Quiescent hepatic stellate cell inducer

Abstract

The present invention addresses the problem of providing: a method for preparing a large amount of quiescent hepatic stellate cells with high purity and in a simple manner; and an organoid that faithfully reproduces the liver in a living body, the organoid including quiescent hepatic stellate cells.　Provided are a quiescent hepatic stellate cell inducer and a quiescent hepatic stellate cell proliferation promoter, each of which comprises an FGF signaling pathway activator.

Description

Resting hepatic stellate cell inducer

[0001] The present invention relates to a quiescent hepatic stellate cell inducer, a quiescent hepatic stellate cell induction and proliferation medium, a method for inducing quiescent hepatic stellate cells, and a method for producing hepatic stellate cells.

[0002] The liver is a complex organ composed of diverse cells such as hepatocytes, vascular (sinusoidal) endothelial cells, Kupffer cells, and bile duct cells, forming a distinctive tissue structure. Despite this complexity, it is a highly regenerative organ capable of recovering from damage. This is due to the plasticity of hepatocytes, which make up approximately 70% of liver tissue. However, if the liver is damaged excessively, exceeding its regenerative capacity, or if damage occurs under conditions where the regenerative capacity itself is reduced or impaired due to aging of hepatocytes, liver function will decline significantly, leading to liver failure. This liver failure is a serious disorder with a high mortality rate.

[0003] Liver transplantation is used as a treatment method for liver failure. Although liver transplantation has long been known as an effective treatment method that can cure liver failure, the shortage of donor livers that can be transplanted is a hindrance, making it difficult for patients to receive treatment when they need it. As a simpler alternative to liver transplantation that does not require major surgery, the use of cell replacement therapy has been explored. However, since cell replacement therapy involves administering hepatocytes derived from a donor liver, including hepatocytes, it also suffers from the problem of being dependent on the number of donor livers. Furthermore, with these methods, depending on the combination of donor liver and recipient, even if the recipient's immune system is suppressed and rejection is reduced, the engraftment rate of transplanted cells one year after transplantation can be low, resulting in limited long-term therapeutic effects.

[0004] Under these circumstances, cell replacement therapy and liver transplantation using human pluripotent stem cells are regarded as promising methods for enabling treatment regardless of the number of donor livers. In particular, iPS cells derived from autologous cells and pluripotent stem cells subjected to genome editing are considered to have low immunogenicity and are less likely to cause rejection reactions in recipients. In addition, liver-like organoids are considered to be particularly useful for transplantation into living organisms, construction of disease models, screening for the effectiveness of drugs and the presence or absence of side effects in the liver, and detailed research on the mechanism of liver development and function. Due to these advantages, various studies have been conducted to date, and the generation of liver organoids and the like using human pluripotent stem cells has been realized. However, since the functions of the liver are realized by a variety of other cells, the identification of the minimum cell types necessary for liver function has not been achieved. As a result, even when organoids are generated, necessary cells are often not included, and thus some tissue structures such as vascular structures are often not fully constructed, making it difficult to faithfully reproduce the complex tissue structure of the liver in vitro (Non-Patent Document 1).

[0005] For these reasons, although they have many advantages, the practical application of organoids derived from pluripotent stem cells has not been achieved, and the establishment of a method for preparing organoids that faithfully reproduce the complex tissue structure of the liver has been desired.

[0006] Feaver RE, et al. JCI Insight. 2016;1(20):e90954.

[0007] It has been reported that hepatic stellate cells contribute significantly to liver angiogenesis. However, hepatic stellate cells isolated from living organisms have a high proportion of activated hepatic stellate cells associated with liver fibrosis and are not practical for use in reproducing normal livers. Although the induction of differentiation of hepatic stellate cells from pluripotent stem cells has also been studied, there is a tendency for the proportion of activated hepatic stellate cells to be high, and a method for efficiently obtaining quiescent hepatic stellate cells that are not activated has not been established. Therefore, with the currently used methods, after induction of differentiation, complex procedures such as cell sorting for separating quiescent hepatic stellate cells are required.

[0008] Furthermore, unlike activated hepatic stellate cells, quiescent hepatic stellate cells generally have poor proliferative ability, so they could not be maintained or proliferated in culture after acquisition. Therefore, there was a problem in that complicated differentiation induction from pluripotent stem cells was required every time they were needed, and the number of finally obtained quiescent hepatic stellate cells was extremely small.

[0009] Therefore, an object of the present invention is to provide a method for simply preparing a large amount of quiescent hepatic stellate cells with high purity, and further to provide an organoid that faithfully reproduces the liver in vivo and contains quiescent hepatic stellate cells.

[0010] As a result of intensive studies by the present inventors to solve the above problems, it was found that quiescent hepatic stellate cells can be prepared with high purity by culturing mesenchymal cells in the transverse septum as raw material cells in a specific culture medium composition. In addition, it was found that the quiescent hepatic stellate cells prepared in this way have fetal-like properties and can proliferate while maintaining an inactivated state. Furthermore, it was found that the organoid containing these quiescent hepatic stellate cells has a complex vascular structure as seen in vivo and can communicate with the internal blood vessels when transplanted into the body. The present invention is based on such novel findings and provides the following.

[0011] [1] A quiescent hepatic stellate cell inducer containing an FGF signaling pathway activator. [1-1] The quiescent hepatic stellate cell inducer according to [1], wherein the FGF signaling pathway is the RAS / MAP kinase pathway and / or the PI3 kinase / AKT pathway. [2] A quiescent hepatic stellate cell inducer medium containing an FGF signaling pathway activator and serum-free medium. [3] The quiescent hepatic stellate cell inducer medium according to [2], further comprising an antioxidant. [3-1] The quiescent hepatic stellate cell inducer medium according to [2] or [3], wherein the FGF signaling pathway is the RAS / MAP kinase pathway and / or the PI3 kinase / AKT pathway. [4] A quiescent hepatic stellate cell proliferation promoter containing an FGF signaling pathway activator. [4-1] The quiescent hepatic stellate cell proliferation promoter according to [4], wherein the FGF signaling pathway is the RAS / MAP kinase pathway and / or the PI3 kinase / AKT pathway. [5] A quiescent hepatic stellate cell proliferation medium containing an FGF signaling pathway activator and serum-free medium. [5-1] The quiescent hepatic stellate cell proliferation promoter according to [5], wherein the FGF signaling pathway is the RAS / MAP kinase pathway and / or the PI3 kinase / AKT pathway. [6] A method for producing quiescent hepatic stellate cells, comprising a quiescent hepatic stellate cell induction step of inducing transverse septal mesenchymal cells in a quiescent hepatic stellate cell induction medium according to any one of [2] to [3-1] to induce differentiation into quiescent hepatic stellate cells. [7] The method for producing quiescent hepatic stellate cells according to [6], further comprising a proliferation step of culturing the induced quiescent hepatic stellate cells in a quiescent hepatic stellate cell proliferation medium according to [5]. [8] Quiescent hepatic stellate cells produced by the method of [6] or [7]. [9] A method for inducing quiescent hepatic stellate cells, comprising a quiescent hepatic stellate cell induction step of inducing transverse septal mesenchymal cells in a quiescent hepatic stellate cell induction medium according to any one of [2] to [3-1] to induce differentiation into quiescent hepatic stellate cells.

[10] A method for producing activated hepatic stellate cells, comprising a quiescent hepatic stellate cell induction step of inducing and culturing transverse septal mesenchymal cells in a quiescent hepatic stellate cell induction medium according to any one of [2] to [3-1] to induce differentiation into quiescent hepatic stellate cells, and an activation step of activating and culturing the induced quiescent hepatic stellate cells.

[11] A method for producing liver organoids, comprising a co-culture step of co-culturing quiescent hepatic stellate cells according to [8] with vascular endothelial cells and hepatic progenitor cells.A cell preparation comprising quiescent hepatic stellate cells as described in

[12] and [8]. A non-human animal comprising quiescent hepatic stellate cells as described in

[13] and [8]. An organoid comprising quiescent hepatic stellate cells as described in

[14] and [8]. A non-human animal comprising an organoid as described in

[15] and

[14] . This specification includes the disclosures of Japanese Patent Application No. 2024-191609, which forms the basis of the priority of this application.

[0012] According to the quiescent hepatic stellate cell inducer, quiescent hepatic stellate cell inducer kit, quiescent hepatic stellate cell inducer medium, and quiescent hepatic stellate cell inducer method of the present invention, quiescent hepatic stellate cells with proliferative capacity can be prepared.

[0013] According to the quiescent hepatic stellate cell proliferation promoter of the present invention, quiescent hepatic stellate cells can be proliferated and cultured while maintaining an inactive state.

[0014] According to the method for producing quiescent hepatic stellate cells of the present invention, it is possible to produce quiescent hepatic stellate cells that can proliferate while maintaining an inactive state.

[0015] According to the organoid manufacturing method of the present invention, liver organoids having a complex vascular structure can be prepared.

[0016] Figure 1 schematically shows the relationship between cells that can be differentiated from transverse septal mesenchymal cells according to the present invention and quiescent hepatic stellate cells that are the target of the present invention. Figure 2 shows the results of qPCR analysis in Example 1. In the figure, "hiPSC" shows the results for human iPS cells (n=10), "hiPSC-MC" shows the results for mesoderm cells induced from human iPS cells (n=10), "hiPSC-STM" shows the results for transverse septal mesenchymal cells induced from human iPS cells (n=7), "hiPSC-HSC" shows the results for hepatic stellate cells induced from human iPS cells (n=5), and "pHSC" shows the results for primary hepatic stellate cells (n=5). In the figure, "Fold change" shows the value normalized to 1 for the expression level obtained from hiPSC. In the figure, error bars indicate the standard deviation, "**" indicates that the p-value compared to the hiPSC results is p < 0.01, "***" indicates that the p-value compared to the hiPSC results is p < 0.005, and "ns" indicates that there is no significant difference from the hiPSC results. Figure 3 shows the results of the transcriptome analysis in Example 1. Each square shows the expression level of each gene (row) in each cell (column) by color, with lighter colors indicating higher expression levels. The label in each row indicates the name of the signature gene of the cell that can be derived from the target transverse septal mesenchymal cells (STM), and the label in each column indicates the number of the target cell. In the figure, "HSC" indicates that it is an HSC signature gene, "mesothelial cell" indicates that it is a mesothelial cell signature gene, and "vascular smooth muscle cell" indicates that it is a vascular smooth muscle cell signature gene. Figure 4 shows the results of the flow cytometry in Example 1. Figure 4A shows the ratio of CD73-expressing cells, Figure 4B shows the ratio of ALCAM-expressing cells, Figure 4C shows the ratio of CD71-expressing cells, Figure 4D shows the ratio of lipid-storing cells, and Figure 4E shows the ratio of vitamin A-storing cells. In Figure 4, "hiPSC-HSC" shows the results for hepatic stellate cells induced from human iPS cells (n=7), and "pHSC" shows the results for primary hepatic stellate cells (n=4). In Figure 4, error bars indicate the standard deviation, and "ns" indicates no significant difference. Figure 5 shows the differences in properties between hepatic stellate cells induced from human iPS cells (hiPSC-HSC) and primary hepatic stellate cells (pHSC). Figure 5A shows the results of transcriptome analysis.Each square indicates the expression level of each gene (column) in each cell (row) using color, with darker colors indicating higher expression levels. The label in each column indicates the name of the target signature gene, and the label in each row indicates the type of cell being targeted. In Figure 5A, "Normal" indicates a normal liver signature gene, and "Cirrhosis" indicates a cirrhosis signature gene. Figure 5B shows the percentage of PDGFRα-positive cells, which are an activated hepatic stellate cell marker. Figure 5C shows the production amount of COL1A1 in the culture medium; in Figure 5C, "Con" indicates the result before TGFβ1 treatment, and "TGFβ1" indicates the result after TGFβ1 treatment. In Figures 5B and 5C, error bars indicate the standard deviation, "ns" indicates no significant difference, "**" indicates a p-value of p < 0.01, and "***" indicates a p-value of p < 0.005. Figure 6 shows the results of the gene set enrichment analysis in Example 1. Figure 6A shows the results for the fetal HSC-related gene set, Figure 6B shows the results for the adult HSC-related gene set, and Figure 6C shows the results for the cell cycle-related gene set. In the figures, the arrows on the horizontal axis indicate that the relative expression level is higher in each cell as the direction of the arrow moves. Figure 7 shows the results of the qPCR analysis in Example 1. Figure 7A shows the relationship between the presence or absence of FGF2 addition and the expression level of activated hepatic stellate cell markers, Figure 7B shows the relationship between the type of culture medium and the expression level of activated hepatic stellate cell markers, and Figure 7C shows the relationship between the type of coating agent and the expression level of activated hepatic stellate cell markers. In Figure 7A, "Fold change" shows the value standardized with the value with FGF2 addition ("FGF2+") set to 1, in Figure 7B, "Fold change" shows the value standardized with the value when DMEM / F12 is used as the culture medium ("DMEM F12") set to 1, and in Figure 7C, "Fold change" shows the value standardized with the value when laminin is used as the coating agent ("Laminin") set to 1. In the figure, error bars indicate the standard deviation, "***" indicates a p-value of p < 0.005, and "ns" indicates no significant difference. Figure 8 shows the relationship between the number of passages and the cumulative cell proliferation rate in Example 2. Figure 8A shows the relationship between the number of passages and the cumulative cell proliferation rate, and Figure 8B shows the relationship between the number of passages and hepatic stellate cell markers.In Figure 8A, the labels for each data series indicate the experiment number performed. In Figure 8B, "P1" shows the results for cells after the first passage, "P3" shows the results for cells after the third passage, and "P6" shows the results for cells after the sixth passage. In Figure 8B, error bars indicate the standard deviation, and "ns" indicates no significant difference. Figure 9 shows the relationship between the number of passages and the expression level of activated hepatic stellate cell markers in Example 2. Figure 9A shows the results for COL1A1 expression, and Figure 9B shows the results for αSMA expression. In the figures, "Fold change" indicates the value standardized to 1, with the expression level obtained from primary hepatic stellate cells ("pHSC") set to 1. In the figures, "P1" shows the results after 1 passage, "P2" after 2 passages, "P3" after 3 passages, "P4" after 4 passages, "P5" after 5 passages, and "P6" after 6 passages. In the figures, error bars indicate the standard deviation, and "***" indicates that the p-value is p < 0.005. Figure 10 shows the vascular structure of liver organoids in Example 3. Figure 10A shows the signaling behavior of human iPS cell-derived vascular endothelial cells (hiPSC-EC) in liver organoids containing HSCs (LO-HSC), and Figure 10B shows the signaling behavior of hiPSC-EC in liver organoids without HSCs (LO-ΔHSC). Figure 11 shows the liver function of liver organoids in Example 3. Figure 11A shows the signaling behavior in LO-HSC, Figure 11B shows the signaling behavior in LO-ΔHSC, and Figure 11C shows the results for NH4 metabolism. In Figures 11A and B, "Albumin" indicates that the image in that column shows the signaling behavior of albumin, and "CYP3A4" indicates that the image in that column shows the signaling behavior of CYP3A4. In Figure 11C, "+HSC" indicates the results of LO-HSC including HSCs, "-HSC" indicates the results of LO-ΔHSC excluding HSCs, error bars indicate the standard deviation, and "**" indicates that the p-value is p < 0.01. Figure 12 shows the appearance of sinusoidal endothelial cell markers in organoids in Example 3.Figures 12A and 12B show the signal in LO-HSC, Figures 12C and 12D show the signal in LO-ΔHSC, Figures 12A and 12C show the signal in CD31, and Figures 12B and 12D show the signal in LYVE1. Figure 13 shows the results of transmission electron microscopy observation of the organoid in Example 3. In the figure, "Lumen" indicates the lumen, "Glycogen" indicates cytoplasmic glycogen, "Microvillus" indicates microvilli, "Tight junction" indicates a tight junction, and "bile canaliculi" indicates bile canaliculi. The scale bars in each figure represent 10 μm (A), 2 μm (B), and 1 μm (C), respectively. Figure 14 shows the blood vessels of the organoid transplanted into the mouse cranial fenestra in Example 4. Figure 14A shows the signal in LO-HSC, and Figure 14B shows the signal in LO-ΔHSC. In the figure, "Blood Vessel" indicates that the image in that column shows the signal of blood vessels, "hiPSC-EC" indicates that the image in that column shows the signal of hiPSC-EC, and "Merge" indicates that the image in that column is an image in which the signals of the two left columns are superimposed. In the figure, arrows indicate the location of blood vessels that are positive for hiPSC-EC signals. Figure 15 shows the blood vessels of organoid LO-HSC transplanted into the mouse cranial fenestra in Example 4. In the figure, "Blood Vessel" indicates the signal of blood vessels, "hiPSC-EC" indicates the signal of hiPSC-EC, and "hiPSC-HSC" indicates the signal of hiPSC-HSC. In the figure, arrows indicate the location of blood vessels that are positive for hiPSC-HSC signals, and arrowheads indicate the location of blood vessels that are negative for hiPSC-HSC signals. Figure 16 shows the results of qPCR analysis in Example 5. Figure 16A shows the relationship between the presence or absence of FGF2 addition, the addition of each inhibitor, and the proliferation rate, while Figure 16B shows the relationship with the expression levels of activated hepatic stellate cell markers. In the figures, "Cell number" indicates the number of cells on day 4 of culture, and "Fold change" is a value standardized to 1 when FGF2 is added to the culture medium and only DMSO without inhibitors is added ("DMSO" in "+FGF2").In the figure, error bars indicate the standard deviation, "*" means the p-value is p < 0.05, "***" means the p-value is p < 0.005, "****" means the p-value is p < 0.001, and "ns" means there is no significant difference. Figure 17 shows the results of the qPCR analysis in Example 6. Figure 17A shows the relationship between the presence or absence of FGF2 addition, the addition of each inhibitor and the proliferation rate, and Figure 17B shows the relationship with the expression level of activated hepatic stellate cell markers. In the figure, "Cell number" indicates the number of cells on day 4 of culture, and "Fold change" shows the value standardized to 1 when FGF2 is added to the culture medium and only DMSO without an inhibitor is added ("DMSO" in "+FGF2"). In the figure, error bars indicate the standard deviation, "*" means the p-value is p < 0.05, "**" means the p-value is p < 0.01, "***" means the p-value is p < 0.005, "****" means the p-value is p < 0.001, and "ns" means there is no significant difference.

[0017] 1. Quiescent Hepatic Stem Cell Inducer 1-1. Overview The first aspect of the present invention is a quiescent hepatic stellate cell inducer. The quiescent hepatic stellate cell inducer of the present invention contains an FGF signaling pathway activator as an active ingredient and induces differentiation from pluripotent stem cells into quiescent hepatic stellate cells. The quiescent hepatic stellate cell inducer of the present invention can be used in methods for inducing quiescent hepatic stellate cells and in methods for producing hepatic stellate cells.

[0018] 1-2. Definitions The terms used herein are defined below. "Hepatic Stellate Cells (HSCs)" are stromal cells located around the blood vessel walls of the sinusoids of the liver. Markers for HSCs include, for example, ALCAM, CD71, CD73, PCDH7, LOX, PDGFRβ, HAND2, RSPO3, HGF, DCN, and desmin, and typically express one or more of these. They are also typically distinguished from mesenchymal cells by being CD271 negative. HSCs are broadly classified into two types: quiescent and activated.

[0019] In this specification, "quiescent hepatic stellate cells (qHSCs)" refers to inactive hepatic stellate cells. qHSCs are HSCs found in the liver of normal individuals, characterized by well-developed lipid droplets and spinous projections on their cell surface. qHSCs possess properties such as the production of extracellular matrix (collagen fibers, etc.), the ability to accumulate lipid droplets and store vitamin A within them, retinoid metabolism, and the function of maintaining extracellular matrix homeostasis, thus significantly contributing to the homeostasis of liver function. Typically, qHSCs are cells that do not express or express low levels of the HSC activation markers described below. Examples of qHSC markers include PCDH7, RGS5, FABP5, FABP4, and BAMBI. In this specification, qHSCs are, for example, αSMA-negative and / or PDGFRα-negative hepatic stellate cells.

[0020] In this specification, "active hepatic stellate cells (aHSCs)" refers to activated hepatic stellate cells. aHSCs are characterized by vitamin A release, high proliferative capacity, high migration, and active secretion of extracellular matrix, and excessive activity of aHSCs can cause hepatic fibrosis. When cells become aHSCs, lipid droplets often shrink. Therefore, cells that express hepatic stellate cell markers and lack lipid droplets can be identified as aHSCs. aHSCs typically express or highly express HSC activation markers. Examples of activation markers include PDGFRα, COL1A1, αSMA, vimentin, fibronectin, IL-6, TGF-β, and collagen I. In this specification, aHSCs are, for example, αSMA-positive and / or PDGFRα-positive hepatic stellate cells.

[0021] The "FGF (fibroblast growth factor) signaling pathway" refers to the signaling pathway activated when FGF binds to its receptor, FGFR, a receptor tyrosine kinase. It is known that a phosphorylation cascade, starting from phosphorylated tyrosine in the receptor after ligand binding, activates pathways such as the RAS / MAP kinase pathway, PI3 kinase / AKT pathway, and PLCγ pathway. In this specification, the FGF signaling pathway is preferably the RAS / MAP kinase pathway and / or the PI3 kinase / AKT pathway.

[0022] "FGF" is a growth factor. In this specification, FGF refers to any protein belonging to the FGF family. Typically, FGF family proteins bind to FGFR and have broad mitogenic and angiogenic activity. These proteins are involved in a wide range of pathways, including limb and nervous system development, wound healing, and tumor growth. Known FGF family proteins include basic FGF (bFGF, FGF2), FGF1 to FGF23, etc. For example, FGF2 can be suitably used in the present invention.

[0023] "Proliferation" refers to the increase in the number of cells. In this specification, proliferation specifically refers to proliferation without differentiation.

[0024] "Differentiation" refers to the specialization and increased specificity of a cell's morphology and / or function. In this specification, differentiation specifically refers to the restriction of a cell's differentiation fate and a reduction in the number of cell types that can arise from it.

[0025] "Pluripotent stem cells" refer to cells that possess the ability to differentiate into all types of cells that make up an organism. Typically, pluripotent stem cells can proliferate indefinitely while maintaining their pluripotency when cultured in vitro under appropriate conditions. Here, "pluripotency" refers to the ability to differentiate into cells of all types of germ layers that make up an individual (in vertebrates, the three germ layers: ectoderm, mesoderm, and endoderm). In this specification, pluripotent stem cells include both naive and primed types.

[0026] 1-3. Composition The inducer of the present invention comprises an FGF signaling pathway activator and optionally includes antioxidants and other components. Typically, the inducer of the present invention induces differentiation of qHSCs from septum transversum mesenchyme cells (STMs). STMs, qHSCs, and each active ingredient will be described in detail below.

[0027] <Transverse Mesenchymal Cells (STMs)> In this specification, "transverse mesenchymal cells" refer to cells located between the cardiac mesoderm and the foregut endoderm in developing liver tissue, which provide differentiation signals to the hepatocyte. Typically, they express HLX, WT1, BMP2, BMP4, FOXF1, GATA4, MRG1, HGF, TBX18, etc.

[0028] The origin of the STM is not particularly limited. For example, it may be cells isolated from animal tissue, organs, or individuals, cells differentiated from pluripotent stem cells, or a mixture thereof.

[0029] The animal species from which STMs and pluripotent stem cells are derived are not particularly limited. For example, any vertebrate, including humans, such as fish (including cartilaginous and bony fish), reptiles, amphibians, birds, mammals, rodents, and primates, may be included. For example, other than humans, examples include pets (tropical fish, frogs, lizards, turtles, small birds such as parakeets, dogs, cats, rabbits, etc.), racehorses, laboratory animals (zebrafish, frogs, chickens, mice, rats, guinea pigs, monkeys, etc.), and livestock (cattle, horses, sheep, goats, pigs, chickens, ostriches, etc.). The animals in question may be healthy or suffering from some disease. For example, cells derived from mammals can be suitably used, and in particular, cells derived from primates such as humans can be suitably used.

[0030] When STMs are differentiated from pluripotent stem cells, any pluripotent stem cells known in the art can be appropriately selected, obtained, or produced and used according to the purpose, and the type is not particularly limited. Examples include somatic cell-derived induced pluripotent stem cells (iPS cells), early embryo-derived embryonic stem cells (ES cells), primordial germ cell-derived embryonic germ cells (EG cells), testicular germline stem cells (GS cells), mesenchymal tissue-derived Muse cells, somatic stem cells (mesenchymal stem cells, neural stem cells, etc. derived from bone marrow, adipose tissue, dental pulp, placenta, amniotic membrane, umbilical cord blood, amnion, chorionic membrane, etc.), or mixtures thereof. For example, iPS cells and ES cells (e.g., iPS cells and ES cells of mammals (especially primates such as humans)) can be suitably used.

[0031] For example, when HSCs induced using the inducer of this embodiment are used for the treatment of a specific individual, STMs with a reduced risk of rejection due to transplantation can be used. In this case, for example, STMs differentiated from iPS cells obtained from somatic cells of the individual to be treated, or STMs derived from an individual whose major histocompatibility complex (MHC) genotype (or human leukocyte antigen (HLA) genotype in the case of a human individual) is the same or substantially the same as that of the individual to be treated, or STMs differentiated from pluripotent stem cells derived from such an individual can be used.

[0032] In this specification, "substantially identical MHC genotype (or HLA genotype)" means that the MHC genotypes match to such an extent that the immune response to the transplanted cells can be suppressed by an immunosuppressant. Specifically, for example, a human individual with substantially identical HLA genotypes can be a human individual whose HLA genotypes match those of the target individual in three types of HLA genotypes (HLA-A, HLA-B, and HLA-DR) or four types of HLA genotypes (HLA-A, HLA-B, HLA-DR, and HLA-C).

[0033] <Quisty Hepatic Stellary Cells (qHSCs)> In this specification, qHSCs are hepatic stellate cells that are negative for HSC activation markers (e.g., αSMA and / or PDGFRα). They are also typically CD271 negative.

[0034] qHSCs differentiated by the inducer of this embodiment have proliferative capacity. Having proliferative capacity means, for example, that they can be subcultured one or more, two or more, three or more, four or more, five or more, or six or more times. The conditions for subculture in this case are not particularly limited, but conditions such as those described in the "subculture step" of the fourth embodiment can be used.

[0035] In this specification, qHSCs are preferably fetal-like qHSCs. Whether or not a qHSC is fetal-like can be determined based on the expression of fetal-HSC-related genes.

[0036] While there are no specific limitations on the fetal HSC-related genes, examples include desmin, p75NTR, RAC1, COTL1, IGFBP2, and S100A16. For example, qHSC in this specification expresses one or more fetal HSC-related genes.

[0037] The origin of qHSC is not particularly limited, as long as it is differentiated using the inducing agent of this embodiment. For example, it may be derived from the biological species or cells mentioned above with respect to STM.

[0038] <FGF Signaling Pathway Activators> In this specification, "FGF signaling pathway activators" refers to agents that activate FGF signaling pathways (particularly the RAS / MAP kinase pathway and / or the PI3 kinase / AKT pathway). The type of agent is not particularly limited. Any agent known in the art can be used as an FGF signaling pathway activator. The specific types of FGF signaling pathway activators used are not particularly limited, but examples include FGF synthesis promoters, FGF secretion promoters, FGFR synthesis promoters, FGFR agonists, RAS / MAP kinase pathway activators (EGF family proteins, insulin, IGF family proteins (IGF-1, etc.), Phorbol 12-myristate 13-acetate, 12-O-tetradecanoylphorbol-13-acetate, etc.), PI3 kinase / AKT pathway activators (EGF family proteins, insulin, IGF family proteins (IGF-1, etc.), SC79, 740 YP peptide, etc.), PLCγ pathway activators, FGF receptor binding promoters, and expression promoters of factors in these pathways. For example, FGF receptor binding promoters (heparan sulfate or its derivatives, etc.), FGFR agonists, and FGF2 signaling pathway activators can be suitably used as FGF signaling pathway activators.

[0039] The target FGFR may be any of FGFR1, FGFR2, FGFR3, FGFR4, or FGFR5, and is not particularly limited. For example, agonists of FGFR1(IIIb), FGFR1(IIIc), FGFR2(IIIc), and / or FGFR4 can be suitably used.

[0040] Examples of FGFR agonists include FGF family proteins or their analogues. Known FGF family proteins include FGF1 to FGF23, which contain basic FGF (bFGF, FGF2). FGF family proteins are classified into seven subfamilies: FGF1 subfamily (FGF1, FGF2), FGF4 subfamily (FGF4, FGF5, FGF6), FGF7 subfamily (FGF3, FGF7, FGF10, FGF22), FGF8 subfamily (FGF8, FGF17, FGF18), FGF9 subfamily (FGF9, FGF16, FGF20), FGF11 subfamily (FGF11, FGF12, FGF13, FGF14), and FGF19 subfamily (FGF19, FGF21, FGF23). In this specification, FGF encompasses proteins belonging to any of these subfamilies. For example, proteins belonging to the FGF1 subfamily, such as FGF2, can be suitably used in the present invention.

[0041] The FGF signaling pathway activator used may be derived from a specific organism, be a recombinant, or be artificially synthesized. If the FGF signaling pathway activator is derived from a specific organism, the species is not particularly limited, but for example, it may be derived from the same or closely related species as the species from which the target cells (e.g., STMs) of the inducer in this embodiment originate. For example, mammalian proteins can be suitably used, and in particular, proteins derived from primates such as humans can be used.

[0042] The final concentration used can be any effective amount and is not particularly limited. Examples of lower limits for the final concentration of FGF signaling pathway activators (e.g., proteins belonging to the FGF1 subfamily such as FGF2) include 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 6 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, etc., and examples of upper limits include 1000 ng / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 30 ng / mL, 25 ng / mL, 20 ng / mL, 15 ng / mL, 12 ng / mL, 10 ng / mL, etc.

[0043] <Antioxidants> In this specification, "antioxidant" refers to a drug that has antioxidant activity and can be used in the culture of animal cells. Antioxidants that can be used in the culture of animal cells are known in the art and are all included in the antioxidants described herein. The antioxidants described herein are preferably water-soluble antioxidants. Specific water-soluble antioxidants are not particularly limited, but examples include glutathione and its precursors, L-ascorbic acid and its derivatives, 2-mercaptoethanol or its substitutes (monothioglycerol), etc. Specific glutathione precursors include, but are not particularly limited, L-cysteine ​​or its derivatives (N-acetyl-L-cysteine, etc.), γ-glutamylcysteine ​​or its derivatives, etc. For example, L-ascorbic acid and its derivatives, L-cysteine ​​or its derivatives (N-acetyl-L-cysteine, etc.), or combinations thereof can be suitably used as antioxidants in this specification.

[0044] The final concentration used can be any effective amount and is not particularly limited. Examples of lower limits for the final concentration of glutathione derivatives (e.g., L-cysteine ​​such as N-acetyl-L-cysteine ​​or its derivatives) include 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 0.6 mM, 0.8 mM, 0.9 mM, 1 mM, etc., and examples of upper limits include 100 mM, 50 mM, 10 mM, 5 mM, 3 mM, 2.5 mM, 2 mM, 1.5 mM, 1.2 mM, 1 mM, etc. Examples of lower limits for the final concentration of L-ascorbic acid and its derivatives (L-ascorbic acid, etc.) include 0.001 mM, 0.005 mM, 0.01 mM, 0.05 mM, 0.09 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.3 mM, 0.35 mM, 0.4 mM, 0.45 mM, 0.5 mM, etc., and examples of upper limits include 100 mM, 75 mM, 50 mM, 25 mM, 20 mM, 10 mM, 5 mM, 1 mM, 0.9 mM, 0.8 mM, 0.7 mM, 0.6 mM, 0.5 mM, etc.

[0045] <Other Components> Any additional components may be included as other components. While not particularly limited, examples include culture medium additives, serum and / or serum substitutes in the third embodiment. For example, B27 TM Supplements and the like can be suitably included.

[0046] <Kit and Culture Medium> The qHSC inducer of this embodiment may be provided in the form of a qHSC inducer kit containing an FGF signaling pathway activator. In this case, the configuration of the kit is the same as described for the kit of the third embodiment.

[0047] The qHSC inducer according to this embodiment can be provided in the form of a qHSC inducer medium by being included in a culture medium. In this case, the basic contents of the culture medium are the same as those described for the culture medium in the third embodiment.

[0048] 1-4. Effects The qHSC inducer of this embodiment can induce differentiation from STM to qHSC. Typically, a homogeneous cell population can be obtained in which more than 90%, 90.5%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.3%, 99.5%, and 99.7% of the differentiated cells are HSCs. In this case, the proportion of HSCs can be determined based on the proportion of cells expressing HSC markers (CD73, ALCAM, CD71, etc.).

[0049] Furthermore, typically, a homogeneous population of qHSC cells can be obtained in which less than 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, and 0.1% of the cells after differentiation induction are PDGFRα-positive.

[0050] As described above, the qHSC inducer of this embodiment can induce qHSCs with high efficiency. Therefore, activated hepatic stellate cells can be obtained with high efficiency by inducing qHSCs from pluripotent stem cells, typically via STM, using this agent, and then activating the qHSCs by any method. The activation method in this case is not particularly limited, and for example, the method described in the fifth embodiment can be used.

[0051] 2. Promoting the proliferation of quiescent hepatic stellate cells 2-1. Overview The second aspect of the present invention is a promoting agent for the proliferation of quiescent hepatic stellate cells. The proliferation promoter of the present invention contains an FGF signaling pathway activator as an active ingredient and promotes the proliferation of hepatic stellate cells while maintaining an inactive state. The promoting agent for the proliferation of quiescent hepatic stellate cells of the present invention can be an active ingredient in a culture medium for the proliferation of quiescent hepatic stellate cells.

[0052] 2-2. Composition The growth promoter of this embodiment contains an FGF signaling pathway activator as an essential component, and optionally contains antioxidants, ROCK inhibitors, etc.

[0053] The growth promoter of this embodiment is applicable to qHSCs with proliferative capacity (such as fetal-like qHSCs). The description of proliferative-like qHSCs and fetal-like qHSCs is the same as that given in the first embodiment. For example, qHSCs differentiated by the inducer described in the first embodiment and the method described in the fourth embodiment, qHSCs produced by the method described in the fifth embodiment, etc., can be suitably used.

[0054] The FGF signaling pathway activators, antioxidants, and other components are described in accordance with the description in the first embodiment. ROCK inhibitors, etc., are described below.

[0055] <ROCK Inhibitors> In this specification, "ROCK inhibitor" refers to an inhibitor of the signaling pathway mediated by Rho-associated coiled-coil forming kinase (Rho). ROCK inhibitors in this specification include both drugs that directly inhibit the function of ROCK and drugs that do not directly inhibit the function of ROCK but inhibit its signaling pathway, and any ROCK inhibitor known in the art can be used.

[0056] There are no specific limitations on the type of ROCK inhibitor used. For example, either ROCK1 inhibitors or ROCK2 inhibitors can be used. Furthermore, ROCK1 inhibitors here do not need to selectively inhibit ROCK1; they broadly encompass any drug that exhibits inhibitory activity against ROCK1. Specifically, for example, thiazovibin, ripasudil, Y-39983 (4-[(1R)-1-aminoethyl]-N-1H-pyrrolo[2,3-b]pyridine-4-ylbenzamide dihydrochloride), AR-13324, Wf-536 ((+)-(R)-4-(1-aminoethyl)-N-(4-pyridyl)benzamide monohydrochloride), AZD-5363, K-155, AR-13503, etc., as well as 4-[(1R)-1-aminoethyl]-N-pyridine-4-ylcyclohexane-1-carboxamide or its salts (e.g., dihydrochloride), hydr Compounds capable of inhibiting ROCK1 include roxifasudil hydrochloride, RKI-1447, GSK-429286A, AT13148, GSK-269962A hydrochloride, BAY-549, and Chroman 1; and compounds capable of specifically inhibiting ROCK2 include Fasudil (1-(5-isoquinoline sulfonyl)homopiperazine) or its salt (e.g., dihydrochloride), H-1152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine) or its salt (e.g., dihydrochloride). Expression inhibitors that suppress the expression of ROCK and its downstream factors can also be used as ROCK inhibitors in this specification. Examples of expression inhibitors in this specification include antisense nucleic acids, RNA interference-inducible nucleic acids (e.g., siRNA), dominant-negative variants, and their expression vectors. For example, ROCK1 inhibitors, more specifically Y-27632 or its salts, can be suitably used as ROCK inhibitors.

[0057] Multiple types of ROCK inhibitors can be used in combination. The types of combinations are not particularly limited, but for example, multiple types of ROCK function inhibitors may be used in combination, or a ROCK function inhibitor may be used in combination with a ROCK expression suppressant.

[0058] The final concentration used can be any effective amount and is not particularly limited. Examples of lower limits for the final concentration of ROCK inhibitors (e.g., ROCK2 inhibitors such as Y-27632) include 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 6 μM, 8 μM, 9 μM, and 10 μM, while examples of upper limits include 1000 μM, 500 μM, 100 μM, 50 μM, 30 μM, 25 μM, 20 μM, 15 μM, 12 μM, and 10 μM.

[0059] ROCK inhibitors may be included for use throughout the entire culture period or for temporary use. If used temporarily, the timing and duration are not particularly limited.

[0060] For example, it may be used at the start of culture (including each subculturing) and / or immediately after a medium change, or on the second day or later. It can also be used for periods of time such as 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 12 hours or more, 15 hours or more, 16 hours or more, 18 hours or more, 20 hours or more, 24 hours or more, etc. It can also be used for periods of time such as 5 days or less, 4 days or less, 3 days or less, 2 days or less, 40 hours or less, 36 hours or less, 30 hours or less, 25 hours or less, 24 hours or less, etc. For example, a ROCK inhibitor may be added to the culture medium on the first day of culture, and the ROCK inhibitor may be removed from the culture medium from the second day onward.

[0061] The growth promoter of this embodiment may include any culture medium additives as described in relation to the culture medium of the third embodiment. Other components may include only one component or a combination of multiple components. When multiple components are combined, they may include multiple components of the same type or components of different types.

[0062] 2-3. Effects: According to the qHSC proliferation promoter of this embodiment, by adding it to a culture medium and using it for culturing qHSCs, qHSCs can be proliferated and cultured for a long period of time while maintaining an inactive state.

[0063] 3. Kits and Culture Mediums 3-1. Overview A third aspect of the present invention is a kit and a culture medium. The kit of the present invention comprises an FGF signaling pathway activator, and the culture medium of the present invention comprises an FGF signaling pathway activator and a serum-free medium. By using the kit of the present invention, differentiation and / or proliferation of qHSCs can be induced and / or promoted.

[0064] 3-2. Composition The kit and culture medium will be described below. <Kit> The kit of the present invention includes an FGF signaling pathway activator as an essential component, and optionally includes one or more additional components. The additional components are not particularly limited as long as they can be used for culturing qHSC, but specifically, examples include antioxidants, serum-free media, media additives, coating agents, and other components. Antioxidants and other components are as described in the first embodiment. Serum-free media and media additives are as described later in relation to the culture medium.

[0065] The coating agent is not particularly limited. Examples include laminin-1 to 12 (including iMatrix-511, etc.), COL1A1, Matrigel, collagen, poly-L-lysine, Gelforce, fibronectin, vitronectin, Geltrex, etc.

[0066] The kit according to this embodiment can be provided as a kit for inducing qHSC, for culturing qHSC, for maintaining qHSC, and / or for producing HSCs (qHSC and / or aHSC).

[0067] When used for the proliferation of qHSCs (for example, when provided for qHSC proliferation, qHSC maintenance culture, or HSC production), the kit of this embodiment may contain a ROCK inhibitor. The ROCK inhibitor is as described in the second embodiment.

[0068] When provided as a manufacturing kit for aHSC, it may include an HSC activator. The HSC activator is as described in the fifth embodiment.

[0069] The kit of the present invention may include a container. The material of the container is not limited as long as it is made of a material that does not contaminate the contents or is not contaminated by the contents. Examples include plastics such as polypropylene and polystyrene, glass, or paper with a special surface coating.

[0070] In the kit of the present invention, each active ingredient can be contained in a container. In the kit of the present invention, each active ingredient can be contained together in one container or divided into multiple containers. When contained in multiple containers, the containers may be separated by ingredient, by other criteria (such as the amount used per dose), or by a combination of these.

[0071] The kit of the present invention may include, for example, means for adding each component to the culture medium (dropper, micropipette, syringe, injection needle, sprayer, etc.). The kit of the present invention may also include instructions for use, if necessary.

[0072] <Culture Medium> The culture medium of the present invention comprises an FGF signaling pathway activator and a culture medium as essential components, and optionally includes one or more additional components. The additional components are not particularly limited as long as they are components that can be used for culturing qHSC, but specifically, examples include antioxidants, culture medium additives and other components. Antioxidants and other components are as described in the first embodiment.

[0073] The culture medium according to this embodiment can be provided as a culture medium for inducing qHSC, for culturing qHSC, for maintaining qHSC, and / or for producing HSCs (qHSC and / or aHSC).

[0074] When used for the proliferation of qHSCs (for example, when provided for qHSC proliferation, qHSC maintenance culture, or HSC production), the culture medium of this embodiment may contain a ROCK inhibitor. The ROCK inhibitor is as described in the second embodiment.

[0075] When provided as a culture medium for the production of aHSC, it may contain an HSC activator. The HSC activator is as described in the fifth embodiment.

[0076] <Type of Culture Medium> The type of basal culture medium used in the culture medium of this embodiment is not particularly limited. For example, any vertebrate cell culture medium, such as a mammalian cell culture medium known in the art, can be used alone or in combination, with culture medium additives added as needed.

[0077] The basal medium used in the culture medium of this embodiment is not particularly limited. For example, Minimum Essential Medium Eagle (MEM) and any modified version thereof can be used. For example, specific mammalian cell culture media include Iscove's Modified Dulbecco's Medium (IMDM), Ham's F-12 medium or its modified form (e.g., Ham's F-12 K medium), Dulbecco's Modified Eagle's Medium (DMEM), Advanced DMEM, Minimum Essential Medium Eagle, Alpha Modification (α-MEM), Improved Minimum Essential Medium, Glasgow Minimum Essential Medium (GMEM), William's E medium, Connaught Medical Research Laboratories Medium 1066, McCoy's 5A medium, RPMI 1640 medium or combinations thereof (e.g., mixed medium of IMDM and Ham's F-12 medium, DMEM / F-12 medium).

[0078] The volume ratio when combining multiple media is not particularly limited. For example, two or more media may be mixed in equal volumes, or one medium may be mixed in a larger amount than others. When the volumes of the mixed media differ, the content and mixing ratio of the main medium (the medium with the largest volume) and other media are not particularly limited. For example, the main medium may be 1 / 4 or more, 1 / 3 or more, 1 / 2 or more, 2 / 3 or more, or 3 / 4 or more of the total. Also, for example, the main medium may be mixed in a ratio of 1 or more, 1.5 or more, 2 or more, 2.5 or more, or 3 or more times the volume of the other media. The type of main medium is not particularly limited. For example, Iskov-modified Dulbecco's medium can be suitably used as the main medium. Specifically, for example, when Ham's F-12 medium is used as the other medium, Ham's F-12 medium may be included in an amount of 3 / 4 or less, 2 / 3 or less, 1 / 2 or less, or 1 / 3 or less of Iskov-modified Dulbecco's medium. Furthermore, for example, Ham's F-12 medium can be included in amounts of 1 / 10, 1 / 8, 1 / 6, 1 / 5, 1 / 4, or 1 / 3 or more of Iskov-modified Dulbecco's medium.

[0079] The basal culture medium used preferably contains an organic buffer and inorganic salts such as sodium selenite (Na2SeO3) and potassium nitrate (KNO3). When using multiple culture media in combination, it is sufficient that these components are present in the combined medium. For example, a medium combining IMDM, which contains the organic buffer HEPES and inorganic salts such as sodium selenite and potassium nitrate, with Ham's F-12 or a modified medium thereof (e.g., Ham's F-12 K medium) that does not contain these components can be suitably used. Furthermore, if the above components are not present or are insufficient in the mixed medium, they can be added externally.

[0080] Any organic compound having pH buffering properties can be used as the organic buffer. Suitable organic buffers include, for example, Good's buffers, specifically HEPES, MOPS, TES, Bis-Tris, PIPES, MES, etc., but are not particularly limited. Any inorganic buffer, such as sodium bicarbonate, may be included as needed.

[0081] The culture medium used may be either a serum-containing medium or a serum-free medium. When using a serum-free medium, the entire medium in this embodiment may be a serum-free medium, or serum may be added to make it a serum-containing medium.

[0082] If serum or serum substitutes are included, the type is not particularly limited. For example, any serum or serum substitute known in the art may be included. Specific examples of serum include, but are not particularly limited, fetal bovine serum (FBS), human serum, sheep serum, or mixtures thereof. Specific examples of serum substitutes include, for example, Knockout. TM serum replacement (KSR), XF212 XerumFree, CDM-HD serum replacement, StemSure serum replacement, Nu-Serum TM These are some examples, but are not limited to any particular category.

[0083] There are no particular limitations on the amount of serum or serum substitutes included. For example, it may contain 0.1(v / v)% or more, 0.5(v / v)% or more, 1(v / v)% or more, 2(v / v)% or more, 3(v / v)% or more, 4(v / v)% or more, or 5(v / v)% or more. Alternatively, it may contain, for example, 20(v / v)% or less, 15(v / v)% or less, 10(v / v)% or less, 8(v / v)% or less, 7(v / v)% or less, 6(v / v)% or less, or 5(v / v)% or less.

[0084] <Culture medium additives> The culture medium of this embodiment may include additional culture medium additives. For example, it may include other components described in the first embodiment. Multiple types of components may be included in combination, even if they have similar functions.

[0085] In addition to the other components described in the first embodiment, the culture medium of this embodiment may further contain culture medium additives such as vitamins, albumin, amino acids, and antibiotics.

[0086] Examples of vitamins include choline chloride, pantothenic acid, folic acid, nicotinamide, pyridoxal hydrochloride, pyridoxine, riboflavin, thiamine, vitamin B12, para-aminobenzoic acid (PABA), thiamine hydrochloride, ascorbic acid, biotin, inositol, ergocalciferol (vitamin D2), vitamin A, retinoic acid, α-tocopherol, niacin, their derivatives, and combinations thereof.

[0087] The concentration to be added is not particularly limited. For example, it is acceptable if it is 0.01 mM or higher, 0.05 mM or higher, 0.1 mM or higher, 0.2 mM or higher, 0.4 mM or higher, or 0.5 mM or higher. For example, it can be added at concentrations such as 0.01 mM to 10 mM, 0.05 mM to 10 mM, 0.1 mM to 10 mM, 0.1 mM to 5 mM, 0.1 mM to 3 mM, 0.1 mM to 1 mM, 0.4 mM to 3 mM, 0.4 mM to 1 mM, or 0.5 mM to 1 mM.

[0088] Albumin can be used from any species, or its variants, and is not particularly limited. Specifically, examples include bovine serum albumin (BSA), human serum albumin, or combinations thereof.

[0089] The concentration to be added is not particularly limited; for example, it is acceptable to use the concentration recommended by the manufacturer. Specifically, concentrations of 0.01(w / v)% or higher, 0.02(w / v)% or higher, 0.04(w / v)% or higher, or 0.05(w / v)% or higher are acceptable, and it can be added at concentrations such as 0.01(w / v)% to 0.5(w / v)%, 0.01(w / v)% to 0.3(w / v)%, 0.05(w / v)% to 0.2(w / v)%, 0.05(w / v)% to 0.1(w / v)%, etc.

[0090] Examples of antibiotics include, but are not limited to, penicillin, streptomycin, penicillin-streptomycin, sulfonamides, pheneticillin, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, metacycline, and minocycline.

[0091] There are no particular restrictions on the amount of antibiotic to be added; for example, it is acceptable to use it at the concentration recommended by the manufacturer. Specific concentrations that are acceptable include, for example, 0.1(v / v)% or higher, 0.5(v / v)% or higher, 0.7(v / v)% or higher, or 1(v / v)% or higher.

[0092] <Other Culture Medium Additives> Other culture medium additives may be included as needed. The type of culture medium additive in this case is not particularly limited, but any additive known in the art may be used. For example, B27 TM In addition to supplements and N2 supplements, the ingredients include sugars (monosaccharides such as glucose, galactose, mannose, and fructose; disaccharides such as sucrose, maltose, and lactose; water-soluble or water-insoluble polysaccharides such as hyaluronic acid, gellan gum, chitin, and chitosan), inorganic salts (calcium chloride, calcium nitrate, copper sulfate pentahydrate, iron(III) nitrate notahydrate, iron(II) sulfate heptahydrate, magnesium chloride hexahydrate, manganese chloride tetrahydrate, magnesium sulfate, potassium chloride, sodium chloride, etc.), stabilizers (e.g., polyethylene glycol), and buffering agents. It may contain (e.g., phosphate buffer, sodium acetate buffer), chelating agents (e.g., EDTA, EGTA, citrate, salicylate), amino acids (e.g., glutamine, alanine, asparagine, serine, aspartic acid, cysteine, glutamic acid, glycine, proline, tyrosine, niacin, and other non-essential amino acids), nucleotides (ATP, UTP, GTP, CTP, or mixtures thereof), antifungal agents (e.g., amphotericin), solubilizers (e.g., dimethyl sulfoxide), preservatives, anti-inflammatory agents (e.g., dexamethasone), etc.

[0093] 4. Method for Inducing Quiescent Hepatic Stem Cells 4-1. Overview The fourth aspect of the present invention is a method for inducing quiescent hepatic stellate cells. The method of this aspect includes a proliferation culture step as an essential step and a pluripotent stem cell preparation step as an optional step. According to the method of this aspect, quiescent hepatic stellate cells with proliferative capacity can be induced.

[0094] 4-2. Process 4-2-1. Pluripotent Stem Cell Preparation Process The pluripotent stem cell preparation process is an optional process of the method of this embodiment, and is a process of preparing pluripotent stem cells from cells isolated from an individual or tissue.

[0095] The method for preparing pluripotent stem cells used in this process can be appropriately selected depending on the type of stem cells used, and is not particularly limited.

[0096] Pluripotent stem cells other than iPS cells are typically prepared by culturing cells isolated from an individual or tissue under culture conditions that maintain their pluripotency.

[0097] For example, numerous methods for preparing iPS cells have been studied, and any of these methods can be used. A typical outline of an iPS cell induction method is described below.

[0098] iPS cells are induced by introducing specific reprogramming factors (DNA or proteins) into somatic cells (including skin cells, bone marrow cells, gastrointestinal cells, hepatocytes, somatic stem cells, etc.).

[0099] Examples of reprogramming factors include Oct family genes (Oct3 / 4, etc.), Klf family genes (Klf4, Klf2, etc.), Sox family genes (Sox1, Sox2, Sox3, Sox15, Sox17, etc.), Myc family genes (c-Myc (including T58A variant), N-Myc, L-Myc, etc.), Nanog family (Nanog, etc.), Lin family genes (Lin28, Lin28b, etc.), and other known genes (Fbx15, ERas, ECAT15-2, Tcl1, β-catenin, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, p53shRNA, Glis1, etc.). Specific combinations of reprogramming factors include, for example, combinations consisting of Oct3 / 4, Sox2, Klf4, and c-Myc; combinations consisting of Oct3 / 4, Sox2, and Klf4; combinations consisting of Oct4, Sox2, Nanog, and Lin28; or combinations consisting of Oct3 / 4, Sox2, Klf4, c-Myc, Nanog, and Lin28.

[0100] The method for culturing pluripotent stem cells is not limited to any known method. For example, they can be cultured in a culture vessel coated with an extracellular matrix together with feeder cells capable of producing factors necessary for maintaining pluripotency and self-renewal.

[0101] Whether or not coating is necessary can be appropriately determined depending on the culture vessel and culture method used, and the cells may be cultured without coating. As an extracellular matrix for coating, for example, the molecules described later in relation to the growth culture step of this embodiment can be used.

[0102] Culture methods include, for example, adherent culture or suspension culture, which can be appropriately selected depending on the purpose. For example, when forming spheroids, suspension culture can be used. Culture may be static culture or a culture method that applies any mechanical stimulation (rotation culture, swirling culture, etc.).

[0103] The culture medium used is not particularly limited. For example, the culture medium exemplified in the third embodiment can be used. In this process, a culture medium provided as being particularly suitable for pluripotent stem cells may be used. For example, as a culture medium for ES cells and iPS cells, mTeSR1 medium, TeSR1 medium (Stem Cell Technologies), Essential 8 TM Culture medium, Essential 6 TM Culture medium (Gibco), StemPro (R) -34 SFM (Life Technologies), StemFlex TM Products such as the Medium (Gibco) and StemFit AK02N (Ajinomoto) are commercially available.

[0104] The necessity of feeder cells can be appropriately selected depending on the culture medium and additives used. For example, feeder cells are not necessary when using feeder-free medium. Specific examples of feeder cells include C3H10T1 / 2 cells, OP9 cells, NIH3T3 cells, ST2 cells, PA6 cells, mouse embryonic fibroblasts (MEF cells), and SL10 cells.

[0105] Although other culture conditions are not particularly limited, for example, culturing can be performed under the culture conditions and subculture conditions described in the proliferation culture step of this aspect.

[0106] Any additional optional treatment can be performed during culturing. Particularly when using feeder cells, a treatment for suppressing cell growth (such as mitomycin C treatment, radiation irradiation, etc.) may be performed.

[0107] 4-2-2. Lateral mesenchymal stromal cell induction step The lateral mesenchymal stromal cell induction step is an optional step of the method of this aspect and is a step for inducing differentiation of pluripotent stem cells into lateral mesenchymal stromal cells. When the pluripotent stem cell preparation step is performed, this step can be performed thereafter.

[0108] The specific method of differentiation induction in this step is not particularly limited. For example, differentiation induction can be achieved by introducing an STM-specific transcription factor or the like. Also, for example, pluripotent stem cells can be induced to differentiate into mesodermal cells, and the mesodermal cells can be induced to differentiate into STM.

[0109] The induction of mesodermal cells from pluripotent stem cells and the induction of STM from mesodermal cells can be performed using any known method. For example, it can be performed by adding an appropriate cytokine to the medium.

[0110] The medium used in this case is not particularly limited. For example, a medium obtained by adding a medium additive to a basal medium (such as DMEM / F12 medium) as exemplified in the third aspect can be used. The medium additive used in this case is not particularly limited, but for example, it can contain the medium additives exemplified in the third aspect (amino acids such as L-glutamine and its substitutes, B27 TM supplement, etc.) and other components exemplified in the first aspect.

[0111] The cytokine to be used may be any cytokine that can promote differentiation into the target cells and is not particularly limited.

[0112] The pluripotent stem cells used in this process may be prepared or obtained, and there are no particular limitations on their type. For example, iPS cells or ES cells can be obtained from the RIKEN BioResource Center Cell Materials Development Laboratory (RIKEN BRC CELL BANK), ATCC (American Type Culture Collection), iPS Academia Japan Co., Ltd., etc. Generally available cell lines include, but are not limited to, WA01(H1), WA09(H9), KhES-1, KhES-2, and KhES-3 for human ES cells, and M48, QHJI, 201B7, 253G1, 409B2, 454E2, 585A1, 606A1, 610B1, 648A1, 1201C1, 1231A3, 1383D2, 1383D6, HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 for human iPS cells.

[0113] Pluripotent stem cells may be used after long-term storage. For example, they can be used after being thawed from cryopreservation. In this case, there are no particular limitations on the storage temperature and period. For example, they can be stored at temperatures below 0°C, below -20°C, below -30°C, below -40°C, below -50°C, below -79°C, below -80°C, below -100°C, below -130°C, below -150°C, below -180°C, and below -196°C. The temperature may remain constant throughout the storage period, change naturally, or be artificially altered. There are no particular limitations on the storage period, but for example, it could be 1 day or more, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 30 days or more, 1 month or more, 2 months or more, 3 months or more, etc.

[0114] <Induction of Mesodermal Cells> For example, in the case of induction of mesodermal cells, Wnt signaling pathway activators, TGFβ family proteins, or combinations thereof can be used.

[0115] The Wnt signaling pathway activator may be a classical Wnt signaling pathway activator, a non-classical Wnt signaling pathway activator, or a mixture thereof, and is not particularly limited.

[0116] In this specification, "non-classical Wnt signaling pathway activator" refers to an inhibitor of a signaling pathway based on Wnt binding to receptors such as Frizzled, which is capable of inhibiting the non-classical Wnt signaling pathway. In this case, Wnt receptors include Frizzled, as well as Celsr, Vangel, and others. In particular, non-classical Wnt signaling pathway activators in this specification do not include classical Wnt signaling pathway-specific activators such as GSK3β inhibitors.

[0117] Any agent known in the art can be used as a non-classical Wnt signaling pathway activator. The type of non-classical Wnt signaling pathway activator used is not particularly limited, but examples include planar cell polarity (PCP) pathway activators and Wnt / Ca 2+ Examples include pathway activators, activators of Wnt binding to receptors, and promoters of the expression of factors in these pathways. For example, Wnt receptor agonists and Wnt3a signaling pathway activators can be suitably used as non-classical Wnt signaling pathway activators.

[0118] Specific non-classical Wnt signaling pathway activators are not particularly limited, but examples include Wnt (WNT3A, etc.) and its substitutes, Notum pectin acetylesterase inhibitors (LP-922056, Notum pectin acetylesterase-1), Notum inhibitors (ARUK3001185, 8BTC, etc.), JNK activators (R-spondin 1), Wnt expression promoters (Wnt expression vectors, γ-Glutamylvaline, etc.), or combinations thereof. Wnt or its analogues can be suitably used as non-classical Wnt signaling pathway activators, and in such cases, they may be derived from a specific organism (e.g., human) or recombinants.

[0119] In this specification, "classical Wnt signaling pathway activator" refers to a drug that activates the classical Wnt signaling pathway, but in particular does not activate the non-classical Wnt signaling pathway.

[0120] The specific type of classical Wnt signaling pathway activator is not particularly limited. For example, any classical Wnt signaling pathway activator known in the art can be used. Examples include GSK3β inhibitors, β-catenin stabilizers (Axin-LRP6 complex formation promoters (Lycorine and its derivatives, etc.), Axin-β-catenin complex formation promoters (SKL2001, etc.), etc.), phosphodiesterase 5 inhibitors (tadalafil, vardenafil, mirodenafil and its derivatives, etc.), inhibitors of the expression of one or more factors of the classical Wnt signaling pathway, other drugs (BML-284, SM-04554, Dalosirvat, methyl vanillate, etc.) or combinations thereof.

[0121] Specific GSK3β inhibitors include, but are not limited to, CHIR99021 (Laduviglusib) and its hydrochloride, CHIR98014, Tideglusib, 6-bromoindilbine-3-oxime (BIO), 6-bromoindilbine-3-acetoxime (BIOacetoxime), SB415286, SB216763, TWS119, Tideglusib, A1070722, LY2090314, AZD1080, 1-Azakenpaullone, AR-A014418, IM-12, Indirubin, small molecule inhibitors of TDZD-8, GSK3β expression suppressors and combinations thereof. For example, CHIR99021 and its hydrochloride can be suitably used.

[0122] The final concentration used should be an effective amount and is not particularly limited. Examples of lower limits for the concentration of Wnt signaling pathway activators (e.g., GSK3β inhibitors such as CHIR99021) include 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 7.5 μM, and 8 μM, while examples of upper limits include 50 μM, 25 μM, 20 μM, 15 μM, 10 μM, 9 μM, and 8 μM.

[0123] The TGFβ family proteins are basically described in the activation step of the fifth embodiment. While there are no specific limitations on the TGFβ family proteins, BMP family proteins such as BMP4 can be suitably used.

[0124] The final concentration used can be any effective amount, and is not particularly limited. Examples of lower limits for the final concentration of BMP family proteins (such as BMP4) include 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 15 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, and 25 ng / mL, while examples of upper limits include 1000 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 90 ng / mL, 70 ng / mL, 50 ng / mL, 40 ng / mL, 30 ng / mL, 29 ng / mL, 28 ng / mL, 27 ng / mL, 26 ng / mL, 25 ng / mL, 20 ng / mL, 19 ng / mL, 17 ng / mL, 15 ng / mL, 14 ng / mL, 13 ng / mL, and 12 ng / mL.

[0125] There is no particular limit to the cell density, but for example, 10^2 cells / cm³ 2 ~10^6 cells / cm 2 , 10^2 cells / cm 2 ~10^5 cells / cm 2 , 10^2 cells / cm 2 ~10^4 cells / cm 2 The density and other factors can be cited.

[0126] The culture period is not particularly limited, but for example, it can be cultured for 1 to 15 days, 2 to 15 days, 3 to 15 days, 1 to 10 days, 2 to 10 days, 3 to 10 days, 1 to 8 days, 2 to 8 days, 3 to 8 days, 1 to 6 days, 2 to 6 days, or 3 to 6 days.

[0127] <Induction of STM> The cytokines used to induce STM are not particularly limited. For example, tyrosine kinase receptor activators, TGFβ family proteins, FGF signaling pathway activators, or combinations thereof can be used.

[0128] STM induction can be carried out in multiple steps, for example. In this case, the types of cytokines used may differ or be partially the same in each step. For example, if STM induction is carried out in a two-step induction reaction, primary induction can be carried out using, for example, a tyrosine kinase receptor activator and a TGFβ family protein, and secondary induction can be carried out using, for example, a TGFβ family protein and an FGF signaling pathway activator.

[0129] The tyrosine kinase receptor activator and TGFβ family protein used in STM induction are not particularly limited, but are similar to those described in the activation step of the fifth embodiment. For example, as the tyrosine kinase receptor activator, cysteine ​​not cytokine superfamily proteins such as PDGF (e.g., PDGF-BB) can be used. Also, as TGFβ family proteins, for example, activin (activin A, etc.) and BMP family proteins (BMP4, etc.) can be used. The final concentration of BMP family protein (BMP4, etc.) is not particularly limited, but can be the concentration described above in relation to the induction of mesodermal cells, for example.

[0130] The final concentration of cysteine-not cytokine superfamily proteins (PDGF-BB, etc.) is not particularly limited, but possible lower limits include, for example, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, etc., and possible upper limits include, for example, 1000 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 90 ng / mL, 70 ng / mL, 50 ng / mL, 40 ng / mL, 30 ng / mL, 25 ng / mL, 20 ng / mL, 15 ng / mL, 14 ng / mL, 13 ng / mL, 12 ng / mL, 11 ng / mL, 10 ng / mL, etc.

[0131] The final concentration of activin (activin A, etc.) is not particularly limited, but examples of lower limits include 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 1.5 ng / mL, 1.6 ng / mL, 1.7 ng / mL, 1.8 ng / mL, 1.9 ng / mL, 2 ng / mL, etc., and examples of upper limits include 100 ng / mL, 50 ng / mL, 25 ng / mL, 10 ng / mL, 9 ng / mL, 7 ng / mL, 5 ng / mL, 4 ng / mL, 3 ng / mL, 2.5 ng / mL, 2.4 ng / mL, 2.3 ng / mL, 2.2 ng / mL, 2.1 ng / mL, 2 ng / mL, etc.

[0132] The type and concentration of FGF signaling pathway activators (such as FGF2) used to induce STM shall be in accordance with the description in the first embodiment.

[0133] The culture period for STM induction is not particularly limited, but it can be carried out over the period exemplified in the quiescent hepatic stellate cell induction process, for example. If STM induction is performed in multiple stages, the induction cultures for each stage may be the same or different from each other. For example, if the total duration of STM induction culture is 4 days, the primary culture may be 2 days and the secondary culture 2 days, or the primary culture may be 1 day and the secondary culture 3 days, or vice versa.

[0134] Whether differentiation to STMs has been induced can be determined based on whether or not the STMs possess any of the properties of STMs and / or whether or not the properties of pluripotent stem cells have been lost. For example, this can be determined by the expression of STM markers and / or pluripotent stem cell markers. Any markers known in the art can be used as STM markers and pluripotent stem cell markers, and are not particularly limited. Specific STM markers include, for example, HLX, WT1, BMP2, BMP4, FOXF1, GATA4, MRG1, HGF, TBX18, etc. Specific pluripotent stem cell markers include, for example, OCT4, NANOG, SOX2, CD9, DNMT3B, GABRB3, GAL, GDF3, IFITM1, PODXL, TDGF1, ZFP42, etc.

[0135] 4-2-3. Quiescent Hepatic Stellary Cell Induction Step The quiescent hepatic stellate cell induction step is an essential step of the method according to this embodiment. It involves inducing the culture of transverse septal mesenchymal cells in quiescent hepatic stellate cell induction medium and inducing their differentiation into quiescent hepatic stellate cells. If the transverse septal mesenchymal cell induction step is to be performed, it can be carried out afterward.

[0136] The quiescent hepatic stellate cell induction medium is as described in the third embodiment. The culture vessel may be coated as needed. The coating agent in this case is not particularly limited. For example, the coating agents exemplified in the third embodiment can be used. For example, laminin (iMatrix-511, etc.), COL1A1, Matrigel, or a combination thereof can be suitably used.

[0137] There is no particular limit to the cell density, but for example, 10^2 cells / cm³ 2 ~10^6 cells / cm 2 , 10^3 cells / cm 2 ~10^5 cells / cm 2 , 10^3 cells / cm 2 ~5×10^4 cells / cm 2 The density and other factors can be cited.

[0138] In this process, cultivation is carried out under appropriate temperature, humidity, and CO2 concentration. While there are no particular limitations on the appropriate temperature, it is usually within the range of approximately 30-40°C, for example, 37°C. Humidity is usually within the range of approximately 70-100%, for example, 95-100%. CO2 concentration is usually within the range of approximately 1-10%, for example, 5%. Furthermore, the O2 concentration is not particularly limited, and cultivation can be carried out at normal oxygen concentrations (18-22%) or low oxygen concentrations (0-10%).

[0139] Furthermore, the oxygen concentration in this step is not particularly limited. For example, it may be a low oxygen concentration, a normal oxygen concentration, or a combination thereof.

[0140] The culture period is not particularly limited and can be extended until the cells reach the desired number or confluence. For example, the cells can be cultured for 1 to 15 days, 2 to 15 days, 3 to 15 days, 4 to 15 days, 2 to 10 days, 3 to 10 days, 4 to 10 days, 2 to 8 days, 3 to 8 days, 4 to 8 days, 2 to 6 days, 3 to 6 days, or 4 to 6 days.

[0141] The culture medium can be changed as needed. There are no particular restrictions on the frequency of culture medium changes. For example, it can be done regularly or irregularly. If done regularly, for example, it can be done once every 1 to 5 days, once every 1 to 4 days, once every 1 to 3 days, once every 1 to 2 days, or once every 2 days. If done irregularly, the timing of the culture medium change can be determined based on, for example, changes in pH or turbidity of the medium. The composition of the culture medium may be the same as or different from the medium used before the change.

[0142] Differentiation into qHSC can be determined based on whether or not qHSC possesses any of the properties of STM and / or whether or not the properties of STM have been lost. For example, this can be determined by the expression of HSC markers (qHSC markers, aHSC markers, etc.) and / or STM markers. Any markers known in the art can be used as HSC markers and aHSC markers, and are not particularly limited. Specifically, for example, HSC markers include ALCAM, CD71, CD73, PCDH7, LOX, PDGFRβ, desmin, etc. For example, qHSC markers include PCDH7, RGS5, FABP5, FABP4, BAMBI, etc. Also, for example, aHSC markers include PDGFRα, COL1A1, αSMA, vimentin, fibronectin, IL-6, TGF-β, collagen I, etc. As an STM marker, for example, any of the above-mentioned STM markers can be used.

[0143] The induced qHSCs typically express the HSC marker and the qHSC marker, but do not express the STM marker and the aHSC marker. Furthermore, the induced qHSCs are proliferative qHSCs, such as fetal-like qHSCs. The description of proliferative qHSCs and fetal-like qHSCs is the same as that given in the first embodiment.

[0144] 4-3. Effects According to the method of this embodiment, qHSCs that are in an inactive state and have proliferative capacity can be induced from STM or pluripotent stem cells. These qHSCs can proliferate while maintaining an inactive state, i.e., a normal state. The proportion of qHSCs after differentiation induction in this step is the same as described in the effects of the first embodiment. The induced qHSCs can be used for the production of aHSCs or liver organoids.

[0145] 5. Method for Producing Hepatic Stem Cells 5-1. Overview The fifth aspect of the present invention is a method for producing hepatic stellate cells (quiescent hepatic stellate cells or activated hepatic stellate cells). The method of this aspect includes a quiescent hepatic stellate cell induction step, and in the case of a method for producing activated hepatic stellate cells, an activation step as essential steps, and includes a pluripotent stem cell preparation step, a transverse septal mesenchymal cell induction step, and a proliferation culture step as optional steps. According to the method of this aspect, it is possible to produce quiescent hepatic stellate cells with proliferative capacity, or a large amount of activated hepatic stellate cells based on the proliferation of quiescent hepatic stellate cells.

[0146] 5-2. Process 5-2-1. Pluripotent Stem Cell Preparation Process The pluripotent stem cell preparation process is an optional process of the method of this embodiment, and is a process of preparing pluripotent stem cells from cells isolated from an individual or tissue. This process can be carried out in accordance with the description of the pluripotent stem cell preparation process of the fourth embodiment.

[0147] 5-2-2. Transverse Mesenchymal Cell Induction Step The transverse mesenchymal cell induction step is an optional step of the method according to this embodiment, and is a step of differentiating pluripotent stem cells into transverse mesenchymal cells. If the pluripotent stem cell preparation step is performed, this step can be performed afterward.

[0148] This process can be carried out in accordance with the description of the transverse septal mesenchymal cell induction process in the fourth embodiment.

[0149] 5-2-3. Quiescent Hepatic Stellary Cell Induction Step The quiescent hepatic stellate cell induction step is an essential step of the method of this embodiment, and is a step of inducing the differentiation of transverse septal mesenchymal cells into quiescent hepatic stellate cells by inducing their culture in quiescent hepatic stellate cell induction medium. If the transverse septal mesenchymal cell induction step is to be performed, it can be performed afterward. This step can be performed in accordance with the description of the quiescent hepatic stellate cell induction step of the fourth embodiment.

[0150] 5-2-4. Proliferation and Culture Step The proliferation and culture step is an optional step of the method according to this embodiment, and is a step of growing and culturing quiescent hepatic stellate cells in a medium for proliferation of quiescent hepatic stellate cells. It can be performed after the quiescent hepatic stellate cell induction step.

[0151] Multiple types of qHSCs may be used in this process, for example, qHSCs differentiated separately, or qHSCs induced in the quiescent hepatic stellate cell induction step may be combined with proliferative qHSCs prepared by a different method.

[0152] In this process, in addition to the essential growth culture step, a subculturing step can be performed as needed.

[0153] <Culturing Step> In this step, qHSCs are cultured in a qHSC culture medium. The qHSC culture medium used in this step is described in detail in the third aspect, so a detailed explanation is omitted here.

[0154] Any culture method known in the art can be used as the culture method, and is not particularly limited. For example, typically, qHSCs seeded at any cell density on a culture plate are cultured in qHSC growth medium under appropriate temperature, humidity, and CO2 concentration.

[0155] There is no particular limit to the cell density, but for example, 10^2 cells / cm³ 2 ~10^6 cells / cm 2 , 10^3 cells / cm 2 ~10^5 cells / cm 2 , 10^5 cells / cm 2 The density and other factors can be cited.

[0156] The plate can be coated on its surface as needed. The coating agent is not particularly limited. For example, coating agents exemplified in the third embodiment, such as iMatrix-511, can be used.

[0157] The temperature, humidity, and CO2 concentration used in this process are not particularly limited, but can be carried out under the conditions exemplified in the resting hepatic stellate cell induction process of the fourth embodiment.

[0158] The culture period is not particularly limited and can be extended until the cells proliferate to the desired number or confluence. For example, it can be carried out over the period exemplified in the quiescent hepatic stellate cell induction step of the fourth embodiment.

[0159] Furthermore, the culture medium can be changed as needed. The frequency of culture medium changes is not particularly limited. For example, it can be changed at the frequency and in the manner exemplified in the quiescent hepatic stellate cell induction step of the fourth embodiment. The composition of the culture medium may be the same as or different from the medium used before the change. For example, a ROCK inhibitor may be added to the culture medium on day 1, and the ROCK inhibitor may be removed from the culture medium from day 2 onwards.

[0160] <Subculture Step> This step is optional and involves subculturing the qHSCs in culture. This step can be performed after the growth culture step.

[0161] The timing of this step can be determined appropriately depending on the culture conditions and is not particularly limited. For example, this step can be performed when the cell confluence reaches a certain level. There is no particular limit to the specific confluence at which subculturing can be performed. Specifically, subculturing can be performed when the confluence is, for example, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 100%.

[0162] The specific method of subculturing is not particularly limited. Typically, cells are detached from the culture dish, dispersed in the culture medium, and re-seed in a new dish. Cell detachment and dispersion may be performed by mechanical stimulation such as pipetting, or by chemical stimulation such as the action of enzymes such as trypsin. Specific detachment agents include, for example, EDTA (e.g., 1-10 mM EDTA) and TrypLE. TM Select, TrypLE TM Express Enzyme, Accutase TM Collagenase, dispase, trypsin, trypsin / EDTA, trypsin / collagenase, ReLeSR TM Other examples include combinations thereof, but are not particularly limited.

[0163] When detachment and / or dispersion is performed by chemical stimulation, any basal culture medium can be used as the solvent for the treatment solution. If the solvent of the treatment solution and / or reaction stop solution is different from the basal culture medium used for subculturing, the cells may be washed with the basal culture medium (or the main medium in the case of a mixed medium) used for subculturing after the detachment and / or dispersion treatment or reaction stop treatment.

[0164] The cell density during seeding, dish coating, etc., can be carried out in accordance with the description of the growth culture step. The composition of the culture medium may be the same as or different from the medium used before subculturing, but preferably the composition of the culture medium before and after subculturing is the same.

[0165] The culture conditions after subculturing can be carried out in accordance with those described in the growth culture step. The conditions at the time of seeding and / or the culture conditions after subculturing may be the same as or different from those used in the growth culture step.

[0166] This step can be performed multiple times. The conditions used in each subculturing step may be the same or different. There is no particular limit to the number of subculturing steps, but for example, it may be subculturred one or more times, two or more times, three or more times, four or more times, five or more times, or six or more times.

[0167] This process can be used as a method for propagating qHSCs by performing it alone. The qHSCs used in this case can be any qHSCs that have the ability to proliferate, and are not particularly limited. Examples include fetal-like qHSCs and qHSCs induced by the method described in the fourth embodiment.

[0168] 5-2-5. Activation Step The activation step is a step in which the induced quiescent hepatic stellate cells are activated and cultured. This step is optional, but in the case of aHSC production methods, this step is mandatory. This step can be performed after the quiescent hepatic stellate cell induction step, and if a proliferation step is performed, it can be performed simultaneously with or after it.

[0169] The conditions for activation culture in this process are not particularly limited, as long as they allow for activation from qHSC to aHSC. Typically, activation culture can be performed by culturing in a culture medium to which an activator has been added.

[0170] The activators used can be any factors that contribute to the activation of qHSCs to aHSCs, and are not particularly limited. Examples include Hippo signaling pathway activators, tyrosine kinase receptor activators, and serine / threonine kinase receptors.

[0171] Specific examples of tyrosine kinase receptor activators include receptor activators such as cystine-knot cytokine superfamily proteins like PGDF (PDGF-BB, etc.) and VEGF.

[0172] Specific examples of serine / threonine kinase receptor activators include, for example, SMAD signaling pathway activators, particularly TGFβ receptor activators such as TGFβ family proteins.

[0173] In this specification, "TGFβ family protein" refers to any protein belonging to the TGFβ superfamily.

[0174] The specific TGFβ family proteins are not particularly limited. Examples include TGFβ protein, infivin (INH) subunit proteins (such as activin (activin A, etc.)), AMH, ARTN, BMP family proteins (such as BMP4), growth / differentiation factors (GDF), glial cell line-derived neurotrophic factor (GDNF), left-right determination factor (LEFTY) family proteins, myostatin, artemin (AMH), NODAL, neurturin, persephin, or combinations thereof. For example, TGFβ proteins such as TGFβ1 can be suitably used.

[0175] The final concentration used can be any effective amount and is not particularly limited. Examples of lower limits for the final concentration of TGFβ family proteins (e.g., TGFβ1 and other TGFβ proteins) include 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 27 ng / mL, 29 ng / mL, 30 ng / mL, etc., and examples of upper limits include 1000 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 90 ng / mL, 70 ng / mL, 50 ng / mL, 40 ng / mL, 35 ng / mL, 30 ng / mL, etc.

[0176] Other conditions are not particularly limited. For example, the culture can be carried out under the culture conditions described in the proliferation step. The culture medium may be the same as or different from the medium used in the proliferation step or the quiescent hepatic stellate cell induction step, but for example, the same medium can be preferably used.

[0177] The culture period is not particularly limited, but for example, it can be cultured for 1 to 15 days, 2 to 15 days, 3 to 15 days, 1 to 10 days, 2 to 10 days, 3 to 10 days, 1 to 8 days, 2 to 8 days, 3 to 8 days, 1 to 6 days, 2 to 6 days, or 3 to 6 days.

[0178] 6. Cell preparation 6-1. Overview The sixth aspect of the present invention is a cell preparation. The cell preparation of the present invention contains quiescent hepatic stellate cells as an essential component, and optionally contains a solvent and a carrier. According to the cell preparation of the present invention, quiescent hepatic stellate cells can be transplanted into an animal.

[0179] 6-2. Composition The following describes each component in detail. <Quiserating hepatic stellate cells> The quiescent hepatic stellate cells contained in the cell preparation of this embodiment preferably include quiescent hepatic stellate cells having proliferative capacity produced by the manufacturing method of the fifth embodiment.

[0180] The description of resting hepatic stellate cells is the same as that given in the first and fifth embodiments. The species from which the resting hepatic stellate cells contained in the cell preparation of this embodiment originate is not particularly limited. For example, the resting hepatic stellate cells may originate from the target species or individual to be administered, or from the target species or individual to be treated.

[0181] The cells used in the cell preparation according to this embodiment may be subjected to any treatment before contact with the preservation solution. Specific treatments are not limited to those described above, but examples include freezing, thawing, culturing, washing, sorting, transformation, genetic manipulation, or combinations thereof.

[0182] The state of cells in the cell preparation according to this embodiment may be single cells or cell aggregates such as spheroids. Preferably, the state is single cells. In this specification, "single cell state" means a state in which cells exist individually and are not aggregated. The percentage of single cells among all cells in the cell preparation is, for example, 70% or more, 90% or more, 95% or more, 99% or more, or 100%. The percentage of single cells in the cell preparation can be measured using any method. For example, it can be measured by dispersing cells in a buffer (e.g., PBS) and observing several randomly selected cells under a microscope to check for aggregation.

[0183] Cells in a cell preparation may be suspended or in contact with the inner wall of the container, etc. Preferably, they are suspended. In this specification, "suspended" means that the cells are not fixed to the inner wall of the container containing the cell preparation by adhesion or the like. For example, the percentage of suspended cells out of the total cells in the cell preparation may be, for example, 40% or more, 50% or more, 60% or more, 70% or more, 90% or more, 95% or more, 99% or more, or 100%.

[0184] The number of cells per unit dose contained in the cell preparation of the present invention is not particularly limited. Generally, the number of cells varies depending on the type of cell, the route of administration, the purpose of administration, and the type of carrier, which is another component described later. Therefore, it should be determined appropriately taking each of these conditions into consideration. For example, it is sufficient that a sufficient number of cells are contained in a single dose of the cell preparation. There is no specific limit on the number of qHSCs per unit dose, but it is sufficient to include qHSCs of 10^7 cells / mL or more, 10^8 cells / mL or more, and 10^9 cells / mL or more. Specifically, it may include qHSCs of 10^7 cells / mL to 10^15 cells / mL, 10^8 cells / mL to 10^15 cells / mL, 10^9 cells / mL to 10^15 cells / mL, 10^7 cells / mL to 10^12 cells / mL, 10^8 cells / mL to 10^12 cells / mL, 10^9 cells / mL to 10^12 cells / mL, 10^7 cells / mL to 10^10 cells / mL, 10^8 cells / mL to 10^10 cells / mL, and 10^9 cells / mL to 10^10 cells / mL. When administered in multiple doses, it is sufficient that the total amount contains a sufficient number of cells. Furthermore, when the cell preparation of the present invention is administered after dilution, it is sufficient that the diluted preparation contains a sufficient number of cells to obtain the desired effect.

[0185] Any additional cells besides qHSCs may be included. The type of additional cells is not particularly limited. Preferably, they are cells of the same species or originating from the same individual as qHSCs.

[0186] <Solvent> The cell preparation according to this embodiment may contain a pharmaceutically acceptable solvent as needed. "Pharmaceutically acceptable solvent" means a solvent commonly used in the pharmaceutical technology field. Examples include water and aqueous solutions. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffers, and sodium acetate buffers. Examples of adjuvants include inorganic salts such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, organic acid salts such as citrate, gluconate, and succinate, as well as low concentrations of nonionic surfactants and polyoxyethylene sorbitan fatty acid esters.

[0187] The pH of the solution is not particularly limited as long as the cells being preserved can survive. Specific pH ranges include, for example, 3.5–8.5, 4–8, 4.5–7.5, or 5–7.5.

[0188] The osmotic pressure of the solution is not particularly limited as long as cells can survive. For example, it can be hypotonic (less than 250 mOsm / L), isotonic (250 mOsm / L to 380 mOsm / L), or hypertonic (greater than 380 mOsm / L). The osmotic pressure may also be expressed as an osmotic pressure ratio with physiological saline (e.g., 306 mOsm / L).

[0189] The solution of the present invention may be prepared in-house or be commercially available. For example, preferred solutions include Ringer's solution (such as lactated Ringer's solution, acetate Ringer's solution, or bicarbonate Ringer's solution), Ringer's basal solution, any other solution used as an intravenous fluid, and culture media (such as serum-free medium as exemplified in the third embodiment).

[0190] The viscosity of the solution is not particularly limited as long as the cells being preserved can survive. For example, it is preferably 8 mPas or higher, and preferably 18 mPas or lower, at the temperature during cell suspension and / or storage. The viscosity of the non-freezing storage solution can be measured, for example, using a TV-20 viscometer (Toki Sangyo Co., Ltd.) at a rotation speed of 10 rpm.

[0191] <Carrier> The cell formulation of the present invention may optionally include a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to an additive commonly used in the pharmaceutical technology field. Examples include excipients, binders, disintegrants, emulsifiers, flow additive modifiers, lubricants, etc.

[0192] Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, and Pluronic acid. (R) Examples include kaolin, silicic acid, or combinations thereof.

[0193] Examples of binders include starch paste made from plant starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone, or combinations thereof.

[0194] Examples of disintegrants include the aforementioned starch, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride stearate, or salts thereof.

[0195] Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0196] Examples of fluid additive regulators and lubricants include silicates, talc, stearates, or polyethylene glycol.

[0197] In addition to the above, if necessary, the composition may also contain solubilizers, suspending agents, diluents, dispersants, surfactants, analgesics, stabilizers, absorption enhancers, bulking agents, humectants, moisturizers, wetting agents, adsorbents, flavoring and deodorizing agents, disintegration inhibitors, coating agents, colorants, preservatives, antioxidants, buffers, pH adjusters, isotonic agents, etc., which are commonly used in pharmaceutical compositions and cell preparations.

[0198] The carrier is used to avoid or inhibit the degradation of the active ingredient by enzymes, etc., within the target body, as well as to facilitate formulation and administration methods, and to maintain the dosage form and efficacy. It should be used as appropriate as needed.

[0199] The cell preparation of the present invention is preferably in a sterile state. Any method known in the art can be used to achieve sterility, and is not particularly limited.

[0200] 6-3. Dosage Form The cell preparation of the present invention is in the form of a liquid. The liquid includes any formulation that has fluidity. Specifically, examples include creams, ointments, gels, injections, suspensions, etc. The specific volume and other details are not particularly limited and should be within the range known in the art for each dosage form. The cell preparation of the present invention can be manufactured according to conventional methods in the art.

[0201] 6-4. Method of Application The cell preparation of the present invention is administered by parenteral administration. Parenteral administration can be further subdivided into systemic administration and local administration. Local administration includes, for example, intrasplenic route, subcutaneous route, intradermal route, intravenous route, intramuscular route, intrafocal route, tissue administration, and organ administration. Systemic parenteral administration includes intracirculatory administration (e.g., intravenous administration (injection), intra-arterial administration, and intralymphatic administration), intraperitoneal administration, etc. For example, the cell preparation of the present invention can be administered locally, in which case it can be administered directly to the target site by, for example, injection. Alternatively, for example, it can be used by being contained in a cream, ointment, gel, suspension, or any other suitable substance at the time of application. For systemic administration, for example, intracirculatory administration such as intravenous injection can be performed. The dosage should be an amount that is effective for the cells to take effect. The effective amount is appropriately selected according to the information of the target, as described above.

[0202] The method and amount of application of the cell preparation of the present invention may vary depending on the information of the subject. In this specification, "information of the subject" refers to various information about the characteristics and condition of the subject. For example, if the subject is a human individual, this may include age, weight, sex, overall health status, presence or absence of disease, progression and severity of disease, drug sensitivity, presence or absence of concomitant drugs, and resistance to treatment.

[0203] The cell preparation of the present invention can be stored before application. The specific storage method, storage period, and storage temperature are not particularly limited.

[0204] Furthermore, the cell preparation of the present invention may be applied as is, or may be applied after any additional processing. Specific additional processing includes, but is not limited to, application by transplantation of a reservoir containing the cell preparation of the present invention, or application by transplantation of tissue, organ, or organoid formed by further culturing the cell preparation. Further culturing can be performed for purposes such as increasing the number of cells, cell differentiation, transformation, or gene introduction into cells.

[0205] Furthermore, the cell preparation of the present invention can be used in combination with one or more known cell preparations.

[0206] 6-5. Applicable Subjects The cell preparation of the present invention is not particularly limited in its application. For example, it can be applied to the tissues, organs, or individuals of organisms exemplified as the species from which the transverse septal mesenchymal cells of the first embodiment originate (mammals including primates such as humans, experimental animals, or livestock).

[0207] The cell preparation of the present invention may be applied to healthy individuals or those suffering from any disease or condition. The diseases and conditions in this case are not particularly limited, but are preferably diseases and conditions related to liver damage. Specifically, for example, the cell preparation of this embodiment can be used as a cell preparation for the improvement, treatment, or prevention of one or more liver disorders selected from the group consisting of end-stage liver disease, cirrhosis, diabetes mellitus, obesity, acute hepatitis, chronic hepatitis, fatty liver, hepatic fibrosis, portal hypertension, regenerative failure, hepatitis, alcoholic hepatitis, non-alcoholic fatty liver disease, autoimmune hepatitis, hepatic dysfunction, impaired hepatic blood flow, acute hepatic failure, hepatic fibrosis, liver cancer, hepatic metabolic disease, and liver damage due to hepatic failure.

[0208] Furthermore, the purpose of application is not particularly limited. For example, it can be used for purposes such as improving, treating, and preventing diseases and conditions.

[0209] 6-6. Effects: By administering the cell preparation according to this embodiment, the introduced qHSCs can stably engraft in the body for a long period of time, and at least a portion of them can function similarly to endogenous cells. Therefore, the cell preparation according to this embodiment can be used in cell replacement therapy.

[0210] For example, introduced qHSCs stably engraft within the organism for a period of two months or more, three months or more, four months or more, five months or more, six months or more, seven months or more, eight months or more, nine months or more, or ten months or more.

[0211] The use of qHSCs prepared by the method described herein offers several advantages compared to cell replacement therapy using primary cultured HSCs. First, since pluripotent stem cell-derived HSCs can be stably manufactured regardless of the donor, a stable supply is possible when treatment is needed. Second, unlike aHSCs, which are abundant in primary cultured HSCs, qHSCs do not promote inflammatory responses or liver fibrosis, and have low expression of cirrhosis-related genes. Therefore, effects such as angiogenesis based on HSCs can be obtained without worsening the disease or condition through administration. Third, since the qHSCs contained in the cell preparation of this embodiment have proliferative capacity, they can be proliferated in vivo, increasing the proportion of qHSCs. Generally, qHSCs present in adults are not proliferative, so HSCs that can proliferate in an inactive state cannot be obtained from adult donors. Due to these characteristics, the present invention provides a new and promising option for the treatment of liver failure.

[0212] 7. Method for Producing Liver Organoids 7-1. Overview The seventh aspect of the present invention is a method for producing liver organoids. The method of this aspect includes a co-culture step as an essential step, and includes a pluripotent stem cell preparation step, a quiescent hepatic stellate cell induction step, a vascular endothelial cell induction step, and a hepatic progenitor cell induction step as optional steps. According to the method of this aspect, liver organoids having a complex vascular structure can be produced based on the function of hepatic stellate cells.

[0213] 7-2. Process 7-2-1. Pluripotent Stem Cell Preparation Process The pluripotent stem cell preparation process is an optional step of the method of this embodiment, and is a process of preparing pluripotent stem cells from cells isolated from an individual or tissue. This process can be carried out in accordance with the description of the pluripotent stem cell preparation process of the fourth embodiment.

[0214] 7-2-2. Preparation of quiescent hepatic stellate cells The preparation of quiescent hepatic stellate cells is an optional step of the method of this embodiment and is a step of preparing quiescent hepatic stellate cells from pluripotent stem cells. If the pluripotent stem cell preparation step is performed, this step can be performed afterward. This step can be performed in accordance with the description of the method for producing quiescent hepatic stellate cells in the fifth embodiment.

[0215] 7-2-3. Vascular Endothelial Cell Induction Step The vascular endothelial cell induction step is an optional step of the method according to this embodiment, and is a step in which pluripotent stem cells are differentiated into vascular endothelial cells. If a pluripotent stem cell preparation step is performed, it can be performed afterward.

[0216] Endothelial cells are the cells that make up capillaries and the innermost layer of blood vessels. Endothelial cells regulate the contraction and relaxation of vascular smooth muscle and are also involved in regulating blood coagulation. Typically, they express markers such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3, and CD31.

[0217] The specific culture conditions are not limited. The basic content of the culture conditions in this process is the same as that described in the proliferation culture process of the fifth aspect.

[0218] The specific method for inducing differentiation into vascular endothelial cells is not particularly limited. For example, any method known in the art can be used. For instance, when using iPS cells as pluripotent stem cells, differentiation induction can be performed using the method described in Takebe et al., Cell Reports, 2017.

[0219] The composition of the differentiation induction medium is not particularly limited, as long as it can induce differentiation into vascular endothelial cells. Specifically, this can be done by combining methods such as culturing in a medium containing a ROCK inhibitor (e.g., a ROCK2 inhibitor such as Y-39983), culturing in a medium containing a Wnt signaling pathway activator (e.g., a GSK3β inhibitor such as CHIR99021) and a TGFβ family protein (e.g., a BMP family protein such as BMP4), or culturing in a medium containing a cAMP pathway activator.

[0220] In this specification, "cAMP pathway activator" refers to a drug that promotes the cAMP-based signaling pathway.

[0221] The type of cAMP pathway activator is not particularly limited, but examples include cAMP synthesis promoters (such as adenylyl cyclase activators), protein kinase A activators on which cAMP acts, or combinations thereof. For example, adenylyl cyclase activators can be suitably used. Specific adenylyl cyclase activators are not particularly limited, but examples include Oct4 or its substitutes (such as forskolin), colfosine or its derivatives (such as colforsine dalopate or its salts), cell-derived toxin proteins (cholera toxin), PACAP-38, SKF83822, or combinations thereof.

[0222] The final concentration of the adenylyl cyclase activator (forskolin, etc.) is not particularly limited, but the lower limit can be, for example, 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, etc., and the upper limit can be, for example, 100 μM, 50 μM, 25 μM, 10 μM, 9 μM, 7 μM, 5 μM, 4 μM, 3 μM, 2.5 μM, 2.4 μM, 2.3 μM, 2.2 μM, 2.1 μM, 2 μM, etc.

[0223] The Wnt signaling pathway activator is as described in the fourth embodiment. The ROCK inhibitor and TGFβ family proteins are as described in the fifth embodiment.

[0224] Furthermore, commercially available kits for inducing vascular endothelial cells can be used, for example. Examples of such kits include, but are not limited to, Miracell. Cultures containing the various factors mentioned above may be used in combination with commercially available kits.

[0225] Whether the induced cells are vascular endothelial cells can be confirmed by examining whether one or more marker proteins, such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3, and CD31, are expressed. The vascular endothelial cells used in this invention may be differentiated or undifferentiated.

[0226] 7-2-4. Hepatic Progenitor Cell Induction Step The hepatic progenitor cell induction step is an optional step of the method according to this embodiment, and is a step in which pluripotent stem cells are differentiated into hepatic progenitor cells. If a pluripotent stem cell preparation step is performed, it can be performed afterward.

[0227] In this specification, "hepatic progenitor cells" refers to cells that have the ability to differentiate into bile duct epithelial cells and mature hepatocytes. Hepatic progenitor cells in this specification broadly include cells that express one or more markers such as HNF4A, AFP, E-cadherin, EpCAM, KRT19, and SOX9. Hepatic progenitor cells may also include hepatic endodermal cells and hepatoblasts, but in this specification, hepatic progenitor cells include any of these.

[0228] In this specification, "hepatic endoderm (HE)" refers to liver-specific endodermal cells that are tissue stem cells derived from the foregut endoderm present during embryonic development and possess the ability to differentiate into bile duct epithelial cells and mature hepatocytes. Hepatoblasts are not typically observed in adult bodies and are only observed in vivo during the development of the liver during embryonic development. Typically, they are known to express markers such as HNF4α, AFP, CD133, SOX17, FOXA2, E-cadherin, EpCAM, KRT19, and SOX9.

[0229] In this specification, "hepatoblast" refers to cells that differentiate from hepatic endodermal cells and are capable of differentiating into both bile duct epithelial cells and mature hepatocytes. Hepatoblasts are not normally observed in adult bodies and are only observed in vivo during the development of the liver during embryonic development. Typically, they express markers such as alpha-fetoprotein (AFP), delta-like non-classical Notch ligand (DLK), and sometimes albumin (ALB), T-box transcription factor 3 (TBX3), hepatocyte growth factor receptor (C-MET), and epithelial cell adhesion molecule (EpCAM), while they do not express markers such as keratin 19 (KRT19), and sometimes OCT4 and CD90.

[0230] The method for inducing differentiation of pluripotent stem cells into hepatocytes and hepatic endoderm cells is not particularly limited, and any method known in the art can be used. For example, the methods described in Gurevich, et al., Biol. Open (2020) (doi:10.1242 / bio.055087) and Li et al., Int. J. Mol. Sci. (2021) (doi:10.3390 / ijms221910471) can be used. For example, differentiation into hepatic endoderm cells or hepatoblasts can be induced after differentiation into embryonic endoderm cells.

[0231] The induction of differentiation into hepatic progenitor cells can be determined, for example, based on the expression of one or more of the various markers exemplified for hepatic progenitor cells, hepatic endodermal cells, and / or hepatoblasts.

[0232] <Induction of Embryonic Endoderm Cells> The specific method for inducing differentiation into embryonic endoderm cells is not particularly limited. For example, differentiation from pluripotent stem cells to embryonic endoderm cells can be induced by culturing pluripotent stem cells in a medium containing a Wnt signaling pathway activator and / or TGFβ family proteins.

[0233] The Wnt signaling pathway activators used to induce endoderm cells may be classical Wnt signaling pathway activators, non-classical Wnt signaling pathway activators, or mixtures thereof, and are not particularly limited.

[0234] The TGFβ family proteins and their concentrations shall conform to the description in the fifth embodiment. For example, activin and / or BMP4 can be suitably used.

[0235] Growth factors may be added to the culture medium for differentiation induction. The growth factors should be in accordance with the description in the co-culture step of this embodiment. For example, FGF2 and VEGF can be suitably used.

[0236] The culture conditions shall be in accordance with the description of the growth culture process in this embodiment. For example, B-27 as a culture medium additive. TMSupplements can be used, and RPMI1640 medium and / or serum-free medium can be suitably used. Other additives such as sodium butyrate may also be used. The culture period is not particularly limited, but can be, for example, 1 day or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, for example, 1 to 150 days, 1 to 90 days, 1 to 50 days, 1 to 30 days, 1 to 20 days, 3 to 10 days, etc.

[0237] During induction culture, the composition of the culture medium may be changed one or more times. For example, as the culture period progresses, the medium may be replaced with one containing a higher variety of TGFβ family proteins and growth factors.

[0238] The induction of differentiation into endoderm cells can be determined based on whether or not the cells possess any of the properties of endoderm cells and / or whether or not the properties of pluripotent stem cells have been lost. For example, this can be determined by the expression of endoderm cell markers and / or pluripotent stem cell markers. Any markers known in the art can be used as endoderm cell markers and pluripotent stem cell markers, and are not particularly limited. Specific endoderm cell markers include, for example, FOXA2, SOX17, CXCR4 (CD184), GATA4, GATA6, CD117, CER1, etc. Specific pluripotent stem cell markers include, for example, OCT4, NANOG, SOX2, CD9, DNMT3B, GABRB3, GAL, GDF3, IFITM1, PODXL, TDGF1, ZFP42, etc.

[0239] The following describes a method for inducing differentiation of endoderm cells into hepatic endoderm cells and hepatoblast cells from embryonic endoderm cells.

[0240] <Induction of Hepatic Endoderm Cells> Hepatic endoderm cells can be induced to differentiate from pluripotent stem cells or endoderm cells of the embryo. Alternatively, hepatic endoderm cells may be induced to differentiate from endoderm cells of the embryo through differentiation into other cell types.

[0241] The specific method for inducing hepatic endodermal cells is not particularly limited. For example, differentiation can be induced by culturing them in a culture medium containing amino acids and antioxidants.

[0242] The amino acids used in this case are not particularly limited, but examples include essential amino acids, non-essential amino acids, glutamic acid, their analogues, or combinations thereof.

[0243] The type of antioxidant used is not particularly limited. For example, antioxidants such as those exemplified in the first embodiment (2-mercaptoethanol, etc.) can be used.

[0244] The culture period is not particularly limited, but can be, for example, 1 day or more, 3 days or more, 5 days or more, 6 days or more, 7 days or more, or, for example, 1 to 150 days, 1 to 90 days, 1 to 50 days, 1 to 30 days, 1 to 20 days, 3 to 10 days, etc.

[0245] The induction of differentiation into hepatic endodermal cells can be determined based on whether or not the cells possess any of the properties of hepatic endodermal cells and / or whether or not the properties of embryonic endoderm cells have been lost. For example, this can be determined by the expression of hepatic endodermal cell markers and / or embryonic endoderm cell markers. Any marker known in the art can be used as the hepatic endodermal cell marker, and there are no particular limitations. Specifically, examples include HNF4α, AFP, CD133, SOX17, FOXA2, E-cadherin, EpCAM, KRT19, SOX9, etc. As the embryonic endoderm cell marker, for example, any of the embryonic endoderm cell markers mentioned above can be used.

[0246] <Induction of Hepatoblasts> Hepatoblasts can be induced from pluripotent stem cells or endoderm cells of the embryo. Alternatively, hepatoblasts may be induced from endoderm cells of the embryo through differentiation into other cell types (e.g., hepatic endoderm cells, foregut progenitor cells).

[0247] The specific method for inducing hepatoblasts is not particularly limited. For example, differentiation into hepatoblasts can be induced by culturing endoderm cells in a serum-free medium containing one or more selected from the group consisting of steroids, TGFβ family proteins, dimethyl sulfoxide, inflammatory cytokines, and growth factors. For example, the medium may contain dimethyl sulfoxide, a growth factor, a TGFβ family protein and a growth factor, a dimethyl sulfoxide and a growth factor, a steroid, an inflammatory cytokine, and a growth factor.

[0248] The culture conditions are in accordance with the description of the proliferation culture step in the fifth embodiment. For example, vitamin B such as nicotinamide or its derivatives, and vitamin C such as L-ascorbic acid or its derivatives can be suitably used as culture medium additives. The culture period is not particularly limited, but for example, culture can be carried out for the period exemplified for hepatic endodermal cells.

[0249] The induction of differentiation into hepatoblasts can be determined based on whether or not hepatoblasts possess any of the properties of hepatoblasts and / or whether or not the properties of endoderm cells of the embryo have been lost. For example, this can be determined by the expression of hepatoblast markers and / or endoderm cell markers of the embryo. Any marker known in the art can be used as hepatoblast marker and is not particularly limited. Specifically, examples include AFP, ALB, CD324, CYP3A7, DLK1, PROX1, TBX3, CDH6, CTNND2, DLK1, EpCAM, FOXA1, GATA4, GATA6, GPRC5B, HHEX, HNF1B, HNF4A, ID3, MCAM, MET, ONECUT1, PROM1, SLC12A2, SOX9, SPP1, STAT1, KRT19, E-cadherin, LIV2, CD13, CD133, etc. As an endoderm cell marker, for example, any of the endoderm cell markers described above in this embodiment can be used.

[0250] 7-2-5. Co-culture step The co-culture step is an essential step of the method according to this embodiment, and is a step of co-culturing quiescent hepatic stellate cells with vascular endothelial cells and hepatic progenitor cells. This step can be performed after the quiescent hepatic stellate cell, vascular endothelial cell induction, and hepatic progenitor cell induction steps if they are to be performed.

[0251] The culture conditions for this process are not particularly limited. It may be suspension culture or adherent culture, and can be carried out, for example, in accordance with the description of the proliferation culture process in the fifth embodiment.

[0252] The cell ratio is not particularly limited. It is preferable that vascular endothelial cells are present in greater numbers than quiescent hepatic stellate cells and hepatic progenitor cells. The specific ratio is not particularly limited. For example, vascular endothelial cells may be present in amounts of 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times more than quiescent hepatic stellate cells and / or hepatic progenitor cells. Furthermore, the ratio of quiescent hepatic stellate cells to hepatic progenitor cells is not particularly limited. For example, quiescent hepatic stellate cells may be present in amounts of 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, or 1 time compared to hepatic progenitor cells. Also, for example, quiescent hepatic stellate cells may be present in amounts of 2 times or less, 1.5 times or less, 1.4 times or less, 1.3 times or less, 1.2 times or less, 1.1 times or less, or 1 time compared to hepatic progenitor cells.

[0253] The culture vessels can be coated as needed. The coating agent is not particularly limited, but for example, Lipidure (R) Even if it is a suspension culture coating, it may be an adhesive culture coating such as Matrigel, as exemplified in the quiescent hepatic stellate cell induction step of the fourth embodiment.

[0254] Preferably, at least a portion of the culture in this step is carried out by gas-liquid interface culture. "Gas-liquid interface culture" refers to a culture method in which a portion of cells or a group of cells in contact with the culture medium are cultured in contact with the gas phase. Typically, this is done by seeding cells on a mesh membrane and supplying the culture medium from below.

[0255] Culture vessels or culture equipment can be coated as needed. The coating agent is not particularly limited, but for example, Lipidure(R) It may be a suspension culture coating such as Matrigel, or an adhesive culture coating such as Matrigel. Alternatively, ultra-low adhesion 6-well micro-dimple Elplasia (R) (Corning) You may also use culture equipment with a surface treatment for suspension culture, such as plates.

[0256] The culture equipment used for gas-liquid interface culture is not particularly limited, but commercially available cell culture inserts can be used, for example. Specific cell culture inserts that can be used include, for example, Falcon cell culture plates (Corning), Falcon multi-cell culture plates (Corning), and Falcon cell culture inserts (Corning).

[0257] The culture medium used in this process can be any medium available for organoid culture and is not particularly limited. For example, the culture medium exemplified in the third embodiment can be used. As a specific basal medium, for example, a mixed medium of DMEM medium and vascular endothelial cell medium can be used.

[0258] As a culture medium for vascular endothelial cells, for example, any culture medium known in the art that can culture any vascular endothelial cells can be used. Specifically, for example, KBM-VEC1 medium, Vascular Cell Basal Medium PCS-100-030 TM EGM TM -2. Endothelial Cell Growth Medium MV 2 or a combination thereof are examples. For example, KBM-VEC1 medium can be suitably used.

[0259] The mixing ratio is not particularly limited. For example, the ratios exemplified for the induction of hepatic stellate cells can be used. For example, an equal-volume mixture of DMEM medium and KBM-VEC1 medium can be suitably used.

[0260] Additives may be added to the culture medium as needed. For example, one or more of the following can be added: JAK-STAT3 pathway activators (such as oncostatin M), growth factors (such as FGF2 and HGF), anti-inflammatory agents (such as steroids like dexamethasone), and ROCK inhibitors (such as Y-27632).

[0261] Examples of JAK-STAT3 pathway activators include FGF19, thrombopoietin (TPO), leukemia suppressor (LIF), FLT-3 ligand, TNF-α, IFN-γ, IL-1, IL-6 family proteins, and IL-17a. Examples of IL-6 family proteins include oncostatin M (OSM), IL-6, IL-11, IL-27, IL-35, IL-39, LIF, CT-1, CNTF, CLCF1, overexpression vectors for JAK-STAT3 pathway-related genes, and combinations thereof. For example, IL-6 family proteins such as OSM can be suitably used.

[0262] "Anti-inflammatory agents" refer to drugs that can suppress the onset and / or progression of inflammatory responses. While there are no specific limitations on the type of anti-inflammatory agent, for example, steroids can be suitably used. "Steroids" refer to drugs that have a steroid skeleton in their chemical structure.

[0263] Examples of steroids usable in this specification include glucocorticoids, glucocorticoids, and their analogs and derivatives. For example, glucocorticoids, or their analogs, derivatives, or substitutes can be suitably used as steroids.

[0264] The specific types of glucocorticoids or their analogs and derivatives used are not particularly limited. Specifically, examples include long-acting steroids with a biological half-life of 36 hours or more (dexamethasone (DEX), betamethasone, paramethasone, etc.), intermediate-acting steroids with a biological half-life of 12 hours or more (prednisolone, methylprednisolone, triamcinolone, etc.), short-acting steroids with a biological half-life of less than 12 hours (cortisone acetate, fludrocortisone acetate, hydrocortisone, cortisone, etc.), natural glucocorticoids (cortisone acetate, fludrocortisone acetate, hydrocortisone, cortisone, etc.) and their salts. For example, the steroid in the hepatoblast proliferation promoter of this embodiment may suitably include long-acting steroids such as dexamethasone (DEX).

[0265] In this specification, multiple steroids may be used in combination. In this case, multiple glucocorticoids may be used in combination, or glucocorticoids may be used in combination with other steroids. The amount of each component is not particularly limited, as long as it is an effective amount.

[0266] The culture period is not particularly limited. For example, it can be cultured for 1 to 30 days, 1 to 20 days, 1 to 15 days, 4 to 30 days, 4 to 20 days, 4 to 15 days, 6 to 30 days, 6 to 20 days, 6 to 15 days, 8 to 30 days, 8 to 20 days, 8 to 15 days, 9 to 30 days, 9 to 20 days, 9 to 15 days, 10 to 30 days, 10 to 20 days, or 10 to 15 days.

[0267] 8. Organoids 8-1. Overview The eighth aspect of the present invention is an organoid. The organoid of the present invention comprises quiescent hepatic stellate cells having proliferative capacity as an essential component, and optionally comprises additional cells. According to the organoid of the present invention, a liver organoid having a complex vascular structure and exhibiting liver function is provided.

[0268] 8-2. Composition The following describes each component in detail. The quiescent hepatic stellate cells contained in the organoid can be any quiescent hepatic stellate cells with proliferative capacity, and are not particularly limited, but are preferably fetal-like quiescent hepatic stellate cells. For example, quiescent hepatic stellate cells produced by the method described in the fifth embodiment can be suitably used.

[0269] If necessary, other cells may be included. The types of other cells in this case are not particularly limited. For example, one or more cells such as vascular endothelial cells, hepatic progenitor cells (hepatoblasts and / or hepatic endodermal cells), and hepatic parenchymal cells may be included.

[0270] The size of the organoid in this embodiment is not particularly limited. For example, in terms of cell number, it could be 10^5 cells / cm². 2 ~10^7 cells / cm 2 , 10^5 cells / cm 2 ~5×10^6 cells / cm 2 , 10^5 cells / cm 2 ~2×10^6 cells / cm 2 , 10^5 cells / cm 2 ~10^6 cells / cm 2 , 2×10^5 cells / cm 2 ~10^7 cells / cm 2 , 2×10^5 cells / cm 2 ~5×10^6 cells / cm 2 , 2×10^5 cells / cm 2 ~2×10^6 cells / cm 2 The density, etc., is sufficient. Also, the length of the long axis can be, for example, 200μm to 10cm, 200μm to 7cm, 200μm to 5cm, 200μm to 2cm, 200μm to 1cm, 400μm to 10cm, 400μm to 7cm, 400μm to 5cm, 400μm to 2cm, 400μm to 1cm, 500μm to 10cm, 500μm to 7cm, 500μm to 5cm, 500μm to 2cm, 500μm to 1cm, 700μm to 10cm, 700μm to 7cm, 700μm to 5cm, 700μm to 2cm, 700μm to 1cm, etc.

[0271] The organoid in this embodiment is preferably an organoid manufactured by the method of the seventh embodiment.

[0272] 8-3. Uses The uses of the organoids of the present invention are not particularly limited. They may be transplanted into non-human animals or humans, and may be used in vitro for experiments such as drug testing. Examples of in vitro experiments include preparing disease model organoids by subjecting them to any treatment. The diseases in this case are not particularly limited. For example, various diseases exemplified in the sixth aspect can be cited.

[0273] 8-4. Properties The organoids of this embodiment have well-developed blood vessels and form a complex vascular structure. Furthermore, they can exhibit liver-specific functions such as albumin production, CYP3A4 production, and NH4 metabolism.

[0274] Specifically, for example, in the case of vascular structures, the number of blood vessel branches per organoid is 50 or more, 75 or more, 100 or more, 125 or more, 150 or more, 175 or more, 200 or more, 225 or more, and 250 or more. Also, for example, in the case of NH4 metabolism, the average metabolic rate is 1.1 times or more, 1.3 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, and 2 times or more compared to organoids that do not contain qHSCs.

[0275] 9. Non-human animals 9-1. Overview The ninth aspect of the present invention is a non-human animal. The non-human animal of this aspect includes qHSC produced by the method of the fifth aspect, or includes the organoid described in the eighth aspect. The non-human animal of the present invention can be used, for example, in drug metabolism tests, safety tests and hepatotoxicity tests, or in the manufacture of artificial livers.

[0276] 9-2. The species of non-human animals used as constituents are not particularly limited. For example, the organisms exemplified as the species from which the transverse septal mesenchymal cells of the first embodiment originate (such as rodents like mice, as well as experimental animals and livestock) can be cited. The non-human animals may be healthy individuals or individuals with one or more diseases or disabilities, and are not particularly limited. For example, immunodeficient individuals can be suitably used.

[0277] The species from which the qHSCs or organoids contained in the non-human animal originate are not particularly limited, but for example, the qHSCs or organoids originate from a species or individual that is the target of the therapeutic administration of the drug being tested in the non-human animal (such as mammals including primates such as humans, or pets, etc.). Preferably, the qHSCs or organoids originate from a species or individual different from the non-human animal of this embodiment. For example, the non-human animal of this embodiment carries a (complete or partial) human liver.

[0278] The non-human animal in this embodiment can carry liver tissue differentiated from qHSCs or organoids. Whether or not the tissue is differentiated from qHSCs or organoids can be determined by any method. For example, it may be determined by distinguishing between cells of qHSCs or organoids and cells of the non-human animal, or by the presence or absence of phenomena detected after application of qHSCs or organoids.

[0279] When distinguishing between qHSC or organoid cells and non-human animal cells, the specific method is not particularly limited. For example, the determination can be made based on distinguishable information. Specific distinguishable information includes, for example, the type of cell surface antigen, the cell's genetic information (including modification information), the presence or absence of labels, or a combination thereof.

[0280] When determining whether or not a phenomenon is detected after the application of qHSC or organoids, the type of phenomenon detected is not particularly limited, as long as it reflects a change in the liver. For example, it may be a phenomenon based on structural changes in the liver, a phenomenon based on functional changes in the liver, or a phenomenon based on changes in the symptoms of liver damage.

[0281] Phenomena based on structural changes in the liver are not particularly limited, but include, for example, changes in liver size, cell number, weight, proportion of fibrotic areas, angiogenesis, and vascular complexity (number of branches, hierarchy, etc.). Phenomena based on functional changes in the liver are not particularly limited, but include, for example, drug metabolism function, albumin production function, CYP3A4 secretion, and NH4 metabolism function. Phenomena based on changes in the symptoms of liver damage are not particularly limited, but include, for example, changes in the number of aHSCs.

[0282] Whether or not a non-human animal carries liver tissue based on qHSCs or organoids can be determined by directly observing the liver through laparotomy, but it is preferable to make this determination using a less invasive method. For example, this can be determined by blood tests. The indicators used in the blood tests can be any indicators that reflect the degree of liver function or the presence or absence of liver damage, and are not particularly limited. For example, if the amount of AST, ALT, γ-GTP etc. released into the blood, the amount of bilirubin and ammonia accumulated, the total protein amount, albumin amount, albumin / globulin ratio etc. are observed after administration of the cell preparation, or if these values ​​are at the same level as those of a normal individual, or if the species of the qHSCs or organoids and the non-human animal are different, and albumin from the organism from which the qHSCs or organoids originated is detected in the blood of the non-human animal, then it can be determined that the non-human animal carries liver tissue based on qHSCs or organoids derived from the cell preparation.

[0283] In this embodiment, the non-human animal preferably has at least a portion of its endogenous liver destroyed. This destruction includes not only physical defects but also functional defects.

[0284] The method of destroying the endogenous liver is not particularly limited, and any method known in the art can be used. For example, the whole or a part of the liver may be resected, liver function may be destroyed using drugs, or the structure and / or function of the liver may be destroyed using genetic modification. Examples of drugs used to destroy liver function include ganciclovir triphosphate, letrolsine, acetaminophen, chlorpromazine, diclofenac, ranitidine, sulindac, trovafloxacin, α-naphthyl isothiocyanate, dicloxacillin, flucloxacillin, flutamide, halothane, methimazole, carbamazepine, phenytoin, azathioprine, 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-collidine, or combinations thereof. The method of administration is not particularly limited and can be appropriately selected depending on the type of drug used. The drug used may be administered in the form of a prodrug. In that case, for example, the administered prodrug may be converted to the active form under any conditions, or only under specific conditions. For example, the prodrug ganciclovir (GCV) is converted to ganciclovir triphosphate in the presence of herpesvirus-derived thymidine kinase.

[0285] The method of administering qHSC to non-human animals is not particularly limited. It can be administered by any of the administration methods exemplified in the sixth embodiment. In the case of organoids, transplantation is preferred.

[0286] 9-3. Applications For example, when human liver cells are supported on a non-human animal according to this embodiment, the effects of a drug being considered for human use on the human liver can be tested in experimental animals such as mice. Examples of such tests include drug metabolism tests, safety tests, and hepatotoxicity tests.

[0287] Furthermore, for example, an artificial liver for transplantation into a human can be manufactured in the body of a large animal, such as livestock, that carries human liver cells.

[0288] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples. Note that all concentrations of additives mentioned in the examples represent the final concentrations.

[0289] <Example 1. Induction of Differentiation into Hepatic Stellary Cells and Evaluation of the Properties of Hepatic Stellary Cells> (Objective) To induce differentiation of iPS cells into hepatic stellate cells and to investigate the properties of the induced hepatic stellate cells.

[0290] (Methods) 1. iPS cells: The human iPS cell (hiPSC) lines used were M48 (CiRA Foundation), 1231A3 (Riken), 1383D2 (Riken), and 1383D6 (Riken). The plate used was iMatrix-511. TM (Nippi, 892011): Cells were coated with iMatrix solution mixed with PBS in a 1:150 ratio and cultured in StemFit AK02N medium (Ajinomoto, RCAK02N) at 37°C in the presence of 5% CO2. Every 7 days, cells were dispersed using acetase (Innovative Cell Technologies, AT104-500) and subcultured.

[0291] Cultured human iPS cells were seeded in StemFit AK02N medium supplemented with 10 μM Y-27632 (Wako, 030-24026) (Day 0). The medium was changed with fresh StemFit AK02N medium on Day 1 of culture, and then every two days thereafter. (MycoAlert) TM Regular mycoplasma testing was performed on all human iPS cell clones using the Mycoplasma Detection Kit (Lonza, LT07-318).

[0292] 2. Differentiation induction into hepatic stellate cells (HSCs) 2-1. Differentiation induction into septum transversum mesenchyme cells (STMs) Same as above using iMatrix-511 TM Human iPS cells (hiPSCs) are placed on a coated dish at a rate of 2 × 10^3 cells / cm². 2~8×10^3 cells / cm 2 Seeds were seeded at a density (day 0), and first cultured for 24 hours in StemFit AKOKO2N medium supplemented with 10M Y-27632 (day 1). Then, mesoderm cells were induced by changing the medium to mesoderm cell induction medium and culturing for 3 days (day 4). The mesoderm cell induction medium consisted of 1% Glutamax and 1% B27 TM DMEM / F12 medium supplemented with 8M CHIR99021 and 25 ng / mL BMP4 was used. Subsequently, cells were cultured for 2 days in STM primary induction medium in which CHIR99021 and BMP4 in the mesoderm cell induction medium were replaced with 2 ng / mL Activin A (Ajinomoto) and 10 ng / mL PDGF-BB (R&D Systems) (day 6). Furthermore, cells were cultured for 2 days in STM secondary induction medium in which Activin A and PDGF-BB in the STM primary induction medium were replaced with 10 ng / mL FGF2 (Fujifilm Wako Pure Chemical Industries) and 12 ng / mL BMP4 to promote the formation of hiPSC-derived STM (hiPSC-STM) (day 8).

[0293] 2-2. Differentiation induction of hepatic stellate cells: iPSC-STM cultured on day 8 is converted to iMatrix-511 TM (Nippi, 892011), 0.2 × 10^4 cells / cm² on plates coated with COL1A1 (Corning) or Matrigel (Corning). 2 ~2×10^4 cells / cm 2 The cells were seeded at the specified cell density and cultured in hepatic stellate cell differentiation medium. The hepatic stellate cell differentiation medium used was serum-free differentiation medium (SFD medium) supplemented with 5(v / v)% fetal bovine serum (FBS; Biowest), 1 mM N-acetylcysteine ​​(NAC; Sigma-Aldrich), and 10 ng / mL FGF2. The medium was changed every other day.

[0294] For serum-free differentiation medium (SFD medium), 375 mL of Iskov-modified Dulbecco's medium (Life Technologies) was mixed with 125 mL of Ham's F-12 K medium (Life Technologies) and B27. TMA culture medium was prepared by mixing 5 mL of supplement (Life Technologies) and 2.5 mL of N2 supplement (Life Technologies), to which 0.05 (w / v)% bovine serum albumin (Sigma-Aldrich), 2 mM L-glutamine (Life Technologies), 1% penicillin-streptomycin (Life Technologies), 0.45 mM monothioglycerol solution (Fujifilm Wako Pure Chemical Industries), and 0.5 mM L-ascorbic acid (Sigma-Aldrich) were added. In some experiments, DMEM / F12 (Thermo Fisher Scientific) was used as a substitute medium for hepatic stellate cell differentiation instead of SFD. Differentiated hepatic stellate cells (hiPSC-HSC) were harvested on day 12 of culture and subjected to characterization analysis.

[0295] 3. Preparation of other cells: Human iPSC-derived mesoderm cells were induced from hiPSCs using the method described in "2-1. Differentiation induction into transverse septal mesenchyme cells (STM)" above.

[0296] Human adherent hepatocytes (BioreclamationIVT, IVT-F00995-P, or IVT-M00995-P) were used as primary hepatocytes (PHH). The PHH were maintained in hepatocyte maintenance medium (Lonza) on plates coated with collagen I (CORNING).

[0297] For primary hepatic stellate cells (pHSCs), human hepatic stellate cells (Adult) (ScienCell Research Laboratories, Cat. No.: 5300) were used. pHSCs were maintained in hepatic stellate cell medium (ScienCell Research Laboratories) on plates coated with poly-L-lysine (CORNING) (Sigma).

[0298] 4. Evaluation of the properties of hepatic stellate cells 4-1. qPCR analysis PureLink TMTotal RNA was isolated from cells using the RNA mini kit (Thermo Fisher Scientific, 12183025). Using the isolated RNA (<2 μg) as a template, single-stranded cDNA was synthesized using a high-volume cDNA reverse transcription kit (Thermo Fisher Scientific, 4368814) according to the manufacturer's protocol. qPCR was performed using a TB Green Premix Ex Taq II (Takara, RR820D) on a CFX96 Real-Time System machine (BioRad) according to the manufacturer's protocol.

[0299] Table 1 shows the primers used to detect the expression of each gene. The expression levels of each gene were calculated using the ΔΔCT method with ACTB as the housekeeping gene, and the magnification change was calculated as a relative value to the expression level obtained from hiPSC.

[0300]

[0301] 4-2. Cells cultured by flow cytometry were dispersed in 0.05% trypsin / EDTA (Gibco), washed with DMEM containing 10% fetal bovine serum (FBS), and then washed with PBS. Next, the cells were stained with labeled antibody on ice for 30 minutes. After that, the samples were washed and resuspended in FACS buffer. Flow cytometry was performed using a BD FACS Celesta Cell Analyzer or Aria III (BD Biosciences), and the collected data were analyzed using FlowJo 10.7.1 (BD Biosciences) software. Antibodies against surface antigens (PDGFRβ, NGFR, ALCAM, PDGFRα, CD73, CD71) and isotype control antibodies were all obtained from BD Pharmingen.

[0302] 4-3. Transcriptome Analysis The total RNA obtained as described above was evaluated for RNA quality using an Agilent 2100 Bioanalyzer (Agilent Technologies), and RNA samples with an RNA Integrity Number (RIN) greater than 9.0 were subjected to RNA sequencing (RNA-Seq) analysis. RNA-Seq libraries were prepared from 100 ng of total RNA using the Ion AmpliSeq Transcriptome Human Gene Expression kit (Thermo Fisher Scientific, A26326) according to the manufacturer's protocol. Library sequencing was performed using the Ion PI Hi-Q Sequencing 200 kit and Ion PI Chip v3 (Thermo Fisher Scientific, A26772) with Ion Proton TM The system performed the sequencing, and the sequenced reads were mapped to hg19_AmpliSeq_Transcriptome_ERCC_v1 using the Torrent Mapping Alignment Program.

[0303] Next, QC metrics and normalized read counts per gene were obtained using the AmpliSeqRNA plugin v5.2.0.3 and Torrent Suite Software v5.2.2 (Thermo Fisher Scientific). Differential expression analysis (DEA) and principal component analysis were performed using GeneSpring (Agilent Technologies). Gene set enrichment analysis (GSEA) was performed using GSEA v4.3.2 (Broad Institute). For annotation in gene ontology analysis, information available from http: / / geneontology.org / was used.

[0304] 4-4. Lipid Accumulation Assay Lipid accumulation was assessed by oil red staining. First, cells were treated with 10 mM palmitic acid (Sigma-Aldrich) and incubated at 37°C for 24 hours. After washing the cells, they were stained with Oil Red O (Fujifilm Wako Pure Chemical Industries). Staining was performed according to the manufacturer's recommended protocol. Stained images were acquired using an All-in-one Fluorescence Microscope BZ-9000 (Biorevo) and quantified using Fiji 3 (NIH).

[0305] 4-5. Vitamin A Storage Assay The amount of vitamin A stored in cells was indirectly quantified by measuring the autofluorescence of retinyl esters in cells after irradiation with excitation light (wavelength: 496 nm). First, cells were incubated in the presence of retinol for 48 hours. After washing the cells, analysis was performed using a SORPAria flow cytometer (BD Biosciences) and FlowJo 10.7.1 (BD Biosciences) software. Measurement results from samples that were not treated with retinol were used as a control.

[0306] 4-6. Activation treatment of HSCs HiPSC-HSCs that had undergone one passage and pHSCs that had undergone two passages were cultured for 3 days in a medium supplemented with TGFβ1 (Peprotech) at a final concentration of 30 ng / mL.

[0307] 5. Statistical Processing GraphPad Prism 9.0 (GraphPad Software) was used for statistical analysis. No data was excluded from the data analysis. The significance level was set at p < 0.05. A t-test was used for two-group comparisons, and one-way ANOVA was used for multi-group comparisons.

[0308] (Results) The results are shown in Figures 2-7. As shown in Figure 2A, the expression of NANOG, a pluripotent stem cell marker, was hardly observed in cells other than human iPS cells (hiPSCs). HLX and WT1, genes specific to hiPSC-derived transverse septal mesenchymal cells (hiPSC-STMs), were highly expressed only in hiPSC-STMs, confirming that hiPSC-HSCs no longer exhibit the gene expression pattern of STMs (Figures 2B and 2C). On the other hand, in hiPSC-derived hepatic stellate cells (hiPSC-HSCs), the expression of HSC-specific genes such as CD73 was high, similar to that of primary hepatic stellate cells (pHSCs) (Figure 2D). This suggests that hiPSC-HSCs exhibit a gene expression profile similar to that of hepatic stellate cells.

[0309] Furthermore, as shown in Figure 3, comprehensive transcriptome analysis confirmed that hiPSC-HSCs strongly express genes specific to hepatic stellate cells (light gray in Figure 3), while conversely, they showed low expression of genes specific to other cell types differentiating from STMs (mesothelial cells, vascular smooth muscle cells) (dark gray in Figure 3). This suggests that the method of the present invention induces differentiation of STMs into HSCs with high purity.

[0310] Furthermore, to clarify the characteristics of hiPSC-HSC as HSCs, a detailed comparison with pHSCs was performed. As a result, it was confirmed that HSC-related genes such as CD73, ALCAM, and CD71 were expressed in almost all cells (CD73: approximately 98.7%; ALCAM: approximately 95.5%; CD71: approximately 97.2%), similar to pHSCs (Figures 4A-C). In addition, the function of hiPSC-HSCs was confirmed. HSCs have characteristic functions such as the accumulation of lipid droplets and the storage of vitamin A in lipid droplets. Therefore, when these were examined, it was confirmed that lipid accumulation and vitamin A storage (approximately 98.9%) were observed in almost all cells, similar to pHSCs (Figures 4E and F). From this, it was suggested that hiPSC-HSCs have a sufficient expression profile and function as HSCs.

[0311] Next, we analyzed the differences between hiPSC-HSC and pHSC using transcriptome analysis. The top 100 differentially expressed genes (DEGs) in hiPSC-HSC were predominantly involved in biological processes typically associated with development, such as circulatory system development, growth, angiogenesis, response to growth factor stimulation, and regeneration. In contrast, pHSCs were predominantly involved in biological processes typical of adult livers, such as tissue remodeling, tissue homeostasis, lipid transport, and acute inflammatory responses. In particular, we compared the expression of genes characteristic of HSCs derived from livers with cirrhosis and those characteristic of HSCs derived from normal livers in both cell types (Figure 5A). In pHSCs, expression of cirrhosis-related genes was high (dark gray in Figure 5A), while expression of normal liver-related genes was low (light gray and white in Figure 5A). Conversely, in hiPSC-HSCs, expression of normal liver-related genes was high, while expression of cirrhosis-related genes was low.

[0312] Furthermore, while many cells in pHSCs expressed PDGFRα, a cirrhosis-related gene specific to inflammatory, activated hepatic stellate cells, hiPSC-HSCs contained almost no cells expressing PDGFRα (approximately 0.1%) (Figure 5B). The secretion of COL1A1, which is characteristic of activated hepatic stellate cells, was also significantly lower in hiPSC-HSCs compared to pHSCs (indicated as "Con" in Figure 5C). When both cells were cultured in the presence of TGFβ1, an activator of quiescent hepatic stellate cells, COL1A1 secretion increased significantly in hiPSC-HSCs, but no significant change was observed in pHSCs (Figure 5C). This suggests that, unlike pHSCs, hiPSC-HSCs contain a large number of quiescent hepatic stellate cells.

[0313] To further investigate the properties of hiPSC-HSC, gene set enrichment analysis was performed on the fetal HSC-related gene set (Figure 6A), the adult HSC-related gene set (Figure 6B), and the cell cycle-related gene set (Figure 6C). In this analysis, the properties of the cells are shown as the bias of genes included in the target gene set (shown as black vertical lines in the lower part of Figure 6) among the genes aligned on the horizontal axis. In this analysis, the horizontal axis was aligned from left to right in order of relative expression level in hiPSC-HSC compared to pHSC (shown as a gradient in the lower part of Figure 6), from highest to lowest relative expression level. As shown in Figure 6A, many of the genes with high relative expression levels in hiPSC-HSC were fetal HSC-related genes, indicating that the expression of fetal HSC-related genes was generally significantly higher compared to pHSC. Similarly, for cell cycle-related genes, the expression of cell cycle-related genes was generally significantly higher compared to pHSC. Conversely, in hiPSC-HSC, there were few adult HSC-related genes among those with high relative expression levels, and the Enrichment Score was low. In contrast, in pHSC, many of the genes with high relative expression levels were adult HSC-related genes, and the distribution was skewed to the right. This indicates that the expression of adult HSC-related genes was generally significantly lower compared to pHSC. This suggests that hiPSC-HSC is an HSC that maintains an immature state similar to that of a fetal HSC.

[0314] When the relationship between culture conditions and the properties of hiPSC-HSCs was investigated, the expression of some activated hepatic stellate cell-related genes, such as LOX, increased when differentiation induction culture was performed under conditions without FGF2 (Figure 7A). This suggests that the presence of FGF2 is important for differentiation induction into quiescent hepatic stellate cells. On the other hand, it was suggested that the properties of quiescent hepatic stellate cells did not significantly change depending on the type of culture medium used (Figure 7B) or the type of coating agent used during culture (Figure 7C). Furthermore, since the results were similar regardless of the strain used as the hiPSC, it was confirmed that differentiation into HSCs is possible regardless of the individual from which the iPS cells originated.

[0315] These findings demonstrate that inducing the differentiation of quiescent hepatic stellate cells, which maintain an immature state, is possible by inducing the culture of transverse septal mesenchymal cells in the presence of FGF2.

[0316] <Example 2. Investigation and evaluation of methods for maintaining hepatic stellate cells> (Objective) To confirm whether hepatic stellate cells (hiPSC-HSC) differentiated from iPS cells can be maintained and to evaluate the changes in the properties of hiPSC-HSC due to maintenance culture.

[0317] (Methods) hiPSC-HSCs were passaged when cell confluence reached 90-100%. TrypLE was used for cell detachment and dispersion. TM Express Enzyme (Gibco, 12604013) was used. 1 × 10^4 cells / cm³ 2 Cells at this density iMatrix-511 TM Seeds were seeded in new coated dishes and cultured in maintenance medium with the same composition as the culture medium before subculturing. 10 μM Y-27632 was added to the medium only on the first day of seeding. The medium was changed every other day. Cell confluence generally reached 100% within 4 days with each subculturing.

[0318] In each passage, the number of seeded cells and the total number of cells immediately preceding the next passage were counted, and the growth rate was calculated by dividing the total number of cells by the number of seeded cells. The cumulative cell growth rate was calculated by setting the number of hepatic stellate cells before passage to 1 and then multiplying this by the growth rate in each passage cumulatively.

[0319] qPCR analysis, flow cytometry, transcriptome analysis, lipid accumulation assay, vitamin A storage assay, and statistical analysis for characterizing hiPSC-HSC were performed in the same manner as in Example 1. In the qPCR analysis, the magnification change was calculated as a relative value to the expression level in pHSC. The experiment was repeated three times.

[0320] (Results) The results are shown in Figures 8 and 9. As shown in Figure 8A, the hiPSC-HSC of the present invention can be reproducibly subcultured more than 6 times, and it was shown that it can grow stably in each subculture (growing 1 × 10^5 to 1 × 10^6 times after 6 subcultures). Furthermore, when HSC markers such as CD73 (ALCAM, CD71, and PDGFRβ are similar), lipid accumulation, and vitamin A storage were examined in the cells, it was confirmed that the characteristics as HSCs were stably maintained even after subculture (Figure 8B). This was supported by the fact that the expression patterns of the top 100 differentially expressed genes (DEGs) did not change with the number of subcultures.

[0321] It has been reported that in vitro proliferation of HSCs such as pHSC tends to increase the activation state of HSCs. Therefore, when we checked the expression of aHSC markers, we found that the expression of aHSC markers such as COL1A1 (Figure 9A) and αSMA (Figure 9B) (and similarly PDGFRα and TIMP1, etc.) remained significantly lower than that of pHSCs. This confirmed that the hiPSC-HSCs of the present invention maintained their properties as qHSCs without activation even after proliferation and passage. This was also true for the expression profiles of cirrhosis-related genes and normal liver-related genes.

[0322] From the above, it has been shown that the hiPSC-HSC of the present invention can be subcultured while maintaining its cellular characteristics, and that it is possible to perform maintenance culture and expansion culture.

[0323] <Example 3. Preparation of liver organoids containing hiPSC-HSCs> (Objective) To prepare liver organoids in vitro from hepatic stellate cells (hiPSC-HSCs) differentiated from iPS cells and to confirm their properties.

[0324] (Methods) 1. Preparation of raw material cells 1-1. Preparation of hepatic endodermal cells (HE) To prepare HE differentiated from hiPSCs (hiPSC-HE), hiPSCs were prepared using iMatrix-511 TM Sow seeds on a coated dish and add 1% B27 TMThe cells were inducibly cultured for 6 days in RPMI1640 medium (Fujifilm Wako Pure Chemical Industries) supplemented with 100 ng / mL human activin A (Ajinomoto) and 50 ng / mL Wnt3a (R&D Systems). 10 μM Y-27632 was added to the medium only on day 0 of culture. Additionally, 500 μM sodium butyrate (NaB: Wako, 193-01522) was added to the medium from day 1 to day 3 of culture. The medium was changed daily.

[0325] From day 7 of culture onward, the culture medium was changed to hepatic endodermal cell differentiation medium. For hepatic endodermal cell differentiation medium, StemFit Basic03 (Ajinomoto) supplemented with 1% non-essential amino acids (NEAA: Life Technologies), 1% DMSO (Nacalai Tesque), 1 mM L-Glutamine (Life Technologies), 0.18% 2-Mercaptoethanol (Life Technologies), and 1% penicillin-streptomycin (Life Technologies) was used. The culture medium was changed daily. Differentiated cells obtained on day 10 of culture were hepatic endodermal cells (HE).

[0326] 1-2. Preparation of vascular endothelial cells (ECs) To prepare ECs differentiated from hiPSCs (hiPSC-ECs), hiPSCs are converted to iMatrix-511 TM Seeds were seeded on coated dishes and inducible culture in StemFit AKOKO2N medium supplemented with 10 μM Y-27632. The medium was changed the following day, and the medium was replaced with 1% Glutamax (Life Technologies) and 1% B27 TM The medium was then replaced with DMEM / F12 (Thermo Fisher Scientific) supplemented with 8 μM CHIR99021 and 25 ng / mL BMP4 (R&D Systems). Three days later, the medium was changed again and replaced with StemPro medium supplemented with 100 ng / mL VEGF (Life Technologies) and 2 μM forskolin (Sigma-Aldrich). TM The culture medium was replaced with -34 SFM medium (Life Technologies). After that, the medium was changed daily.

[0327] On the 10th day of culture, TrypLE TM hiPSC-ECs were dispersed using Express Enzyme (1X). The dispersed cells were then treated with iMatrix-511. TM Cells were seeded at a density of 1 × 10^6 cells in coated 10 cm dishes. For the first 24 hours, the cells were cultured in a medium containing equal volumes of StemPro-34 SFM (GIBCO) and Miracell (TAKARA) supplemented with 50 ng / mL of VEGF (Life Technologies). After that, the medium was replaced with a medium consisting only of Miracell, and the cells were cultured for 6 days. The medium was changed every other day.

[0328] 2. Preparation of Liver Organoids (LO) For organoid preparation, 5 × 10^5 cells of hiPSC-HE, 1 × 10^5 cells of hiPSC-EC, and 1 × 10^5 cells of hiPSC-HSC were co-cultured on 24-well plates. The 24-well plates contained 5% lipidure. (R) The organoids used were treated with a low-adhesion coating (NOF) by NOF Corporation. Culture was performed using the following procedure. First, the cells were suspended in a total of 50 μL of liver organoid culture medium and incubated for 1 hour to allow the cells to adhere to each other. Next, 450 μL of medium was added to each well, and the cells were cultured for 24 hours to allow self-assembly and form organoids (LO-HSCs). In some experiments, organoids (LO-ΔHSCs) were prepared under conditions that did not include iPSC-HSCs.

[0329] For the liver organoid culture medium, an equal mixture of DMEM and KBM-VEC1 basal medium (Kohjin Bio) was used, supplemented with 10 μM Y-27632, 0.25% FBS, 10 ng / mL Oncostatin M (OSM; R&D Systems), and 50 nM Dexamethasone (DEX; Merck). The culture medium was changed every other day.

[0330] In some experiments, organoids were transferred onto cell culture inserts and cultured for an additional 10 days in a gas-liquid interface (ALI) culture system. ALI culture was performed using the following method.

[0331] First, a cell culture insert (Falcon, 353090) was placed in a 6-well flat-bottom plate. The prepared microliver organoids were seeded in each well so that the hepatic endodermal cells numbered 1 × 10^6 cells, and cultured in 1.2 mL of liver organoid culture medium. Culture was carried out for 10 days at a CO2 concentration of 5% and 37°C (D10). The culture medium was changed every other day.

[0332] 3. Evaluation of Organoid Properties The properties of the organoids were evaluated using the various analyses described in Example 1, as well as immunohistochemistry, human albumin assay, CYP3A activity assay, ammonia metabolism assay, and transmission electron microscopy.

[0333] 3-1. Immunostaining: Sections of organoids, 5 μm thick, were fixed with 4% PFA at room temperature for 30 minutes. After fixation, the samples were wrapped in paraffin or Tissue-Tek. (R) The sections were embedded in OCT Compound (Sakura Fintech). Paraffin-embedded sections were treated with antigen retrieval using citrate buffer (pH 6.0) before application of the primary antibody. On the other hand, OCT Compound-embedded sections were post-fixed with 4% PFA at room temperature for 20 minutes. The primary antibody reaction was performed by incubating the blocked sections on a slide with the primary antibody reaction solution at 4°C overnight, and the secondary antibody reaction was performed by incubating at room temperature for 60 minutes.

[0334] The organoids were fixed with 4% paraformaldehyde at room temperature for 30 minutes. After fixation, the organoids were blocked for 1 hour at 4°C with organoid blocking buffer consisting of PBS containing 0.2% bovine serum albumin (BSA) and 0.1% Triton X-100. Primary and secondary antibody reactions were performed by incubation overnight at 4°C. Before observation, the organoids were washed and incubated at room temperature for 20 minutes in 50% tissue removal reagent CUBIC-R+(M) (TCI).

[0335] Primary antibodies used included anti-human ALB antibody (Bethyl), anti-CYP3A4 antibody (Santa Cruz Biotechnology), anti-CD31 antibody (Agilent), and anti-LYVE1 antibody (Abcam). Images were acquired using a Leica TCS SP8 confocal microscope (Leica).

[0336] 3-2. Human albumin assay: The levels of human albumin were measured in the culture medium of organoids recovered and stored at -30°C using a human albumin ELISA quantification kit (Bethyl Laboratories).

[0337] 3-3. CYP3A Activity Assay To perform the assay, organoids were incubated in fresh medium containing the luminescent CYP substrate luciferin-PFBE (Promega) at a concentration of 50 μM at 37°C for 4 hours. After incubation, the supernatant was collected, and CYP3A4 activity was measured by reacting and detecting luciferin using the CYP450-Glo Assay Kit (Promega) according to the manufacturer's protocol. The measured values ​​were calculated as relative luminescence units (RLU / mL) per unit volume of medium.

[0338] 3-4. Ammonia metabolism assay: To perform the assay, use 1% B27 and 2 mM NH4 + Organoids were incubated at 37°C for 24 hours in 1 mL of RPMI-1640 medium (Fujifilm Wako Pure Chemical Industries) to which [substance name] was added. Before incubation (C0) and after incubation (C0) 24 The ammonia concentration per 1 mL in the recovered culture medium was measured using a PocketChem BA Blood Ammonia Analyzer (Arkray) according to the manufacturer's protocol. Ammonia metabolism was calculated based on the following formula: Ammonia metabolism (% / 24h) = {(C0 - C 24 ) / C0} × 100.

[0339] 3-5. Transmission electron microscopy observation: Organoids were fixed with 4% paraformaldehyde and 4% glutaraldehyde solutions in 0.1 M phosphate buffer. The fixed samples were cut into 1 mm × 1 mm blocks and incubated in equal volumes of 4% paraformaldehyde and 4% glutaraldehyde solutions at 4°C for 1 hour, followed by incubation overnight in 2% glutaraldehyde solution in 0.1 M phosphate buffer at 4°C. The fixed samples were post-fixed with 2% tetrooxide osmium, dehydrated using a series of ethyl alcohols, and embedded in fresh 100% resin.

[0340] Ultrathin sections of 70 nm were cut with an ultramicrotome (Leica, Ultracut UCT) and stained with 2% uranyl acetate. The sections were washed with distilled water and stained with lead staining solution. Observation was performed using a JEM-1400Plus transmission electron microscope (JEOL Ltd) at an acceleration voltage of 80 kV. A CCD camera (Olympus Soft Imaging Solutions GmbH, VELETA) was used to acquire digital images.

[0341] (Results) The results are shown in Figures 10-13. As shown in Figure 10, developed vascular structures were observed in liver organoids containing HSCs (LO-HSC; Figure 10A), whereas no clear vascular structures were observed in liver organoids without HSCs (LO-ΔHSC; Figure 10B). Furthermore, LO-HSCs showed higher expression levels of vascular endothelium-related genes CD34 and sinusoid-related genes (FVIII, MRC1), and a decrease in caspase 3 / 7-positive cells. This indicates that including HSCs leads to active induction of vascular structures and an increase in cell viability.

[0342] Similar results were supported from the perspective of liver function. Assays on albumin production and CYP3A4 secretion confirmed that the production and secretion levels of each were significantly increased by including HSCs. As shown in Figures 11A and 11B, LO-HSC (Figure 11A) showed a large number of albumin-producing cells (left column in Figures 11A and 11B) and CYP3A4-producing cells (left column in Figures 11A and 11B), and these cells were also organized, whereas LO-ΔHSC (Figure 11B) showed a small amount of both types of cells and they were not organized. Furthermore, as shown in Figure 11C, the metabolic rate of NH4 also significantly increased, by about three times, when HSCs were included ("+HSC") compared to when HSCs were not included ("-HSC" in the figure). Furthermore, as shown in Figure 12, LO-HSCs showed expression of CD31 (Figure 12A) and LYVE1 (Figure 12B), which are markers for hepatic sinusoidal endothelial cells, which are vascular endothelial cells unique to the liver, and a complex sinusoidal structure was formed. In contrast, LO-ΔHSCs (Figures 12C and D) showed little expression of either marker and no sinusoidal formation was observed.

[0343] In LO-HSCs, including HSCs, the formation of liver-specific ultrastructures, similar to those found in in vivo liver tissue, was confirmed. As shown in Figures 13A and 13B, hepatocytes exhibited a polygonal morphology, and a lumen (indicated as "Lumen" in Figure 13A) was observed. Further detailed observation revealed cytoplasmic glycogen (indicated as "Glycogen" in Figure 13B) in some cells, and numerous microvilli were observed between hepatocytes (indicated as "Microvillus" in Figure 13B). Furthermore, the formation of fine and complex structures that contribute to the performance of liver-specific functions, such as tight junctions between hepatocytes (indicated as "Tight junction" in Figure 13C) and bile canaliculi (indicated as "bile canaliculi" in Figure 13C), was confirmed. Furthermore, when activated iPSC-HSCs or pHSCs were used instead of quiescent iPSC-HSCs, vascular structure formation occurred, but the number of vascular branches and the branch depth were significantly lower compared to quiescent iPSC-HSCs. This indicates that complex vascular structures are less likely to form when activated HSCs are used.

[0344] From the above, it was demonstrated that liver organoids with liver-specific functions and structures can be prepared by including HSCs.

[0345] <Example 4. Transplantation of liver organoids containing hiPSC-HSCs> (Objective) Liver organoids were prepared in vitro from hepatic stellate cells (hiPSC-HSCs) differentiated from iPS cells, and transplanted into mice to confirm that the prepared liver organoids could engraft after transplantation.

[0346] (Methods) Six-week-old type 1 diabetes mellitus / severe combined immunodeficiency (NOD / SCID) mice (CREA Japan) were used as transplantation subjects. The mice were reared and maintained in accordance with the guidelines established by the University of Tokyo for the use of experimental animals.

[0347] Organoids (LO-HSC or LO-ΔHSC) were transplanted into the anteform cranial fenestra of NOD / SCID mice after 15 hours of co-culture. In vivo imaging was performed using a Leica TCS SP8 confocal microscope to monitor the transplanted organoids in real time. Blood samples were collected 28 days after transplantation to measure albumin production. In addition, Alexa647-conjugated mouse-specific CD31 (BD Pharmingen) was administered to the transplanted mice to visualize vascular endothelial cells.

[0348] Organoid staining and other procedures were carried out in accordance with Example 3. Albumin production was measured and calculated in the same manner as in Example 3 for serum samples collected by centrifuging blood samples at 400 × g at 4°C for 11 minutes and stored at -30°C. Blood cells and coagulation factors were excluded from the serum samples.

[0349] (Results) The results are shown in Figures 14 and 15. Three days after transplantation, angiogenesis was confirmed in all sites in the LO-HSC transplantation group, while angiogenesis was not confirmed in some sites in the LO-ΔHSC transplantation group. In vivo imaging showed extensive formation of organoid-derived human blood vessels both inside and outside the organoid seven days after transplantation in the LO-HSC transplantation group (Figure 14A). On the other hand, the formation of organoid-derived human blood vessels was poor in the LO-ΔHSC transplantation group (Figure 14B).

[0350] At 28 days post-transplantation, the transplanted LO-HSCs formed a multilayered and highly branched vascular structure and successfully engrafted. In contrast, the engraftment rate was poor when LO-ΔHSC transplants were received. In particular, as shown in Figure 15, continuous engraftment of mouse and human blood vessels was confirmed, and hiPSC-HSCs adhered closely to these newly formed human blood vessels, showing a typical cell distribution observed in natural liver tissue (white arrows in the figure). This is further supported by the significant observation of human albumin produced in organoids from mouse blood.

[0351] Furthermore, the transplanted organoids also showed the presence of albumin-positive cell clusters, CYP3A4-positive cell clusters, and bile ducts, as well as the formation of complex sinusoids.

[0352] From the above, it has been shown that by including the quiescent hepatic stellate cells of the present invention, a functional organoid is provided that has a high engraftment rate even after transplantation into vivo and can form a complex vascular structure together with the endogenous cells of the transplanted individual.

[0353] <Example 5. Effect of the FGF2 downstream signaling pathway on hiPSC-HSC differentiation> (Objective) The effect of the FGF2 downstream signaling pathway on HSC differentiation was investigated by using various inhibitors of the FGF2 downstream signaling pathway.

[0354] (Methods) Differentiation induction of iPS cells into hepatic stellate cells (HSCs) and qPCR analysis were performed in the same manner as in Example 1. For differentiation induction into hepatic stellate cells, SFD medium with or without FGF2 was used, and in the SFD medium with FGF2, an inhibitor of the FGF2 downstream signaling pathway was added. As a control, conditions with only the same amount of DMSO solvent added were used. The experiment was repeated four times.

[0355] As inhibitors of the FGF2 downstream signaling pathway, we used BGJ398 (APExBIO; final concentration: 100 nM), an FGFR inhibitor; trametinib (MedChemExpress; final concentration: 10 nM), a RAS / MAP kinase pathway inhibitor; copanlisib (MedKoo Biosciences, Inc.; final concentration: 100 nM), a PI3 kinase / AKT pathway inhibitor; U-73122 (MedChemExpress; final concentration: 1 μM), a PLCγ pathway inhibitor; and Static (MedChemExpress; final concentration: 100 nM), a STAT inhibitor.

[0356] The cell count was measured on day 4 of differentiation induction from hiPSC-STM to hepatic stellate cells.

[0357] The multiplier change in gene expression levels was calculated as a relative value, with the condition using SFD medium supplemented with FGF2 and only DMSO solvent added set as 1.

[0358] (Results) The results are shown in Figure 16. When no inhibitor was used (indicated as "DMSO" in Figure 16), the number of cells increased significantly when FGF2 was added compared to when it was not added, and the expression of hepatic stellate cell activation markers such as CAL1A1 and LOX was significantly reduced. These results are consistent with those of Example 1.

[0359] When various inhibitors were added, the groups treated with BGJ398 or trametinib (in Figure 16, labeled "BGJ398" and "Trametinib") showed a decrease in cell count to a level comparable to that of the group without FGF2, and a significant increase in the expression of hepatic stellate cell activation markers. Furthermore, in the group treated with copanlisib (in Figure 16, labeled "Copanlisib"), only the cell count showed a change to a level comparable to that of the group without FGF2.

[0360] This suggests that the activity of the RAS / MAP kinase pathway, in particular, contributes to the differentiation of hepatic stellate cells into quiescent cells, and that the PI3 kinase / AKT pathway also contributes, especially to cell proliferation during the differentiation process.

[0361] <Example 6. Effect of the FGF2 downstream signaling pathway on hiPSC-HSC proliferation> (Objective) The effect of the FGF2 downstream signaling pathway on HSC proliferation was investigated by using various inhibitors of the FGF2 downstream signaling pathway.

[0362] (Methods) The procedure for hepatic stellate cell differentiation was the same as in Example 1. In the subculture of hepatic stellate cells (culture from the first subculture onward), the culture medium was changed to SFD medium supplemented with or without the inhibitor of FGF2, or SFD medium without FGF2. The procedure was the same as in Example 5. The experiment was repeated four times.

[0363] (Results) The results are shown in Figure 17. When no inhibitor was used (in Figure 17, labeled "DMSO"), the number of cells increased significantly when FGF2 was added compared to when it was not added, and the expression of hepatic stellate cell activation markers such as CAL1A1 and LOX was significantly reduced.

[0364] When various inhibitors were added, the groups treated with BGJ398, trametinib, or copanlisib (in Figure 17, labeled "BGJ398," "Trametinib," and "Copanlisib") showed a decrease in cell count to a level comparable to that of the group not treated with FGF2, while simultaneously significantly increasing the expression of hepatic stellate cell activation markers.

[0365] This suggests that the proliferation of quiescent hepatic stellate cells is influenced by the RAS / MAP kinase pathway and the PI3 kinase / AKT signaling pathway, both part of the FGF2 signaling pathway. All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A quiescent hepatic stellate cell inducer containing an FGF signaling pathway activator.

2. A quiescent hepatic stellate cell induction medium containing an FGF signaling pathway activator and serum-free medium.

3. The quiescent hepatic stellate cell induction medium according to claim 2, further comprising an antioxidant.

4. A quiescent hepatic stellate cell proliferation promoter containing an FGF signaling pathway activator.

5. A quiescent hepatic stellate cell proliferation medium containing an FGF signaling pathway activator and serum-free medium.

6. A method for producing quiescent hepatic stellate cells, comprising a quiescent hepatic stellate cell induction step of inducing and culturing transverse septal mesenchymal cells in the quiescent hepatic stellate cell induction medium described in claim 2, thereby inducing differentiation into quiescent hepatic stellate cells.

7. The production method according to claim 6, further comprising a proliferation step of growing and culturing the induced quiescent hepatic stellate cells in the quiescent hepatic stellate cell proliferation medium according to claim 5.

8. Restless hepatic stellate cells produced by the method of claim 6 or 7.

9. A method for inducing quiescent hepatic stellate cells, comprising a step of inducing and culturing transverse septal mesenchymal cells in the quiescent hepatic stellate cell induction medium described in claim 2 or 3, and inducing differentiation into quiescent hepatic stellate cells.

10. A method for producing activated hepatic stellate cells, comprising a quiescent hepatic stellate cell induction step of inducing and culturing transverse septal mesenchymal cells in the quiescent hepatic stellate cell induction medium described in claim 2 or 3 to induce differentiation into quiescent hepatic stellate cells, and an activation step of activating and culturing the induced quiescent hepatic stellate cells.

11. A method for producing liver organoids, comprising a co-culture step of co-culturing quiescent hepatic stellate cells described in claim 8 with vascular endothelial cells and hepatic progenitor cells.

12. A cell preparation comprising quiescent hepatic stellate cells as described in claim 8.

13. A non-human animal comprising quiescent hepatic stellate cells as described in claim 8.

14. An organoid comprising quiescent hepatic stellate cells as described in claim 8.

15. A non-human animal comprising the organoid described in claim 14.

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