preferably 1.0 × 10 ^{− It is in} the range of ^{6 to} 0.1% by weight, more preferably in the range of 1.0 × 10 ^{−5 to} 0.1% by weight.

  The concentration of glucose in the differentiation induction medium of the present invention is not particularly limited, and the ratio of glucose (dry weight) to the total solid content (dry weight) of the differentiation induction medium is, for example, in the range of 0.2 to 10% by weight. Preferably in the range of 0.5 to 5 wt%, more preferably in the range of 0.8 to 3.2 wt%, still more preferably in the range of 1 to 2.2 wt%, more preferably 1.3 to Within the range of 2% by weight.

  The ratio of the total molar concentration of glycolytic inhibitors in the differentiation-inducing medium of the present invention to the molar concentration of glucose is, for example, in the range of 1: 185 to 1: 222000, preferably in the range of 1: 222 to 1: 92500. Inside is mentioned.

  In the differentiation-inducing medium of the present invention, the components other than the glycolytic inhibitor and glucose include one or more inorganic salts (s), one or more amino acids (s), and one or mores. Vitamin (s) and one or more other ingredients. The hepatic progenitor cell induction medium may further contain one or two or more kinds of sugar (s) in addition to glucose, but may not contain. The aspect and concentration of the glycolytic inhibitor (preferably 2DG, 3BP), glucose, and other medium components are the same as those described above for the “hepatic progenitor cell induction medium” in the present invention.

  As a suitable aspect of the differentiation-inducing medium of the present invention, the composition (parts by weight) of each component of the solid content of the differentiation-inducing medium is different from the weight part (Table 3) of each component of the solid content of the WE medium. A medium containing each component in parts by weight within a range of 70 to 130% independently for each component may be mentioned.

  The differentiation-inducing medium of the present invention can be used as a hepatic progenitor cell-inducing medium as it is or after being appropriately diluted depending on the mode. The mode and concentration of the glycolytic inhibitor and glucose when used as a hepatic progenitor cell induction medium are the same as those described above for the “hepatic progenitor cell induction medium” in the present invention.

In addition, a culture kit can be obtained by combining the differentiation-inducing medium of the present invention and the HSM medium. Such a culture kit includes at least the differentiation induction medium of the present invention and an HSM medium. Such a culture kit is:
Step A of culturing pluripotent stem cells in a hepatic progenitor cell induction medium containing a glycolytic inhibitor and glucose, and
Step R of culturing hepatic progenitor cells in HSM medium after step A
Can be suitably used in the method for producing hepatic progenitor cells of the present invention. That is, this culture kit is a culture kit for inducing differentiation from pluripotent stem cells to hepatic progenitor cells and selecting hepatocyte cells (preferably hepatic progenitor cells). The composition of the HSM medium is shown in Table 4 above.

[Reference Example 1]
[Effects of the presence or absence of glucose in the medium on the expression of liver-specific genes]
In order to examine the effects of culture in a medium containing no galactose and containing galactose (medium containing no glucose and containing galactose) on the expression of liver-specific genes in human iPS cells, the following experiment was conducted. went. The method of the experiment is described in 3. (Method) of Examples described later. ~ 6. According to the method.

  The HSM medium and HDI medium do not contain glucose, contain galactose, do not contain arginine, and contain ornithine. ReproFF (manufactured by Reprocell) is a medium for culturing pluripotent stem cells such as iPS cells while maintaining undifferentiation. The composition of ReproFF has not been published and is unknown, but it is thought to contain at least glucose.

  In order to analyze the expression level of liver-specific genes, 201B7 cells (RIKEN cell bank human iPS cells) were cultured in ReproFF medium, HSM medium, or HDI medium for 2 days. RNA is isolated from the cultured cells, subjected to real-time quantitative PCR, cytosolic aspartate aminotransferase (Cyto AST) (FIG. 1A), mitochondrial aspartate aminotransferase (mitochondrial aspartate aminotransferase; Mito The expression levels of AST) (FIG. 1B), alanine aminotransferase (ALT) (FIG. 1C), and glycogen synthase (GS) (FIG. 1D) were analyzed. The results are shown in FIGS.

  As can be seen from the results shown in FIG. 1, when cultured in HSM medium or HDI medium, the expression levels of mitochondrial AST, ALT, and glycogen synthase are significantly higher than those cultured in ReproFF (P <0.05). Increased (FIGS. 1B, C, D). In addition, when cultured in HSM medium or HDI medium, the expression level of cytoplasm AST was markedly increased (P <0.05) as compared with the case of culturing with ReproFF (FIG. 1A). From these results, it was shown that the liver-specific gene and glycogen synthase increase when the medium does not contain glucose, or does not contain arginine, and contains galactose and ornithine.

[Reference Example 2]
[Effect of the presence or absence of galactose in the medium on the expression of α-fetoprotein gene]
In order to examine the possibility that galactose affected hepatocyte differentiation of human iPS cells (201B7 cells) in the results of the test of Reference Example 1 (FIG. 1), the following experiment was performed. The method of the experiment is described in 3. (Method) of Examples described later. ~ 6. According to the method. In addition, when DF12 culture medium (Dulbecco modified Eagle culture medium / Nutrient F-12 Ham culture medium) was used, it replaced with WE culture medium in the (method) of the below-mentioned Example, and used DF12 culture medium.

  201B7 cells were cultured in L15 medium, WE medium and DF12 medium with or without galactose added. RNA was isolated from the cultured cells and subjected to real-time quantitative PCR, and the expression level of α-feto protein (AFP), which is a marker of hepatic progenitor cells, was analyzed. The results are shown in FIG.

  As can be seen from the results of FIG. 2, galactose was added in any of the L15 medium (Leibovitz 15 medium), WE medium (William E medium), and DF12 medium (Dulbecco's modified Eagle medium / Ham F-12 mixed medium). There was almost no difference in the expression level of AFP depending on the presence or absence. From these results, it was shown that galactose has no influence on the differentiation of 201B7 cells into the hepatocyte lineage.

[Reference Example 3]
[Influence of GALK1 promoter activity and GALK2 promoter activity by containing galactose without glucose in the medium]
The present inventors have already found that the expression level of GALK1 (galactokinase 1) gene and GALK2 (galactokinase 2) gene is increased in 201B7 cells cultured in HDI medium supplemented with galactose without glucose. (Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S and Ishige N: AnOptimal Medium Supplementation Regimen for Initiation of Hepatocyte Differentiation in Human Induced Pluripotent Stem Cells. J Cell Biochem 116: 1479-1489, 2015). HDI medium is supplemented with Oncostatin M and low molecular weight compounds. Therefore, oncostatin M and small molecule compounds may affect the expression of GALK1 and GALK2. Therefore, in order to clarify changes in the promoter activity of GALK1 and GLAK2 in a medium supplemented with galactose without adding glucose, Metridia luciferase activity assay was performed after culturing 201B7 cells in HSM medium. As a comparative control, the Metridia luciferase activity assay was performed on 201B7 cells cultured in ReproFF medium or WE medium instead of HSM medium. As described above, the ReproFF medium is a medium for culturing pluripotent stem cells such as iPS cells while maintaining undifferentiation. In addition, WE medium was originally constructed for primary hepatocyte culture (Wu D, Ramin SA and Cederbaum AI: Effect of pyridine on the expression of cytochrome P450 isozymes in primary rat hepatocyte culture. Mol Cell Biochem. 173: 103-111, 1997; Takeba Y, Matsumoto N, Takenoshita-Nakaya S, et al .: Comparative study of culture conditions for maintaining CYP3A4 and ATP-binding cassette transporters activity in primary cultured human hepacytes. J Pharmacol Sci 115: 516 -524, 2011). The Metridia luciferase activity assay was performed as follows.

(Plasmid construction used for Metridia luciferase activity assay)
From the GALK1 promoter of pLightSwitch Prom (manufactured by SwitchGear Genomics), the Sac1-Hind3 fragment of the GALK1 promoter was subcloned into pMetLuc2-reporter (manufactured by Promega Corporation) to prepare pMetLuc2 / GALK1. A Sac1-Hind3 fragment of the GALK2 promoter was subcloned from the GALK2 promoter of pLightSwitch Prom (manufactured by SwitchGear Genomics) into pMetLuc2-reporter (manufactured by Promega Corporation) to prepare pMetLuc2 / GALK2. As a control plasmid, the CMV promoter was subcloned into pMetLuc2-reporter (Promega Corporation) to prepare pMetLuc2-control.

(Transfection and Metridia luciferase activity assay)
201B7 cells were seeded on 96-well plates (manufactured by Asahi Techno Glass) coated with Matrigel (registered trademark). According to the instructions of the product, 100 ng of pMetLuc2-control, pMetLuc2 / GALK1 or pMetLuc2 / GALK2 was transfected into 201B7 cells with FuGENE HD (Clontech) (Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S and Sueishi M: Dual gene expression in embryoid bodies derived from human induced pluripotent stem cells using episomal vectors. Tissue Eng Part A 20: 3154-3162, 2014). Reporter plasmids pMetLuc2-control, pMetLuc2 / GALK1 or pMetLuc2 / GALK2 express Metridia luciferase secreted into the medium. pMetLuc2-control is driven by the cytomegalovirus immediate early promoter (CMV promoter) and expresses Metridia luciferase. The luciferase assay was performed in ReproFF medium, WE medium, or HSM medium for 2 days, followed by Ready-To-Glow secretory luciferase reporter assay (Clontech) and Gene Light (GL-200A) luminometer (Microtech Nichion). ). To monitor transfection efficiency, 10 ng of pSEAP2 control vector (Clontech) was used in each well of a black 96 well FIA plate, and secreted embryonic alkaline phosphatase (SEAP) chemiluminescence kit (Clontech) and Transcriptional activity was measured using a Gene Light luminometer according to the product instructions. The luciferase activity was calculated by dividing the Metridia luciferase activity by the SEAP activity (luminescence amount). Metridia luciferase activity was normalized to the results (CMV) when pMetLuc2-control was used. Metridia luciferase activity was shown as a relative luminescence amount (%) with respect to the luminescence amount (CMV) when pMetLuc2-control was used. The relative expression level of the AFP luminescence amount of Metridia luciferase was analyzed by one-way analysis of variance using JMP5.0J software (manufactured by SAS Institute). Differences with P values <0.05 were considered statistically significant.

  The results of such a Metridia luciferase activity assay are shown in FIG. As can be seen from FIG. 3, in both the result using pMetLuc2 / GALK1 (GALK1) and the result using pMetLuc2 / GALK2 (GALK2), when cultured in the HSM medium, the ReproFF medium is used. It was shown that the Metridia luciferase activity was significantly (P <0.05) higher than when cultured. That is, it is shown that when human iPS cells are cultured in a medium supplemented with galactose without adding glucose, the increase in the expression level of GALK1 gene and GALK2 gene is due to the increase in GALK1 promoter activity and GALK2 promoter activity. It was done.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.

(Method)
1. [Cell culture]
ReproFF medium (Feeder Asahi Techno Glass Co., Ltd.) coated with Matrigel (registered trademark) (manufactured by Becton Dickinson) on a human iPS cell line 201B7 (RIKEN Cell Bank) under feeder-free conditions (ReproFF medium) Cultured by Reprocell). Such culturing was carried out in a wet chamber under conditions of 37 ° C. and 5% carbon dioxide. When the cells became confluent, the cells were rinsed with phosphate buffered saline and harvested with Accutase® (Innovative Cell Technologies). When subcultured, the collected cells were spread on a new culture dish Matrigel every 4 to 5 days, and the culture was continued. When observing the collected cells, the cells were observed with a microscope (CKX41N-31PHP, manufactured by Olympus).

2. [reagent]
100 × NEAA (Non-Essential Amino Acids Solution) and 100 × sodium pyruvate were purchased from Life Technologies. Apoptosis inhibitor (M50054) (2,2′-methylenepis (1,3-cyclohexanedione)) was purchased from Merck. FPH1 (2- (N- (5-chloro-2-methylphenyl) methylsulfonamide) -N- (2,6-difluorophenyl) acetamide) was purchased from XcessBio (Shan J, Schwartz RE, Ross NT, et al .: Identification of small molecules for human hepatocyte expansion and iPS differentiation. Nat Chem Biol 9: 514-520, 2013). Oncostatin M (20 ng / mL), nicotinamide (1.2 mg / mL), proline (30 ng / mL) and L-glutamine (0.3 mg / mL) were purchased from Wako Pure Chemical Industries, Ltd. 2-Deoxy-d-glucose (2DG) was purchased from Sigma-Aldrich.

3. [Hepatocyte selection medium and hepatocyte differentiation initiation medium]
A human iPS cell line 201B7 (RIKEN Cell Bank) was cultured in a 6-well plate coated with Matrigel (registered trademark) in HSM medium or HDI medium for 2 days and subjected to real-time quantitative PCR (polymerase chain reaction). did. HSM medium was prepared from amino acids according to the formulation of Leibovits-15 medium (Life Technologies), except that arginine, tyrosine, glucose and sodium pyruvate were omitted and galactose (900 mg / L) ( Wako Pure Chemical Industries), ornithine (1 mM) (Wako Pure Chemical Industries), glycerol (5 mM) (Wako Pure Chemical Industries) and proline (260 mM) (Wako Pure Chemical Industries) were added. (Tomizawa M, Shinozaki F, Sugiyama T, Yamamoto S, Sueishi M and Yoshida T: Survival of primary human hepatocytes and death of induced pluripotent stem cells in media lacking glucose and arginine. PLoS One 8: e71897, 2013). Proline (30 mg / L) was added because it is necessary for DNA synthesis (Nakamura T, Teramoto H, Tomita Y and Ichihara A: L-proline is an essential amino acid for hepatocyte growth in culture. Biochem Biophys Res Commun 122: 884-891, 1984). Aspartic acid was one of the ornithine metabolites and was not added because it is a substrate for arginine synthesis. Instead of FCS, Knockout® Serum Replacement (Life Technologies) was added at a final concentration of 10% to establish xeno-free limiting conditions. The HDI medium was prepared by adding Oncostatin M, FPH1, M50054, 1 × NEAA, 1 × sodium pyruvate, nicotinamide, proline and glutamine. Proline and nicotinamide are found in primary hepatocyte growth (Nakamura T, Teramoto H, Tomita Y and Ichihara A: L-proline is an essential amino acid for hepatocyte growth in culture.Biochem Biophys Res Commun 122: 884-891, 1984, Mitaka T, Sattler CA, Sattler GL, Sargent LM and Pitot HC: Multiple cell cycles occur in rat hepatocytes cultured in the presence of nicotinamide and epidermal growth factor. Hepatology 13: 21-30, 1991). The concentration of each reagent is as described above.

4). [Culture in conventional medium with or without 3BP or 2DG]
201B7 cells were collected, seeded in 6-well plates (Asahi Techno Glass), and cultured in ReproFF medium, Reebovitz-15 (L15) medium, or William E medium (WE). ReproFF medium, L15 medium and WE medium were supplemented with 1.2 mg / mL nicotinamide, 30 ng / mL proline and 10% Knockout (registered trademark) Serum Replacement (manufactured by Life Technologies). Since nicotinamide and proline are necessary for primary hepatocyte growth, they were added to each medium (Nakamura T, Teramoto H, Tomita Y and Ichihara A: L-proline is an essential amino acid for hepatocyte growth in culture. Biochem Biophys Res Commun. 122: 884-891, 1984; Mitaka T, Sattler CA, Sattler GL, Sargent LM and Pitot HC: Multiple cell cycles occur in rat hepatocytes cultured in the presence of nicotinamide and epidermal growth factor. Hepatology 13: 21-30, 1991) . In some experiments, 0.1, 1.0 or 10 μM 2DG (Sigma-Aldrich) and / or 10 μM 3BP (Sigma-Aldrich) were added to the medium. In some experiments, 900 mg / mL galactose was also added to the medium. After culturing for 7 days, the cells were observed with a microscope (CKX41N-31PHP; manufactured by Olympus) or subjected to real-time quantitative PCR (qPCR).

5. [Real-time quantitative polymerase chain reaction (qPCR)]
5 μg (micrograms) of total RNA isolated with Isogen (Nippon Gene) was used together with SuperScript III reverse transcriptase and oligo (dT) primer (Life Technologies) for the synthesis of single-stranded cDNA. Used according to the instructions. Total RNA derived from human fetal liver was purchased from Clontech. Real-time qPCR was performed with Fast SYBR Green Master Mix (Life Technologies) and the results were analyzed using the Mini Opticon system (Bio-Rad). For real-time qPCR, 40 cycles of denaturation 5 seconds and annealing 5 seconds were performed. The annealing temperature was 60 ° C. Table 8 shows primer sequences corresponding to each gene. In addition, although not used in this Example, the primer sequences corresponding to the γ-glutamyl transpeptidase (G-GTP) gene, which is a bile duct epithelial cell marker, are also shown in Table 8.

  Since the RPL19 gene is a constitutively expressed housekeeping gene, it was used as an endogenous control to monitor the amount of mRNA (Davies B and Fried M: The L19 ribosomal protein gene (RPL19): gene organization, chromosomal mapping, and novel promoter region. Genomics 25: 372-380, 1995). Gene expression levels were automatically analyzed by the Mini Opticon system based on the delta-delta cycle threshold (ddCt) method (Tam S, Clavijo A, Engelhard EK and Thurmond MC: Fluorescence-based multiplex real-time RT- PCR arrays for the detection and serotype determination of foot-and-mouth disease virus. J Virol Methods 161: 183-191, 2009). The relative expression level of each liver-specific gene with respect to the RPL19 gene was calculated by dividing the expression level of each liver-specific gene by the expression level of RPL19.

6). [Statistical analysis]
The relative expression level of the genes was analyzed by one-way analysis of variance using JMP5.0J software (SAS Institute). Differences with P values <0.05 were considered statistically significant.

(Result 1) [Effect of presence or absence of 2DG in medium on expression of α-fetoprotein gene]
In order to analyze the effect of 2DG, a glucose analog, on hepatocyte differentiation of 201B7 cells, 201B7 cells were cultured in ReproFF medium with or without 2DG (0.1 μM, 1.0 μM, or 10 μM) The expression level of cellular AFP gene was analyzed. In addition, the effect of 201B7 cells on hepatocyte differentiation when 2DG and galactose (900 mg / ml) were used in combination was also analyzed.

  Specifically, the above 4. 201B7 cells are cultured by the above method, and RNA is isolated from the cultured cells. Real-time quantitative PCR was performed by the method described above, and the expression level of AFP was analyzed. The expression level of the AFP gene in 201B7 cells cultured in ReproFF medium without 2DG and without galactose was used as the control expression level.

  The results of such analysis are shown in FIG. As can be seen from FIG. 4, when the medium without galactose was used, the expression level of AFP increased remarkably (P <0.05) at 0.1 μM or 10 μM. When a galactose-added medium was used, the expression level of AFP increased remarkably (P <0.05) at 0.1 μM. From these results, it was shown that 2DG promotes hepatocyte differentiation of 201B7 cells. In addition, the expression level of AFP was significantly increased when 0.1 μM 2DG was added and galactose was added, compared to the case where 0.1 μM 2DG was not added and galactose was not added.

(Result 2) [Effects of presence or absence of 2DG or presence or absence of 3BP on morphological characteristics and number of cells]
3BP is an analog of pyruvate and 2DG is an analog of glucose. These reagents are known as glycolytic inhibitors. How does the presence or absence of 2DG or 3BP in the medium affect the morphological characteristics of 201B7 cells, and how does the presence or absence of 2DG or 3BP in the medium affect the transition of the number of cells In order to analyze the following, the following culture experiment was conducted.

  Specifically, the above 4. According to the method, 201B7 cells were cultured in L15 medium or WE medium supplemented with 3BP and / or 2DG. As a control, 201B7 cells were cultured in L15 medium or WE medium without addition of 3BP or 2DG. Further, as another control, 201B7 cells were cultured in ReproFF medium or HDI medium to which neither 3BP nor 2DG was added. FIG. 5 shows the results of observation of the cells of each group with a microscope on the seventh day from the start of the culture.

  As can be seen from these results, the morphological characteristics of cells cultured in L15 medium or WE medium supplemented with 3BP and / or 2DG were compared with the morphological characteristics of cells cultured in ReproFF medium. Was not recognized. In addition, in the case of an HDI medium containing no galactose and not containing glucose, it was also found that the number of cells after culture would be extremely small.

In addition, when this culture experiment was conducted for 10 days, the number of cells (cells / cm ² ) of each group was determined immediately before the start of culture (D0), 7 days after the start of culture (D7), and 10 days after the start of culture (D10). The measurement results are shown in Table 9 below.

  As can be seen from the results in Table 9, in the L15 medium containing no glucose and containing galactose, the number of cells was greatly reduced to D7 and D10. On the other hand, when the WE medium was used, the decrease in the number of cells in D7 and D10 was suppressed as compared with the case where the L15 medium was used. This was thought to be mainly due to the fact that the WE medium contained glucose, whereas the L15 medium did not contain glucose. When WE medium was used, in D7, WE 3BP, WE 2DG, and WE 3BP 2DG had fewer cells than WE (-), but in D10, WE 3BP, WE 2DG, and WE 3BP 2DG were WE ( There were more cells than-). That is, it was shown that when 3BP and / or 2DG was added to the WE medium, the decrease in the number of cells was suppressed. Therefore, when a medium containing glucose and a glycolytic inhibitor (3BP and / or 2DG) is used as a medium for inducing hepatic progenitor cells from pluripotent stem cells, the number of cells can be reduced. It was shown that it can be suppressed.

(Result 3) [Effect of presence or absence of 2DG in medium or presence or absence of 3BP on expression of α-fetoprotein gene]
In order to analyze how the presence or absence of 2DG or the presence or absence of 3BP in the medium affects the expression of the α-fetoprotein gene in 201B7 cells, the following culture experiment was performed.

  Specifically, the above 4. According to the method, 201B7 cells were cultured in L15 medium or WE medium supplemented with 3BP and / or 2DG. As a control, 201B7 cells were cultured in L15 medium or WE medium without addition of 3BP or 2DG. Furthermore, as another control, 201B7 cells were cultured in ReproFF medium to which neither 3BP nor 2DG was added. After culturing for 7 days, RNA was isolated from each group of cells, and 5. Real-time quantitative PCR was performed by the method described above, and the expression level of AFP was analyzed. The expression level of the AFP gene in 201B7 cells cultured in ReproFF medium to which neither 3BP nor 2DG was added was used as the control expression level.

  The results of such analysis are shown in FIG. In the WE medium, when either 3BP or 2DG was added, the expression level of AFP was slightly lower than that in the case of no addition, but the expression level of AFP was significantly higher than that in the case of ReproFF medium. In addition, when both 3BP and 2DG were added to the WE medium, the expression level of AFP was higher than when both were not added. Thus, it was found that the addition of both 3BP and 2DG is more preferable because a higher AFP expression level can be obtained.

  On the other hand, when the L15 medium containing no glucose and containing galactose was used, the expression level of AFP was significantly higher than that of the ReproFF medium regardless of the addition or absence of 3BP or 2DG. However, as shown in the results of (Result 2) above (Table 9), when L15 medium is used, since the degree of decrease of 201B7 cells in D7 and D10 is large, hepatic progenitor cells are removed from pluripotent stem cells. It is not preferable as a culture medium.

  According to the present invention, when producing hepatic progenitor cells from pluripotent stem cells, hepatic progenitor compared to conventional media (particularly HDI medium or L15 medium) that can induce hepatic progenitor cells from pluripotent stem cells. Cells can be cultured for a longer period and / or more hepatic progenitor cells can be obtained by suppressing the decrease in hepatic progenitor cells.

Claims (11)

  A method for producing hepatic progenitor cells, comprising a step of culturing pluripotent stem cells in a hepatic progenitor cell induction medium containing a glycolytic inhibitor and glucose.   The method for producing hepatic progenitor cells according to claim 1, wherein the glycolytic inhibitor is an analog substance of a substance constituting a glycolytic system or an inhibitory substance of an enzyme constituting the glycolytic system.   The method for producing hepatic progenitor cells according to claim 1 or 2, wherein the glycolytic inhibitor includes any one or both of 2-deoxy-D-glucose and 3-bromopyruvic acid.   The method for producing hepatic progenitor cells according to claim 3, wherein the concentration of 2-deoxy-D-glucose in the hepatic progenitor cell induction medium is 0.05 to 30 µM. The method for producing hepatic progenitor cells according to claim 3 or 4, wherein the concentration of 3-bromopyruvic acid in the hepatic progenitor cell induction medium is 0.05 to 30 µM.   The method for producing hepatic progenitor cells according to any one of claims 1 to 5, wherein the hepatic progenitor cell induction medium is William E medium containing a glycolytic inhibitor. The method for producing hepatic progenitor cells according to any one of claims 1 to 6, wherein the cells are cultured for at least 7 days.   A medium additive for adding to a differentiation-inducing medium from pluripotent stem cells to hepatic progenitor cells, comprising a glycolytic inhibitor.   9. The culture medium additive according to claim 8, wherein the glycolytic inhibitor is an analog substance of a substance constituting the glycolytic system or an inhibitor of an enzyme constituting the glycolytic system.   Furthermore, glucose is included, The culture medium additive of Claim 8 or 9 characterized by the above-mentioned.   A differentiation-inducing medium from pluripotent stem cells to hepatic progenitor cells, comprising a glycolytic inhibitor and glucose.