Method for producing multilayered liver organoid

Abstract

The present invention addresses the problem of providing a method for forming a complex tissue structure and preparing a liver organoid that faithfully reproduces a healthy liver in which a pathological state can be mimicked by a simple process, and further providing a simple method for preparing a steatotic liver disease model organoid in which it is possible to reproduce the degeneration and complex hepatopathy that are specific to steatotic liver disease.　Provided is a method for producing a multilayered liver organoid, said method including: a cocultivation step for cocultivating source cells; a differentiation inducement step for inducing differentiation of the source cells; and a tissue construction step for constructing a tissue structure that is composed of a plurality of layers of cells, wherein the source cells include hepatoblasts, vascular endothelial progenitor cells, and mesenchymal cells.

Description

Method for producing multi-layered liver organoids

[0001] The present invention relates to multi-layered liver organoids and fatty liver disease model organoids, and a method for producing the same.

[0002] The liver is a complex organ composed of various cells such as hepatocytes, vascular (sinusoid) endothelial cells, Kupffer cells, and bile duct cells, and forms a characteristic tissue structure. Nevertheless, it is a highly regenerable organ that can recover from damage. This is due to the plasticity of hepatocytes, which account for about 70% of liver tissue. However, when the liver is damaged excessively beyond its regenerative capacity, or when it is damaged under circumstances where its regenerative capacity itself has been reduced or impaired due to aging of hepatocytes or the like, liver function is significantly reduced and liver failure ensues. This liver failure is a serious disorder with a high mortality rate.

[0003] Liver transplantation is used as a treatment method for such liver failure. Although liver transplantation has conventionally been known as an effective treatment method capable of curing liver failure, there is a problem that the shortage of transplantable donor livers is a bottleneck and it is difficult for patients to receive treatment at the necessary timing. The use of cell replacement therapy has been explored as an alternative means that does not require a large-scale operation and is simpler than liver transplantation. However, since cell replacement therapy is a method of administering donor liver-derived hepatocytes including hepatocytes, etc., there is also a problem that it is also affected by the number of donor livers. In addition, in these methods, depending on the combination of the donor liver and the recipient, even in a state where the recipient's immunity is suppressed and the rejection reaction is reduced, the engraftment rate one year after transplantation of the transplanted cells is low, and the long-term therapeutic effect may be limited.

[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 efficacy of drugs and the presence or absence of side effects in the liver, and detailed research on the mechanisms of liver development and functions. 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, the necessary cells are not included, and thus some tissue structures such as the vascular structure are often not sufficiently constructed, and it has been 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 can faithfully reproduce the complex tissue structure of the liver has been desired.

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

[0007] In particular, it has been difficult to construct disease models with conventional organoids. This is because diseases are caused by abnormalities in homeostasis based on a variety of cells and complex tissue structures, and sufficient imitation cannot be achieved without faithfully reproducing the liver in the adult body.

[0008] For example, in organoids containing hepatocytes, hepatic stellate cells, and Kupffer cells described in Non-Patent Document 1, a fatty liver disease model has been constructed by loading high glucose, oleic acid, and palmitic acid. However, degenerations characteristic of fatty liver diseases such as ballooning degeneration and Mallory bodies have not been observed, and since the cells are in a single layer and there is no tissue structure, degenerations of tissue structures such as the migration of inflammatory cells cannot be reproduced (Figure 1A).

[0009] Furthermore, for example, in Ouchi R, et al. Cell Metab. 2019;30(2):374-384.e6., organoids containing hepatocytes, Kupffer cells, and hepatic stellate cells were prepared, and a fatty liver disease model was constructed by loading lipopolysaccharide for inflammation induction in addition to oleic acid. However, even here, only a planar structure made up of a single layer of cells was constructed, and a complex tissue structure was not established. Therefore, degenerations characteristic of fatty liver disease, such as balloon-like degeneration and Mallory bodies, were not observed, and the inflammatory response was limited to an increase in inflammatory factors known to be induced by lipopolysaccharide (Figure 1A).

[0010] While these model organoids require complex processing to induce disease-like states, the changes observed are limited to individual changes in the cells within the organoids. Furthermore, the absence of sinusoidal endothelial cells and complex tissue structures means that characteristic liver lesions and complex liver damage are not reproduced.

[0011] Furthermore, since hepatic stellate cells isolated from living organisms and hepatic stellate cells prepared in vitro are almost always pathological activated hepatic stellate cells, it can be inferred that these organoids containing such hepatic stellate cells are partially pathological even before the addition of various disease-inducing factors.

[0012] Therefore, the object of the present invention is to provide a method for preparing liver organoids that faithfully reproduce a healthy liver, forming a complex tissue structure and enabling the mimicry of disease conditions through simple processing, and further, to provide a simple method for preparing fatty liver disease model organoids that can reproduce the degeneration and complex liver damage characteristic of fatty liver disease.

[0013] To address the above challenges, the inventors diligently conducted research based on the strategy of using progenitor cells rather than differentiated cells in organoid preparation, and increasing the variety of cells used. As a result, they discovered that by using hepatic progenitor cells, mesenchymal cells, and vascular endothelial progenitor cells, and particularly by using hepatoblasts as hepatic progenitor cells, it is possible to prepare liver organoids that exhibit improved liver function and possess complex tissue structures such as liver-specific sinusoidal structures and surrounding cell accumulations. These hepatoblasts are cells that can be mass-produced in vitro using the inventors' unique method.

[0014] Furthermore, we discovered that by adding hepatic stellate cells and monocyte cells, it is possible to prepare liver organoids containing Kupffer cells and inactive, normal hepatic stellate cells. We found that this liver organoid can be used to produce a fatty liver disease model organoid that can reproduce characteristic lesions such as liver dysfunction and ballooning degeneration simply by adding lipid molecules (Figure 1B). The present invention is based on these novel findings and provides the following.

[0015] [1] A method for producing a multilayer liver organoid, comprising a co-culture step of co-culturing raw material cells, a differentiation induction step of inducing differentiation of the raw material cells, and a tissue construction step of constructing a tissue structure composed of multiple layers of cells, wherein the raw material cells include hepatoblasts, vascular endothelial progenitor cells, and mesenchymal cells. [2] The method according to [1], wherein the raw material cells further comprise hepatic stellate cells. [3] The method according to [2], wherein the hepatic stellate cells are quiescent hepatic stellate cells. [4] The method according to any one of [1] to [3], wherein the raw material cells further comprise monocyte cells. [5] The method according to any one of [1] to [4], wherein the tissue construction step comprises the construction of a vascular structure. [6] The method according to any one of [1] to [5], further comprising a proliferation culture step of culturing the raw material cells. [7] The method according to any one of [1] to [6], further comprising a raw material cell preparation step of preparing the raw material cells from pluripotent stem cells. [8] A multilayer liver organoid produced by any one of [1] to [7]. [9] A multilayer liver organoid comprising hepatic parenchymal cells, hepatic stellate cells, hepatic sinusoidal endothelial cells, and Kupffer cells.

[10] The multilayer liver organoid according to [9], comprising a vascular structure.

[11] A method for producing a fatty liver disease model organoid, comprising a lipid addition step of adding lipid molecules to a multilayer liver organoid comprising hepatic parenchymal cells, hepatic stellate cells, hepatic sinusoidal endothelial cells, and Kupffer cells, and a disease progression step of culturing the multilayer liver organoid to which lipid molecules have been added to advance the disease state.

[12] The method according to

[11] , wherein the lipid molecules comprise free fatty acids.

[13] The method according to

[11] or

[12] , wherein the multilayer liver organoid is the organoid according to [8].

[14] A fatty liver disease model organoid produced by any of the methods of

[11] to

[13] .

[15] A fatty liver disease model organoid comprising hepatic parenchymal cells, hepatic stellate cells, hepatic sinusoidal endothelial cells, and Kupffer cells that have accumulated lipids intracellularly.

[16] The fatty liver disease model organoid according to

[15] , further comprising balloon-like degenerated liver parenchymal cells.

[17] A non-human animal comprising the organoid according to any one of [8] to

[10] .

[18] A method for producing a fatty liver disease model non-human animal, comprising a lipid administration step of administering lipid molecules to the non-human animal according to

[17] .This specification includes the disclosures of Japanese Patent Application No. 2024-191727, which forms the basis of the priority claim of this application.

[0016] According to the method for producing multilayer liver organoids of the present invention, it is possible to produce liver organoids having a complex tissue structure.

[0017] The present invention provides a method for producing fatty liver disease model organoids that exhibit the characteristic pathological features of fatty liver disease.

[0018] Figure 1 schematically shows the differences between the conventional method and the method of the present invention. Figure 1A shows an overview of the conventional method for preparing liver organoids and the method for constructing a fatty liver disease model from those organoids, as well as its problems. Figure 1B shows an overview of the method for preparing liver organoids and the method for constructing a fatty liver disease model from those organoids according to the present invention. In the figures, dashed arrows indicate raw material cells that are not essential or differ depending on the method. Figure 2 shows the results of qPCR analysis comparing the properties of liver organoids containing hepatic endodermal cells (HE) and liver organoids containing hepatoblasts (HB) in Example 1. In the figure, "HE" indicates the results for liver organoids containing HE (n=6), and "HB" indicates the results for liver organoids containing HB (n=6). In the figure, "Fold Change" shows the value standardized with the results for liver organoids containing HE set to 1. In the figure, error bars indicate the standard deviation, "**" indicates that the p-value is p < 0.01, and "****" indicates that the p-value is p < 0.0001. Figure 3 shows the results of a comparison of the properties of liver organoids containing hepatic endodermal cells (HE) and liver organoids containing hepatoblasts (HB) in Example 1. Figure 3A shows the results for albumin production, Figure 3B shows the results for urea production, Figure 3C shows the results for CYP3A4 activity, and Figure 3D shows the results for ammonia metabolism. In the figures, "HE" indicates the results for liver organoids containing HE (n=6), and "HB" indicates the results for liver organoids containing HB (n=6). Error bars indicate the standard deviation, "*" indicates a p-value of p < 0.05, "**" indicates a p-value of p < 0.01, and "****" indicates a p-value of p < 0.0001. Figure 4 shows the appearance of hepatic stellate cells in liver organoids in Example 2. Figure 4A shows the αSMA signal, and Figure 4B shows the GFP signal of hepatic stellate cells. In the figures, arrowheads indicate exemplary αSMA-negative hepatic stellate cells. Figure 5 shows the results of immunohistochemical staining in Example 2. Figure 5A shows an overlay image of Figures 5B and 5C, where Figure 5B shows the signaling pattern of FCGR2β and Figure 5C shows the signaling pattern of CD31. In the figures, the arrows indicate the regions where FCGR2β and CD31 are co-expressed. Figure 6 is a scanning electron microscope image of the cell membrane of hepatic sinusoidal endothelial cells in Example 2.The white dashed frame indicates the location of the cribriform plate structure on the cell membrane. Figure 7 shows the results of qPCR analysis of monocyte cells before co-culture and monocyte cells derived from liver organoids in Example 2. In the figure, "D0" indicates the results for monocyte cells before co-culture (n=3), and "D10" indicates the results for monocyte cells derived from liver organoids (n=3). In the figure, "Fold Change" indicates the value standardized to 1 for the results of monocyte cells before co-culture including HE. In the figure, error bars indicate the standard deviation, "**" indicates that the p-value is p < 0.01, "***" indicates that the p-value is p < 0.001, and "****" indicates that the p-value is p < 0.0001. Figure 8 is a transmission electron microscope image showing the ultrastructure of liver organoids in Example 2. Figure 8A shows the ultrastructure around the sinusoidal lumen, and Figure 8B shows the ultrastructure between hepatocytes. In the figure, "Hep" indicates hepatocytes. In Figure 8A, "Lu" indicates the sinusoidal lumen, "*" indicates hepatic sinusoidal endothelial cells, "†" indicates Kupffer cells, "SD" indicates the space of Disse, and "\" indicates hepatic stellate cells. In Figure 8B, "BC" indicates bile canaliculi, and "TJ" indicates tight junctions. Figure 9 shows the lipid accumulation in liver organoids in Example 3. Figure 9A shows the results for liver organoids without free fatty acid (FFA) loading, and Figure 9B shows the results for liver organoids with FFA loading. Figure 10 is a bright-field image showing balloon-like degeneration of liver organoids in Example 3. Figures 10A and 10B show the results for liver organoids without FFA loading, and Figures 10C and 10D show the results for liver organoids with FFA loading. Figures 10A and 10C show enlarged images of the black-framed areas in Figures 10B and 10D, respectively. In the figures, "Lu" indicates the sinusoidal lumen. Figure 11 is a fluorescence-stained image showing the Mallory bodies of liver organoids in Example 3. Figure 11A shows the results for liver organoids without free fatty acid (FFA) loading, and Figure 11B shows the results for liver organoids with FFA loading. Figure 12 shows the results of balloon-like degeneration and quantification of reactive oxygen species in liver organoids in Example 3. Figure 12A shows the results for the amount of the p62 signal, a Mallory body marker, and Figure 12B shows the results for the amount of reactive oxygen species (ROS).In the figures, "-FFA" indicates the results for liver organoids that were not subjected to FFA loading (Figure 12A: n=6; Figure 12B: n=10), and "+FFA" indicates the results for liver organoids that were subjected to FFA loading (Figure 12A: n=6; Figure 12B: n=10). In the figures, error bars indicate the standard deviation, "**" indicates that the p-value is p < 0.01, and "***" indicates that the p-value is p < 0.001. Figure 13 shows the changes in inflammatory factors in liver organoids in Example 3. Figure 13A shows the changes in the expression levels of inflammation-related genes, and Figure 13B shows the changes in IL6 secretion. In the figures, "-FFA" indicates the results for liver organoids that were not subjected to FFA loading (n=4 to 6), and "+FFA" indicates the results for liver organoids that were subjected to FFA loading (n=5 to 7). In Figure 13A, "Fold change" shows the value standardized to 1 for the results of liver organoids that were not subjected to FFA loading. In the figure, error bars indicate the standard deviation, "*" indicates that the p-value is p < 0.05, and "**" indicates that the p-value is p < 0.01. Figure 14 shows the hepatic stellate cells in liver organoids in Example 3. Figure 14A shows the αSMA signal, and Figure 14B shows the GFP signal of hepatic stellate cells. In the figure, arrows indicate exemplary αSMA-positive hepatic stellate cells. Figure 15 shows the change in the amount of hepatic stellate cells ("HSC Area") in liver organoids in Example 3. In the figure, "-FFA" shows the results for liver organoids that were not subjected to FFA loading (n=6), and "+FFA" shows the results for liver organoids that were subjected to FFA loading (n=6). In the figure, "D0" shows the results before FFA loading, "D4" shows the results 4 days after the start of FFA loading, "D7" shows the results 7 days after the start of FFA loading, and "D10" shows the results 10 days after the start of FFA loading. In the figure, error bars indicate the standard deviation, "**" indicates that the p-value when comparing the results with and without FFA loading is p < 0.01, and "***" indicates that the p-value when comparing the results with and without FFA loading is p < 0.001. Figure 16 shows the results of liver function measurements of liver organoids in Example 3. Figure 16A shows the results of albumin secretion, Figure 16B shows the results of urea production, and Figure 16C shows the results of CYP3A4 activity.In the figure, "-FFA" indicates the results for liver organoids without FFA loading (n=6-10), and "+FFA" indicates the results for liver organoids with FFA loading (n=6-10). In the figure, error bars indicate the standard deviation, "*" indicates that the p-value is p < 0.05, and "**" indicates that the p-value is p < 0.01. Figure 17 shows the evaluation results of fatty liver disease treatment drugs using fatty liver disease model organoids in Example 4. In the figure, "Control" shows the results for fatty liver disease model organoids prepared without using drugs, and each of the other series shows the results for fatty liver disease model organoids prepared using the drugs described in each series. In the figure, error bars indicate the standard deviation, and "***" indicates that the p-value is p < 0.001 when compared with the results for fatty liver disease model organoids prepared without using drugs. Figure 18 shows the evaluation results of fatty liver disease treatment drugs using fatty liver disease model organoids in Example 4. In the figure, "Control" shows the results of fatty liver disease model organoids prepared without the use of drugs, while each of the other series shows the results of fatty liver disease model organoids prepared using the drugs described in each series. In the figure, error bars indicate the standard deviation, "*" indicates that the p-value compared to the results of fatty liver disease model organoids prepared without the use of drugs is p < 0.05, "**" indicates that the p-value compared to the results of fatty liver disease model organoids prepared without the use of drugs is p < 0.01, and "***" indicates that the p-value compared to the results of fatty liver disease model organoids prepared without the use of drugs is p < 0.001. Figure 19 shows the evaluation results of fatty liver disease treatment drugs using fatty liver disease model organoids in Example 4. In the figure, "Control" shows the results of fatty liver disease model organoids prepared without the use of drugs, while each of the other series shows the results of fatty liver disease model organoids prepared using the drugs described in each series. In the figure, error bars indicate the standard deviation, "**" indicates that the p-value is p < 0.01 when compared to the results of fatty liver disease model organoids prepared without the use of drugs, and "ns" indicates that there is no significant difference compared to the results of fatty liver disease model organoids prepared without the use of drugs.Figure 20 shows the evaluation results of UMAP (Uniform Manifold Approximation and Projection) analysis of the cellular composition of liver organoids (Figure 20A) and human liver (Figure 20B) in Example 5. In the figure, "Hep" represents a cluster of hepatocytes, "Cholangiocyte" represents a cluster of bile duct epithelial cells, "LSEC" represents a cluster of hepatic sinusoidal endothelial cells, "HSC" represents a cluster of hepatic stellate cells, "VSMC" represents a cluster of vascular smooth muscle cells, "Macrophage" represents a cluster of macrophages, "Kupffer" represents a cluster of Kupffer cells, "B_cell" represents a cluster of B cells, "T_cell" represents a cluster of T cells, "NK_cell" represents a cluster of natural killer cells, "Vascular_EC" represents a cluster of vascular endothelial cells, "Monocyte" represents a cluster of monocyte cells, and "Fibroblast" represents a cluster of fibroblasts. Each axis in the figure represents the axis in the UMAP analysis. Figure 21 shows the expression of marker genes by cell type in liver organoids (left in the figure) and human liver (right in the figure) in Example 5. In the figure, "Hep" shows the results for hepatocytes, "LSEC" shows the results for hepatic sinusoidal endothelial cells, "Kupffer" shows the results for Kupffer cells, "Mac" shows the results for macrophages, "HSC" shows the results for hepatic stellate cells, and "VSMC" shows the results for vascular smooth muscle cells. Each row in the figure shows a violin plot of the marker genes for the various cells described on the right. Figure 22 shows the results of immunohistochemistry in Example 5. Figure 22A shows the signal for ALB, and Figure 22B shows the signal for CYP3A4. In the figure, the arrows indicate representative cells in which ALB and CYP3A4 are co-expressed. Figure 23 shows the results of immunohistochemistry in Example 5. Figure 23A shows the signal for CK19, and Figure 23B shows the signal for SOX9. In the figure, the arrows indicate representative sites in which CK19 and SOX9 are co-expressed. Figure 24 shows the results of immunohistochemical staining in Example 5. Figure 24A shows the signal for LYVE-1, and Figure 24B shows the signal for CD31.In the figure, the arrows indicate representative sites where LYVE-1 and CD31 are co-expressed. Figure 25 shows the results of immunohistochemistry in Example 5. Figure 25A shows the signal of CD31, and Figure 25B shows the signal of CD68. In the figure, the dashed ellipse indicates the region containing a cross-section of the hepatic sinusoids bordered by CD31-positive cells. Figure 26 shows the evaluation results of UMAP analysis of the cellular composition of human liver in Example 6. Figure 26A shows the evaluation results of a normal human liver, and Figure 26B shows the evaluation results of a human liver that corresponds to classification 6 by NAS. In the figure, "Hep" represents a cluster of hepatocytes, "Cholangiocyte" represents a cluster of bile duct epithelial cells, "LSEC" represents a cluster of hepatic sinusoidal endothelial cells, "HSC" represents a cluster of hepatic stellate cells, "Macrophage" represents a cluster of macrophages, "Kupffer" represents a cluster of Kupffer cells, "B_cell" represents a cluster of B cells, "T_cell" represents a cluster of T cells, "NK_cell" represents a cluster of natural killer cells, "Vascular_EC" represents a cluster of vascular endothelial cells, "Monocyte" represents a cluster of monocyte cells, "VSMC" represents a cluster of vascular smooth muscle cells, and "Fibroblast" represents a cluster of fibroblasts. Each axis in the figure represents the axis in UMAP analysis. Figure 27 shows the evaluation results of the UMAP analysis of the cell composition of human liver in Example 6. Figure 27A shows the evaluation results of a human liver corresponding to classification 2 by NAS, and Figure 27B shows the evaluation results of a human liver corresponding to classification 4 by NAS.In the figure, "Hep" represents a cluster of hepatocytes, "Cholangiocyte" represents a cluster of bile duct epithelial cells, "LSEC" represents a cluster of hepatic sinusoidal endothelial cells, "HSC" represents a cluster of hepatic stellate cells, "Macrophage" represents a cluster of macrophages, "Kupffer" represents a cluster of Kupffer cells, "B_cell" represents a cluster of B cells, "T_cell" represents a cluster of T cells, "NK_cell" represents a cluster of natural killer cells, "Vascular_EC" represents a cluster of vascular endothelial cells, "Monocyte" represents a cluster of monocyte cells, "VSMC" represents a cluster of vascular smooth muscle cells, and "Fibroblast" represents a cluster of fibroblasts. Each axis in the figure represents the axis in UMAP analysis. Figure 28 shows the evaluation results of the UMAP analysis of the cellular composition of liver organoids in Example 6. Figure 28A shows the evaluation results of liver organoids before MASH induction, and Figure 28B shows the evaluation results of liver organoids after MASH induction. In the figure, "Hep" represents a cluster of hepatocytes, "Cholangiocyte" represents a cluster of bile duct epithelial cells, "LSEC" represents a cluster of hepatic sinusoidal endothelial cells, "HSC" represents a cluster of hepatic stellate cells, "Macrophage" represents a cluster of macrophages, "Kupffer" represents a cluster of Kupffer cells, "B_cell" represents a cluster of B cells, "T_cell" represents a cluster of T cells, "NK_cell" represents a cluster of natural killer cells, "Vascular_EC" represents a cluster of vascular endothelial cells, "Monocyte" represents a cluster of monocyte cells, "VSMC" represents a cluster of vascular smooth muscle cells, and "Fibroblast" represents a cluster of fibroblast cells. Each axis in the figure represents the axis in UMAP analysis. Figure 29 shows the evaluation results of various cell ratios as the disease progresses in Example 6. In the figure, "Monocyte" represents a cluster of monocyte cells, "Macrophage" represents a cluster of macrophages, "LSEC" represents a cluster of hepatic sinusoidal endothelial cells, and "Hep" represents a cluster of hepatocytes.In the figure, "NAS0" shows the results for normal human liver, "NAS2" shows the results for human liver classified as 2 by NAS, "NAS4" shows the results for human liver classified as 4 by NAS, "NAS6" shows the results for human liver classified as 6 by NAS, "Con_LO" shows the results for liver organoids before MASH induction, and "MASH_LO" shows the results for liver organoids after MASH induction. The vertical axis of each row shows the proportion of each cell type in the sample of each column. Figure 30 shows the evaluation results of changes in gene expression levels in hepatocytes accompanying the progression of the disease in Example 6. Figure 30A shows the differences in genes whose expression levels increased with the progression of MASH, and Figure 30B shows the results of gene ontology analysis of genes that showed increased expression in both (1) and (4) in Figure 30A. In the figure, (1) shows the gene group whose expression level increased in liver organoids to which MASH was induced compared to liver organoids to which MASH was induced, (2) shows the gene group whose expression level increased in human livers classified as 2 by NAS compared to normal human livers, (3) shows the gene group whose expression level increased in human livers classified as 4 by NAS compared to normal human livers, and (4) shows the gene group whose expression level increased in human livers classified as 6 by NAS compared to normal human livers. In Figure 30B, "(a)" indicates a gene involved in inflammation, "(b)" indicates a gene involved in cytotoxicity, and "(c)" indicates a gene involved in fibrosis. "-log10 (Adj p value)" indicates that the higher the reciprocal of the natural logarithm of the standardized p-value related to the expression level of the gene belonging to that classification, the darker the color of the circle. "Gene Ratio" indicates that the larger the ratio of the number of genes belonging to that classification to the total number of genes analyzed, the larger the circle. Figure 31 shows the evaluation results of the changes in gene expression levels in Kupffer cells accompanying the progression of the disease in Example 6. Figure 31A shows the differences in genes whose expression levels increased with the progression of MASH, and Figure 31B shows the results of gene ontology analysis of genes that showed increased expression in both (1) and (4) in Figure 31A.In the figure, (1) shows the gene group whose expression level increased in liver organoids that underwent MASH compared to liver organoids before MASH induction, (2) shows the gene group whose expression level increased in human livers classified as 2 by NAS compared to normal human livers, (3) shows the gene group whose expression level increased in human livers classified as 4 by NAS compared to normal human livers, and (4) shows the gene group whose expression level increased in human livers classified as 6 by NAS compared to normal human livers. In Figure 30B, "(a)" indicates a gene involved in inflammation, "(b)" indicates a gene involved in cytotoxicity, and "(c)" indicates a gene involved in fibrosis. "-log10 (Adj p value)" indicates that the higher the reciprocal of the natural logarithm of the standardized p-value related to the expression level of the gene belonging to that classification, the darker the color of the circle. "Gene Ratio" indicates that the larger the ratio of the number of genes belonging to that classification to the total number of genes analyzed, the larger the circle. Figure 32 shows the evaluation results of the changes in gene expression levels in macrophages accompanying the progression of the disease in Example 6. Figure 32A shows the differences in genes whose expression levels increased with the progression of MASH, and Figure 32B shows the results of gene ontology analysis of genes whose expression levels increased in both (1) and (4) in Figure 32A. In the figures, (1) shows the gene population whose expression levels increased in liver organoids with MASH induced compared to liver organoids with MASH induced; (2) shows the gene population whose expression levels increased in human livers classified as 2 by NAS compared to normal human livers; (3) shows the gene population whose expression levels increased in human livers classified as 4 by NAS compared to normal human livers; and (4) shows the gene population whose expression levels increased in human livers classified as 6 by NAS compared to normal human livers.In Figure 30B, "(a)" indicates a gene involved in inflammation, "(b)" indicates a gene involved in cell damage, and "(c)" indicates a gene involved in fibrosis. "-log10(Adj p value)" indicates that the higher the reciprocal of the natural logarithm of the standardized p-value related to the expression level of the gene belonging to that classification, the darker the color of the circle. "Gene Ratio" indicates that the larger the ratio of the number of genes belonging to that classification to the total number of genes analyzed, the larger the circle. Figure 33 shows the evaluation results of changes in gene expression levels in monocyte cells accompanying the progression of the disease in Example 6. Figure 33A shows the differences in genes whose expression levels increased with the progression of MASH, and Figure 33B shows the results of gene ontology analysis of genes that showed increased expression in both (1) and (4) in Figure 33A. In the figure, (1) shows the gene group whose expression level increased in liver organoids to which MASH was induced compared to liver organoids to which MASH was induced, (2) shows the gene group whose expression level increased in human livers classified as 2 by NAS compared to normal human livers, (3) shows the gene group whose expression level increased in human livers classified as 4 by NAS compared to normal human livers, and (4) shows the gene group whose expression level increased in human livers classified as 6 by NAS compared to normal human livers. In Figure 30B, "(a)" indicates a gene involved in inflammation, "(b)" indicates a gene involved in cell damage, and "(c)" indicates a gene involved in fibrosis. "-log10 (Adj p value)" indicates that the higher the reciprocal of the natural logarithm of the standardized p-value related to the expression level of the gene belonging to that classification, the darker the color of the circle. "Gene Ratio" indicates that the larger the ratio of the number of genes belonging to that classification to the total number of genes analyzed, the larger the circle. Figure 34 shows the evaluation results of the changes in gene expression levels in hepatic stellate cells accompanying the progression of the disease in Example 6. Figure 34A shows the differences in genes whose expression levels increased with the progression of MASH, and Figure 34B shows the results of gene ontology analysis of genes that showed increased expression in both (1) and (4) in Figure 34A.In the figure, (1) shows the gene group whose expression level increased in liver organoids to which MASH was induced compared to liver organoids to which MASH was induced, (2) shows the gene group whose expression level increased in human livers classified as 2 by NAS compared to normal human livers, (3) shows the gene group whose expression level increased in human livers classified as 4 by NAS compared to normal human livers, and (4) shows the gene group whose expression level increased in human livers classified as 6 by NAS compared to normal human livers. In Figure 30B, "(a)" indicates a gene involved in inflammation, "(b)" indicates a gene involved in cell damage, and "(c)" indicates a gene involved in fibrosis. "-log10 (Adj p value)" indicates that the higher the reciprocal of the natural logarithm of the standardized p-value related to the expression level of the gene belonging to that classification, the darker the color of the circle. "Gene Ratio" indicates that the larger the ratio of the number of genes belonging to that classification to the total number of genes analyzed, the larger the circle. Figure 35 shows the evaluation results of the changes in gene expression levels in liver fibroblasts accompanying the progression of the disease in Example 6. Figure 35A shows the differences in genes whose expression levels increased with the progression of MASH, and Figure 35B shows the results of gene ontology analysis of genes whose expression levels increased in both (1) and (4) in Figure 35A. In the figure, (1) shows the gene population whose expression levels increased in liver organoids in which MASH was induced compared to liver organoids before MASH induction, (2) shows the gene population whose expression levels increased in human livers classified as 2 by NAS compared to normal human livers, (3) shows the gene population whose expression levels increased in human livers classified as 4 by NAS compared to normal human livers, and (4) shows the gene population whose expression levels increased in human livers classified as 6 by NAS compared to normal human livers.In Figure 30B, "(a)" indicates a gene involved in inflammation, "(b)" indicates a gene involved in cell damage, and "(c)" indicates a gene involved in fibrosis. "-log10(Adj p value)" indicates that the higher the reciprocal of the natural logarithm of the standardized p-value related to the expression level of the gene belonging to that category, the darker the color of the circle. "Gene Ratio" indicates that the larger the ratio of the number of genes belonging to that category to the total number of genes analyzed, the larger the circle.

[0019] 1. Method for Producing Liver Organoids 1-1. Overview The first aspect of the present invention is a method for producing liver organoids (hereinafter often referred to as "the organoids of the present invention"). The method of this aspect includes a co-culture step, a differentiation induction step, and a tissue construction step as essential steps, and includes a pluripotent stem cell preparation step, a raw material cell preparation step, and a proliferation culture step as optional steps. According to the method of this aspect, liver organoids having a complex vascular structure can be produced.

[0020] 1-2. Definitions The terms used herein are defined below. In this specification, “liver organoid” means a cellular structure composed of a population of cells containing liver-specific cell types and having one or more tissue structures characteristic of the liver.

[0021] In this specification, "multilayer liver organoid" refers to a liver organoid in which cells are stacked in multiple layers in the height direction, and a tissue structure is constructed in the height direction.

[0022] In this specification, "tissue structure" refers to a structure constructed by the combination of multiple types of tissues, in which each tissue can cooperate to perform a certain function. In this specification, a tissue structure is composed of multiple tissues and constitutes an organ or tissue. In this specification, "each tissue can cooperate to perform a certain function" means that the exchange of cells, signal transduction, and biomolecules between tissues is possible in order to perform one or more functions of the liver.

[0023] In this specification, “raw material cells” refers to cells used for the preparation of liver organoids.

[0024] In this specification, "hepatoblast" refers to tissue stem cells derived from the foregut endoderm that exist during embryonic development and are capable of differentiating into both bile duct epithelial cells and mature hepatocytes. Hepatoblasts are not typically observed in adult bodies and are only found in vivo during the development of the liver during embryonic development. Typically, hepatoblasts 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), CER1, and epithelial cell adhesion molecule (EpCAM), while they do not express markers such as keratin 19 (KRT19), and sometimes OCT4 and CD90.

[0025] Endothelial progenitor cells are cells present in fetal organs and blood during fetal development and in adult organs, and are capable of differentiating into vascular endothelial cells. They are recruited to angiogenesis sites and function as a source of new vascular endothelial cells necessary for angiogenesis. They are generally known to be CD34-positive. Endothelial progenitor cells are sometimes classified based on the presence or absence of CD133 expression, but in this specification, endothelial progenitor cells encompass all of these classifications. In addition to CD133, other known markers include CD31, CD144, CD34, VEGFR1, VEGFR2, and VEGFR3.

[0026] In this specification, "mesenchymal cell" refers to cells that constitute mesodermal tissue. Mesenchymal cells include, but are not limited to, all cells that make up mesodermal tissue, such as osteoblasts, adipocytes, muscle cells, and chondrocytes. Typically, they express markers such as CD90 and PDGFRβ.

[0027] In this specification, "hepatic stellate cells" refer to stromal cells located around the blood vessel walls of the sinusoidal regions of the liver. Markers for hepatic stellate cells 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 CD271-negative, which distinguishes them from mesenchymal cells. Hepatic stellate cells are broadly classified into two types: quiescent and activated.

[0028] In this specification, "quiescent hepatic stellate cells" refer to inactive hepatic stellate cells. Quiescent hepatic stellate cells are HSCs found in the liver of normal individuals, characterized by well-developed lipid droplets and spinous projections on their cell surface. Quiescent hepatic stellate cells have 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 capacity, and the function of maintaining extracellular matrix homeostasis, thus greatly contributing to the homeostasis of liver function. Quiescent hepatic stellate cells are typically cells that do not express or express low levels of the activation markers for activated hepatic stellate cells described below. Examples of markers for quiescent hepatic stellate cells include PCDH7, RGS5, FABP5, FABP4, and BAMBI. In this specification, activated hepatic stellate cells are, for example, αSMA-negative and / or PDGFRα-negative hepatic stellate cells.

[0029] In this specification, "active hepatic stellate cell" refers to activated hepatic stellate cells. Activated hepatic stellate cells are characterized by vitamin A release, high proliferative capacity, high migration, and active secretion of extracellular matrix. Excessive activity of activated hepatic stellate cells can lead to hepatic fibrosis. In activated hepatic stellate cells, lipid droplets often contract. Therefore, cells that express hepatic stellate cell markers and lack lipid droplets can be identified as activated hepatic stellate cells. Activated hepatic stellate cells typically express or highly express hepatic stellate cell activation markers. Examples of activation markers include PDGFRα, COL1A1, αSMA, vimentin, fibronectin, IL-6, TGF-β, and collagen I. In this specification, activated hepatic stellate cells are, for example, αSMA-positive and / or PDGFRα-positive hepatic stellate cells.

[0030] A monocyte is a type of white blood cell, making up 2-10% of white blood cells, excluding lymphocytes and granulocytes. Monocytes are larger than lymphocytes and granulocytes, measuring approximately 13-21 μm. They can be distinguished morphologically from other white blood cells by features such as a constricted nucleus, the absence of small or large granules, and the presence of vacuoles. Monocytes are known to express CD14, and are sometimes classified into three types based on the amount of CD14 expression and the presence and amount of CD16 expression; however, the term "monocyte" in this specification encompasses all of these classifications. Monocytes can differentiate into macrophages and dendritic cells, and also function as antigen-presenting immune cells, taking in pathogens and foreign substances through phagocytosis, breaking them down, and presenting a portion of them as antigens outside the cell. In humans, markers such as CD14, CD45, HLA-DR, TNFR1, TNFR2, LYZ, CD300E, and VCAN are expressed, while markers such as CD206 and CD86 are not.

[0031] Hepatocytes are cells that make up approximately 60% of the liver and constitute the liver parenchyma. In this specification, hepatocytes include not only mature hepatocytes but also cells at all differentiation stages in which differentiation into hepatocytes has been determined, such as hepatic progenitor cells. Mature hepatocytes are terminally differentiated cells that perform various functions specific to the liver, such as protein and cholesterol synthesis, metabolism of biomolecules such as proteins, carbohydrates, and lipids, and drug detoxification. Typically, they express markers such as ALB, and sometimes α-1 antitrypsin (A1AT), carbamoyl phosphate synthase 1 (CPS1), cytochrome P450 3A4 (CYP3A4), and glucose-6-phosphatase catalytic subunit (G6PC), and are known not to express markers such as AFP, DLK, and sometimes KRT19, EpCAM, and CD90.

[0032] Liver sinusoidal endothelial cells (SEMs) are cells specific to the liver and are a type of vascular endothelial cell. They have a distinctive morphology that differs from other vascular endothelial cells, such as having a cribriform plate structure on their cell membrane and lacking a basement membrane. They are located between the Disse space and hepatocytes and mediate the exchange of substances between them. In addition to expressing common markers in vascular endothelial cells such as pecam and VE-cadherin, they are also known to express FCGR2β, FVIII, CD36, Stab1, Stab2, LYVE-1, KDR, VEGFR3, and CD105.

[0033] Kupffer cells are non-parenchymal cells that make up the liver and are liver-specific tissue macrophages. Kupffer cells are responsible for biological defense functions such as the recognition of foreign substances and the induction of immune responses, and they also play an important role in the onset or suppression of liver damage. They are generally known to express CD68, ADORA3, C1QA, TIMD4, MARCO, CD5L, TIM4, F4 / 80, Clec4f, Il18bp, CD169, VCAM1, etc.

[0034] A "lipid molecule" refers to a compound that is separated into a lipid-soluble fraction. Lipid molecules include simple lipids, which are formed by the bonding of glycerol and fatty acids; complex lipids (phospholipids, glycolipids, sulfolipids, etc.) that further contain phosphates, sugars, nitrogen compounds, etc.; derived lipids; and steroid lipids that have a steroid skeleton.

[0035] "Derived lipids" refer to lipid molecules produced by the hydrolysis of simple lipids or complex lipids. While not specifically limited, examples include free fatty acids, alcohols, and hydrocarbons.

[0036] "Free fatty acids" refer to fatty acids that make up lipid molecules and are in a free state, not esterified with glycerol or the like. In this specification, free fatty acids also include molecules in the salt state. Specific examples of free fatty acids include oleic acid, palmitic acid, stearic acid, and linoleic acid.

[0037] "Steatotic Liver Disease (SLD)" refers to a condition characterized by the observation of fat accumulation in 5% or more of the hepatic parenchymal cells, either by imaging or histological examination. In this specification, SLD specifically refers to non-alcoholic liver disease or liver failure due to metabolic dysfunction. SLD includes liver failure due to metabolic dysfunction such as MASLD and MASH, MetALD (where alcohol consumption is not sufficient for alcoholic liver disease but some criteria for metabolic syndrome are met), and specific aetiology SLD (non-alcoholic liver disease that does not meet any of the criteria for metabolic syndrome). It also includes both conventional NAFLD and NASH.

[0038] Non-alcoholic fatty liver disease (NAFLD) refers to a condition within SLD that excludes secondary fatty liver disease caused by alcohol, drugs, or genetic disorders. The standard ethanol intake for NAFLD is, for example, 30g or less per day for men and 20g or less per day for women in Japan. Typically, it occurs due to the accumulation of excess lipids in the liver, associated with metabolic syndrome such as obesity, diabetes, dyslipidemia, and hypertension. NAFLD is classified using various methods, including the FLIP algorithm, NAS classification, Brunt classification, NASHCRN classification, and Matteoni classification, but in this specification, NAFLD encompasses conditions classified according to any of these methods.

[0039] Non-alcoholic steatohepatitis (NASH) refers to a condition of NAFLD characterized by ballooning degeneration of hepatocytes and lobular inflammation. Since conditions with ballooning degeneration but without lobular inflammation are rare, NASH in this specification broadly refers to NAFLD accompanied by ballooning degeneration. Typical pathological findings of NASH include ballooning degeneration of hepatocytes, macrodroplet-like fatty degeneration, infiltration of inflammatory cells such as neutrophils, Mallory bodies, giant mitochondria, eosinophil necrosis, nuclear vacuolation, large and small lipogranulomas, pericellular fibrosis, and perisinusoidal fibrosis.

[0040] "Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD)" refers to a type of SLD that meets one or more of the diagnostic criteria for metabolic syndrome, while excluding secondary fatty liver disease caused by alcohol, drugs, or genetic disorders. It was recently introduced as a disease classification to replace NAFLD.

[0041] Metabolic Dysfunction Associated Steatotic Liver Disease (MASH) refers to a type of NASLD characterized by ballooning degeneration of hepatocytes and lobular inflammation. Since the presence of ballooning degeneration without lobular inflammation is rare, in this specification, MASH broadly refers to NASLD accompanied by ballooning degeneration. It is a recently introduced classification of disease states, replacing NASH.

[0042] "Proliferation" refers to the increase in the number of cells. In this specification, proliferation includes both proliferation without differentiation and proliferation with differentiation.

[0043] "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.

[0044] "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.

[0045] 1-3. Process 1-3-1. 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.

[0046] 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.

[0047] The animal species from which pluripotent stem cells are derived is 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 target animals 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.

[0048] The type of pluripotent stem cell is not particularly limited. For example, any pluripotent stem cell known in the art can be appropriately selected, obtained, or produced and used depending on the purpose. 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.

[0049] For example, when organoids produced by the method of this embodiment are used for the treatment of a specific individual, pluripotent stem cells with a reduced risk of rejection due to transplantation can be used. In this case, for example, iPS cells obtained from the somatic cells of the individual to be treated, or pluripotent stem cells 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, can be used.

[0050] 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).

[0051] 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.

[0052] 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.

[0053] 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.).

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.).

[0058] The culture medium used is not particularly limited. For example, the following media can be used. In this process, media specifically provided as suitable for pluripotent stem cells may be used. For example, for ES cells and iPS cells, mTeSR1 medium, TeSR1 medium (Stem Cell Technologies), and Essential 8 TM Culture medium, Essential 6 TM Culture medium (Gibco), StemPro (R) -34 SFM (Life Technologies), StemFlex TM Medium (Gibco), StemFit (R) The AK02N (Ajinomoto), among others, is commercially available.

[0059] 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.

[0060] Other culture conditions are not particularly limited, but for example, culture can be carried out using the culture conditions and subculturing conditions described in the growth culture step of this embodiment.

[0061] Additional optional treatments can be performed during culture. In particular, when using feeder cells, treatments to suppress cell proliferation (such as mitomycin C treatment or irradiation) may be performed.

[0062] 1-3-2. Raw material cell preparation step The raw material cell preparation step is an optional step of the method of this embodiment and is a step of preparing raw material cells from pluripotent stem cells. If a pluripotent stem cell preparation step is performed, this step can be performed afterward. If vascular endothelial progenitor cells are to be prepared, this step can be performed as a vascular endothelial progenitor cell preparation step; if hepatoblasts are to be prepared, this step can be performed as a hepatoblast cell preparation step; if mesenchymal cells are to be prepared, this step can be performed as a mesenchymal cell preparation step; if hepatic stellate cells are to be prepared, this step can be performed as a hepatic stellate cell preparation step; and if monocytes are to be prepared, this step can be performed as a monocyte cell preparation step.

[0063] This process can be performed multiple times. The purpose of performing it multiple times is not particularly limited, but for example, it can be done to prepare multiple types of raw material cells or to replenish deficient raw material cells.

[0064] In this process, differentiation of pluripotent stem cells into the target raw material cells is induced. Any known method can be used for any cell type, and the specific method is not limited, but the methods for inducing differentiation into each type of raw material cell are described below.

[0065] <Induction of Vascular Endothelial Progenitor Cells> The specific method for inducing differentiation into vascular endothelial progenitor 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.

[0066] Differentiation induction can be performed by culturing in a differentiation-inducing medium for a certain period of time in a coated culture vessel.

[0067] The coating agent used is not particularly limited, but examples include laminin-1 to 12 (including iMatrix-511, etc.), COL1A1, Matrigel, collagen, poly-L-lysine, Gelforce, fibronectin, vitronectin, Geltrex, etc.

[0068] The material of the culture vessel 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.

[0069] The composition of the differentiation-inducing medium is not particularly limited, as long as it can induce differentiation into vascular endothelial progenitor cells. Specifically, for example, this can be done by combining culture in a medium containing a ROCK inhibitor, culture in a medium containing a Wnt signaling pathway activator and TGFβ family proteins, or culture in a medium containing a cAMP pathway activator.

[0070] (1) ROCK inhibitors In this specification, "ROCK inhibitor" refers to an inhibitor of the signaling pathway mediated by Rho-associated coiled-coil forming kinase. 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.

[0071] 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.

[0072] 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.

[0073] 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-39983) 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.

[0074] 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.

[0075] For example, it may be used at the start of culture (including each subculturing) and / or immediately after a medium change, or from the second day onward. It can also be used for periods of 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 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. When added in the early stages of culture, the basal medium used is not particularly limited, but for example, a medium such as those exemplified in the pluripotent stem cell preparation process (StemFit medium, etc.) can be used.

[0076] (2) Wnt signaling pathway activators The Wnt signaling pathway activators may be classical Wnt signaling pathway activators, non-classical Wnt signaling pathway activators, or mixtures thereof, and are not particularly limited.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] (3) TGFβ family proteins In this specification, "TGFβ family proteins" refers to any protein belonging to the TGFβ superfamily.

[0085] 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.

[0086] 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.

[0087] (4) cAMP pathway activators In this specification, "cAMP pathway activators" refers to drugs that promote cAMP-based signaling pathways.

[0088] 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.

[0089] 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.

[0090] (5) Basic culture medium The type of basic culture medium used herein is not particularly limited. For example, any vertebrate cell culture medium known in the art, such as a mammalian cell culture medium, can be used alone or in combination, with culture medium additives added as needed.

[0091] Specifically, for example, in the pluripotent stem cell preparation process, the aforementioned StemPro (R) -34 SFM (Life Technologies), StemFit (R)In addition to culture media suitable for pluripotent stem cells such as AK02N (Ajinomoto), Minimum Essential Medium Eagle (MEM) and any modified versions 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). In this process, for example, DMEM / F-12 medium, StemPro-34 SFM medium, etc., can be suitably used.

[0092] 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.

[0093] When containing serum or a serum substitute, its type is not particularly limited. For example, any serum or serum substitute known in the art can be included. Specific sera include, for example, fetal bovine serum (FBS), human serum, sheep serum, or mixtures thereof, etc., but are not particularly limited. Also, specific serum substitutes include, for example, Knockout TM serum replacement (KSR), XF212 XerumFree, CDM-HD serum substitute, StemSure serum substitute, Nu-Serum TM etc., but are not particularly limited.

[0094] When containing serum or a serum substitute, the content is not particularly limited. For example, it can be included at 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, 5 (v / v)% or more. Also, for example, it can be 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, 5 (v / v)% or less.

[0095] (6) Other components The medium used in this specification can include any other components. The specific types of other components are not particularly limited. For example, antioxidants, growth factors, vitamins, albumin, amino acids, antibiotics, etc. can be included.

[0096] In this specification, "antioxidant" refers to a drug that has antioxidant properties 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 definition of antioxidant in this specification. The antioxidants in this specification 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.

[0097] 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.

[0098] A "growth factor" is a molecule that promotes cell proliferation and differentiation within an organism, and the concept encompasses both low-molecular-weight molecules such as vitamins and high-molecular-weight molecules such as proteins. In this specification, "growth factor" specifically refers to proteins that promote cell proliferation and differentiation within an organism.

[0099] Any growth factor known in the art can be used as the growth factor. Specifically, examples include, but are not limited to, fibroblast growth factor (FGF: including basic FGF (bFGF, FGF2), FGF1 to FGF23, etc.), epidermal growth factor (EGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), their substitutes, or combinations thereof. The growth factor may be derived from a specific organism (e.g., human) or may be a recombinant. For example, the growth factor in the hepatoblast growth promoter of this embodiment may suitably include EGF or its substitute.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] Examples of antibiotics include, but are not limited to, penicillin, streptomycin, penicillin-streptomycin, sulfonamides, pheneticillin, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, metacycline, and minocycline.

[0105] 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.

[0106] (7) Other culture conditions Other culture conditions (such as medium exchange, cell density, temperature, humidity, and CO2 concentration) can be those of normal culture conditions and are not particularly limited.

[0107] 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.

[0108] 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.

[0109] 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%).

[0110] 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.

[0111] The culture period is not particularly limited, and culture can be continued until differentiation into the target raw material cells is induced. For example, it can be 1 day or more, 3 days or more, 5 days or more, 7 days or more, 9 days or more, or 10 days or more. Specifically, for example, the culture period can be 1 to 150 days, 1 to 90 days, 5 to 50 days, 6 to 30 days, 7 to 20 days, or 7 to 15 days.

[0112] (8) Confirmation of Differentiation Induction Differentiation into vascular endothelial progenitor cells can be determined based on whether or not the cells possess any of the properties of vascular endothelial progenitor 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 vascular endothelial progenitor cell markers and / or pluripotent stem cell markers. Any marker known in the art can be used as vascular endothelial progenitor cell markers and pluripotent stem cell markers, and there are no particular limitations. Specifically, for example, vascular endothelial progenitor cell markers include CD34, CD31, CD144, CD34, VEGFR1, VEGFR2, VEGFR3, etc. Specific pluripotent stem cell markers can include, for example, OCT4, NANOG, SOX2, CD9, DNMT3B, GABRB3, GAL, GDF3, IFITM1, PODXL, TDGF1, ZFP42, etc.

[0113] <Induction of Hepatoblasts> The method for inducing differentiation of hepatoblasts from pluripotent stem 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 hepatoblasts can be induced after differentiation into endoderm cells of the embryo.

[0114] (1) Induction of Endoderm Cells in Embryoembryonic Cells: The specific method for inducing differentiation in endoderm cells in embryoembryonic cells is not particularly limited. For example, differentiation from pluripotent stem cells to endoderm cells can be induced by culturing pluripotent stem cells in a medium containing a Wnt signaling pathway activator and / or TGFβ family proteins.

[0115] 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. The type and concentration should be the same as described for vascular endothelial progenitor cells. For example, Wnt3a and other WNTs, or GSK3β inhibitors such as CHIR99021, can be suitably used.

[0116] The TGFβ family proteins and their concentrations should be the same as those described for vascular endothelial progenitor cells. For example, activin and / or BMP4 can be suitably used.

[0117] Growth factors may be added to the culture medium for differentiation induction. The growth factors should be the same as those described for vascular endothelial progenitor cells. For example, FGF2 and VEGF can be suitably used.

[0118] 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. TM Supplements 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.

[0119] 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.

[0120] 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.

[0121] (2) Induction of hepatoblasts Hepatoblasts can be induced to differentiate from pluripotent stem cells or endoderm cells of the embryo. Alternatively, hepatoblasts may be induced to differentiate from endoderm cells of the embryo through differentiation into other cell types (e.g., hepatic endoderm cells, foregut progenitor cells).

[0122] 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.

[0123] In this specification, "steroid" refers to a drug having a steroid skeleton in its chemical structure. Examples of steroids usable in this specification include glucocorticoids and their analogs and derivatives. For example, glucocorticoids, or their analogs, derivatives, or substitutes can be suitably used as steroids.

[0124] 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 this embodiment may suitably include long-acting steroids such as dexamethasone (DEX).

[0125] 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.

[0126] The inflammatory cytokines used are not particularly limited. Specific examples of inflammatory cytokines include 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, and combinations thereof. For example, oncostatin M can be suitably used.

[0127] The concentration of inflammatory cytokines (e.g., IL-6 family proteins such as OSM) is not particularly limited, as long as it is an effective amount. Examples of lower limits for concentration include 0.001 nM, 0.005 nM, 0.01 nM, 0.1 nM, 0.15 nM, 0.2 nM, 0.25 nM, 0.3 nM, and 0.33 nM, while examples of upper limits include 10 nM, 5 nM, 1 nM, 0.6 nM, 0.5 nM, 0.45 nM, 0.4 nM, and 0.35 nM.

[0128] For other components, the same instructions as those given for vascular endothelial progenitor cells apply. 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.

[0129] The basal culture medium used is not particularly limited. For example, the medium exemplified in relation to vascular endothelial progenitor cells, or a mixed medium combining multiple media, can be used.

[0130] 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 volume 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 the 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 medium can be suitably used as the main medium.

[0131] The culture medium used preferably contains an organic buffer, L-glutamine or its derivative, and sodium pyruvate. When using multiple culture media in combination, it is sufficient that these components are present after the combination. For example, a culture medium combining IMDM, which contains the organic buffer HEPES but does not contain L-glutamine, and Ham's F-12 or a modified medium thereof (e.g., Ham's F-12 K medium), which contains L-glutamine but does not contain HEPES, can be suitably used. Furthermore, if the above components are not present or are insufficient in the culture medium after mixing, they can be added externally.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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 endoderm cell markers, for example, any of the endoderm cell markers mentioned above can be used.

[0136] <Induction of Mesenchymal Cells> Methods for preparing mesenchymal cells are known in the art, and the specific method is not particularly limited. For example, when inducing differentiation from iPS cells, the method described in Takebe et al., Cell Reports, 2017 can be used.

[0137] For example, this can be carried out by appropriately modifying the induction of mesodermal cells and transverse septal mesenchymal cells, as described later.

[0138] Specifically, for example, the culture of mesoderm cells in a medium supplemented with a ROCK inhibitor can be performed for a longer period. There is no particular limit to the specific duration, but for example, it can be 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, or 6 days or more. Specifically, for example, the culture period can be 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, or 4 to 6 days. The conditions such as concentration during this period can be the same as those described above for ROCK inhibitors in relation to vascular endothelial progenitor cells.

[0139] Furthermore, mesenchymal cells can be induced using a procedure similar to that used for inducing transverse septal mesenchymal cells. For example, BMP family proteins (such as BMP4) can be suitably used as TGFβ family proteins in secondary induction. While the concentration is not particularly limited, the concentrations mentioned above can be used, for example, in relation to vascular endothelial progenitor cells.

[0140] The basal culture medium used for inducing mesenchymal cells is not particularly limited. For example, media such as those exemplified in relation to vascular endothelial progenitor cells (DMEM / F12 medium, StemPro-34 SFM medium, etc.) can be used.

[0141] The induction of differentiation into mesenchymal cells can be determined based on whether or not the cells possess any of the properties of mesenchymal 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 mesenchymal cell markers and / or pluripotent stem cell markers. Any markers known in the art can be used as mesenchymal cell markers and pluripotent stem cell markers, and are not particularly limited. Specific examples of mesenchymal cell markers include CD90 and PDGFRβ. Specific examples of pluripotent stem cell markers include the markers mentioned above in relation to induction into pluripotent stem cells.

[0142] <Induction of Hepatic Stellary Cells> The method for inducing quiescent hepatic stellate cells is not particularly limited, as long as it allows for differentiation into quiescent hepatic stellate cells. For example, it can be carried out using any known method. Specifically, for example, quiescent hepatic stellate cells can be prepared by inducing the culture of transverse septal mesenchymal cells and inducing their differentiation into quiescent hepatic stellate cells.

[0143] The specific method for inducing differentiation into transverse septal mesenchymal cells is not particularly limited. For example, pluripotent stem cells can be differentiated into mesodermal cells, and then mesodermal cells can be differentiated into transverse septal mesenchymal cells.

[0144] The induction of mesodermal cells from pluripotent stem cells, and the induction of transverse septal mesenchymal cells from mesodermal cells, can be carried out using any known method. For example, this can be done by adding appropriate cytokines to the culture medium.

[0145] The culture medium used in this case is not particularly limited. For example, a basal medium (such as DMEM / F12 medium) to which a culture medium additive has been added can be used. The culture medium additive used in this case is not particularly limited, but for example, other components as exemplified above (amino acids such as L-glutamine and its substitutes, B27) can be used. TM It may include supplements, etc.

[0146] The cytokines used can be any cytokine capable of promoting differentiation into the target cells, and are not particularly limited.

[0147] (1) Induction of mesodermal cells For example, in the case of induction of mesodermal cells, Wnt signaling pathway activators, TGFβ family proteins, or a combination thereof can be used.

[0148] For Wnt signaling pathway activators (e.g., GSK3β inhibitors such as CHIR99021) and TGFβ family proteins (e.g., BMP family proteins such as BMP4), the same guidelines as those for pluripotent stem cells apply.

[0149] The basal culture medium used is not particularly limited. For example, the mediums exemplified in relation to vascular endothelial progenitor cells can be used. Specifically, for example, MEM such as DMEM / F12 medium and any modified thereof can be used.

[0150] 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.

[0151] ROCK inhibitors as described above can be used for vascular endothelial progenitor cells. There are no particular limitations on the timing of their application, but they can be used temporarily, for example, at the start of culture. The application period should be the same as that described for vascular endothelial progenitor cells. The basal medium used may be the same as or different from the basal medium used for induction. For example, a medium suitable for pluripotent stem cells, such as StemFit AKOKO2N medium, can be suitably used.

[0152] (2) Induction of transverse septal mesenchymal cells The cytokines used to induce transverse septal mesenchymal cells are not particularly limited. For example, tyrosine kinase receptor activators, TGFβ family proteins, FGF signaling pathway activators, or combinations thereof can be used.

[0153] Transverse septal mesenchymal cells can be induced 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 performed in a two-step induction reaction, primary induction can be performed using, for example, a tyrosine kinase receptor activator and a TGFβ family protein, and secondary induction can be performed using, for example, a TGFβ family protein and an FGF signaling pathway activator.

[0154] The tyrosine kinase receptor activators and TGFβ family proteins used in the induction of transverse septal mesenchymal cells are not particularly limited. For example, cysteine-not cytokine superfamily proteins such as PDGF (e.g., PDGF-BB) can be used as tyrosine kinase receptor activators. Also, for example, activin (activin A, etc.) and BMP family proteins (BMP4, etc.) can be used as TGFβ family proteins. The final concentration of BMP family proteins (BMP4, etc.) is not particularly limited, but for example, the concentrations mentioned above can be used in relation to vascular endothelial progenitor cells.

[0155] 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.

[0156] 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.

[0157] The "FGF (fibroblast growth factor) signaling pathway" refers to the signaling pathway activated when FGF binds to its receptor, the receptor tyrosine kinase FGFR. 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 hepatic stellate cell induction, the FGF signaling pathway is preferably the RAS / MAP kinase pathway and / or the PI3 kinase / AKT pathway.

[0158] "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.

[0159] In this specification, "FGF signaling pathway activator" refers to a drug that activates the FGF signaling pathway (particularly the RAS / MAP kinase pathway and / or the PI3 kinase / AKT pathway). The type of drug is not particularly limited. Any drug known in the art can be used as the FGF signaling pathway activator. The specific type of FGF signaling pathway activator used is not particularly limited, but examples include FGF synthesis promoters, FGF secretion promoters, FGFR synthesis promoters, FGFR agonists, RAS / MAP kinase pathway activators, PI3 kinase / AKT pathway activators, PLCγ pathway activators, FGF receptor binding promoters, and expression promoters of factors in these pathways. For example, FGF receptor binding promoters (such as heparan sulfate or its derivatives), FGFR agonists, and FGF2 signaling pathway activators can be suitably used as FGF signaling pathway activators.

[0160] 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.

[0161] 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.

[0162] 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 can 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.

[0163] 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.

[0164] The basal medium may be the same medium used for inducing mesoderm cells, or a different medium may be used. For example, the same basal medium used for inducing mesoderm cells can be used.

[0165] The culture period for the induction of transverse septal mesenchymal cells is not particularly limited, but it can be carried out for the period exemplified, for example, in the induction of quiescent hepatic stellate cells. If the induction of transverse septal mesenchymal cells is carried out in multiple stages, the induction cultures for each stage may be the same or different from each other. For example, if the total period of induction culture of transverse septal mesenchymal cells is 4 days, the primary culture may be carried out for 2 days and the secondary culture for 2 days, or the primary culture may be carried out for 1 day and the secondary culture for 3 days, or vice versa.

[0166] Differentiation into transverse mesenchymal cells can be determined based on whether or not the cells possess any of the properties of transverse mesenchymal 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 transverse mesenchymal cell markers and / or pluripotent stem cell markers. Any markers known in the art can be used as transverse mesenchymal cell markers and pluripotent stem cell markers, and are not particularly limited. Specific transverse mesenchymal cell markers include, for example, HLX, WT1, BMP2, BMP4, FOXF1, GATA4, MRG1, HGF, TBX18, etc. Specific pluripotent stem cell markers can include, for example, the markers mentioned above in relation to induction into pluripotent stem cells.

[0167] (3) Induction of hepatic stellate cells The method for inducing hepatic stellate cells is not particularly limited, as long as it is a method capable of culturing hepatic stellate cells, preferably quiescent hepatic stellate cells or fetal-like quiescent hepatic stellate cells. For example, they can be induced from transverse septal mesenchymal cells by culturing in a medium containing an FGF signaling pathway activator and optionally containing antioxidants and other components.

[0168] As a culture medium, for example, a medium made by combining multiple basic media as described above can be used. The volume ratio when combining 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 the 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 medium can be suitably used as the main medium. Specifically, for example, when using Ham's F-12 medium as another culture medium, the amount of Ham's F-12 medium can be 3 / 4 or less, 2 / 3 or less, 1 / 2 or less, or 1 / 3 or less of Iskov-modified Dulbecco's medium. Also, for example, the amount of Ham's F-12 medium can be 1 / 10 or more, 1 / 8 or more, 1 / 6 or more, 1 / 5 or more, 1 / 4 or more, or 1 / 3 or more of Iskov-modified Dulbecco's medium.

[0169] 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.

[0170] 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.

[0171] The induction of differentiation into hepatic stellate cells, particularly quiescent hepatic stellate cells, can be determined based on whether or not the cells possess any of the properties of quiescent hepatic stellate cells and / or whether or not the properties of transverse septal mesenchymal cells have been lost. For example, this can be determined by the expression of quiescent hepatic stellate cell markers (quiescent hepatic stellate cell markers, activated hepatic stellate cell markers, etc.) and / or transverse septal mesenchymal cell markers. Any markers known in the art can be used as hepatic stellate cell markers and activated hepatic stellate cell markers, and are not particularly limited. Specifically, for example, hepatic stellate cell markers include ALCAM, CD71, CD73, PCDH7, LOX, PDGFRβ, desmin, etc. For example, quiescent hepatic stellate cell markers include PCDH7, RGS5, FABP5, FABP4, BAMBI, etc. Also, for example, activated hepatic stellate cell markers include PDGFRα, COL1A1, αSMA, vimentin, fibronectin, IL-6, TGF-β, collagen I, etc. As a transverse septal mesenchymal cell marker, for example, any of the transverse septal mesenchymal cell markers mentioned above can be used.

[0172] The induced quiescent hepatic stellate cells typically express hepatic stellate cell markers but not transverse septal mesenchymal cell markers or activated hepatic stellate cell markers. Furthermore, the induced quiescent hepatic stellate cells are proliferative quiescent hepatic stellate cells, such as fetal-like quiescent hepatic stellate cells. Whether or not a cell is fetal-like quiescent hepatic stellate cell can be determined based on the expression of fetal hepatic stellate cell-related genes.

[0173] While there are no specific limitations on the fetal hepatic stellate cell-related genes, examples include desmin, p75NTR, RAC1, COTL1, IGFBP2, and S100A16. For example, the quiescent hepatic stellate cells described herein express one or more fetal hepatic stellate cell-related genes.

[0174] <Induction of Monocytes> Methods for preparing monocytes are known in the art, and the specific method is not particularly limited. For example, monocytes can be induced from erythrocyte bone marrow progenitor cells.

[0175] The origin of the erythrocyte bone marrow progenitor cells is not particularly limited. For example, they may be isolated from living organisms or differentiated from pluripotent stem cells.

[0176] (1) Induction of erythrocytes and bone marrow progenitor cells: Methods for inducing erythrocytes and bone marrow progenitor cells are known in the art, and the specific method used is not particularly limited. For example, any method used for differentiation induction into hematopoietic progenitor cells can be used.

[0177] For example, differentiation induction may be performed using FLT3 and FLT3L, or a commercially available kit may be used. An example of a commercially available kit is STEMdiff. TM Examples include the Hematopoietic Kit (StemCell Technologies).

[0178] The culture period is not particularly limited. For example, it can be cultured for the period exemplified for vascular endothelial progenitor cells.

[0179] (2) Methods for inducing differentiation of erythrocytes into monocytes from bone marrow progenitor cells are known in the art, and the specific methods are not particularly limited. For example, methods for inducing differentiation of hematopoietic progenitor cells into monocytes can be used.

[0180] Any of the following can be used: a coating agent for adhesive culture, a coating agent for suspension culture, or culture equipment with a surface treatment for suspension culture. For example, a coating agent for suspension culture or culture equipment with a surface treatment for suspension culture can be suitably used. Specifically, for example, the culture equipment exemplified in the co-culture process can be used.

[0181] Differentiation can be induced by culturing in the presence of anti-inflammatory agents (e.g., IL-10 pathway activators such as M-CSF).

[0182] The final concentration of the anti-inflammatory agent (e.g., IL-10 pathway activators such as M-CSF) is not particularly limited, but the lower limit can be, for example, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, etc., and the upper limit can be, for example, 10000 ng / mL, 5000 ng / mL, 1000 ng / mL, 500 ng / mL, 300 ng / mL, 250 ng / mL, 200 ng / mL, 150 ng / mL, 100 ng / mL, 90 ng / mL, 70 ng / mL, 60 ng / mL, 55 ng / mL, 50 ng / mL, etc.

[0183] As a culture medium to which an anti-inflammatory agent is added, for example, the culture medium described above can be used for the induction of hepatoblasts.

[0184] There are no specific limitations on the culture period, but for example, it can be 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, or 6 days or more. Specifically, for example, the culture period can be 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, or 4 to 6 days.

[0185] Induction of differentiation into monocytes can be determined based on whether or not the cells possess any of the properties of monocytes and / or whether or not the properties of pluripotent stem cells have been lost. For example, this can be determined by the expression of monocyte cell markers and / or pluripotent stem cell markers. Any markers known in the art can be used as monocyte cell markers and / or pluripotent stem cell markers, and are not particularly limited. Specific monocyte cell markers include, for example, CD14, CD45, HLA-DR, TNFR1, and TNFR2. Specific pluripotent stem cell markers can include, for example, the markers mentioned above in relation to induction into pluripotent stem cells.

[0186] 1-3-3. Growth and Culture Step The growth and culture step is an optional step of the method according to this embodiment, and is a step of growing and culturing raw material cells. If the raw material cell preparation step is to be performed, it can be performed afterward.

[0187] In this process, in addition to the essential growth culture step, a subculturing step can be performed as needed.

[0188] <Proliferation and Culture Step> In this step, the raw material cells are cultured and grown in a growth medium. For the growth medium used in this step, a medium suitable for each cell type as described above in the raw material cell preparation step can be used.

[0189] Any culture method known in the art can be used, and is not particularly limited. For example, the culture can be performed under the above-described culture conditions during the raw material cell preparation step.

[0190] The culture period is not particularly limited and can be extended until the cells reach the desired number or confluence.

[0191] Furthermore, the culture medium can be changed as needed. There are no particular limitations on the frequency of culture medium changes. For example, it can be changed at the frequency and in the manner exemplified in the raw material cell preparation process. 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 of culture, and the ROCK inhibitor may be removed from the culture medium from day 2 onwards.

[0192] <Subculture Step> This step is optional and involves subculturing the raw material cells during culture. This step can be performed after the growth culture step.

[0193] 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%.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 1-3-4. Co-culture step The co-culture step is an essential step of the method according to this embodiment, and is a step in which raw material cells are co-cultured. This step can be performed after the raw material cell preparation step if one is to be performed.

[0200] 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 growth culture process.

[0201] The cell ratio is not particularly limited. It is preferable that hepatoblasts are present in greater numbers than vascular endothelial progenitor cells, mesenchymal cells, and hepatic stellate cells. The specific ratio is not particularly limited. For example, hepatoblasts 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 vascular endothelial progenitor cells and / or mesenchymal cells. Furthermore, the ratio of vascular endothelial progenitor cells to mesenchymal cells is not particularly limited. For example, vascular endothelial progenitor 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 or more than mesenchymal cells. Also, for example, vascular endothelial progenitor 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 or less than mesenchymal cells.

[0202] Furthermore, the ratio of hepatic stellate cells to hepatoblasts is not particularly limited. For example, hepatoblasts may be 2 times, 3 times, 4 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, or 10 times more than hepatic stellate cells. Also, the ratio of hepatic stellate cells to mesenchymal cells is not particularly limited. For example, hepatic stellate cells may be 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.45 times, 0.5 times, etc., of mesenchymal cells. Also, for example, hepatic stellate cells may be 2 times or less, 1.5 times or less, 1.2 times or less, 1 time or less, 0.9 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, etc., of mesenchymal cells.

[0203] The ratio of monocytes to other cells is not particularly limited. For example, all non-monocyte cells (e.g., hepatoblasts, vascular endothelial progenitor cells, mesenchymal cells, and hepatic stellate cells) can be included in such a way that their total number is 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times greater than the number of monocytes. Alternatively, all non-monocyte cells can be included in such a way that their total number is 15 times or less, 10 times or less, 9 times or less, 8 times or less, 7 times or less, 6 times or less, 5.5 times or less, or 5 times or less than the number of monocytes.

[0204] This process can be performed multiple times. For example, co-culture of cells other than monocytes can be performed first, and then monocytes can be added later for further co-culture.

[0205] The culture method for this process is not particularly limited. For example, it may be suspension culture or adherent culture. For instance, the culture in this process can be preferably carried out by adherent culture.

[0206] The culture vessels 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.

[0207] The culture medium used in this process can be any medium available for organoid culture and is not particularly limited. For example, the culture media exemplified in the raw material cell preparation process can be used. As a specific basal medium, for example, a mixed medium of DMEM medium and vascular endothelial cell medium can be used.

[0208] 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.

[0209] 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.

[0210] 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).

[0211] 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.

[0212] 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.

[0213] 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.

[0214] In this specification, "TGFβ signaling pathway inhibitor" refers to an inhibitor of a signaling pathway based on the binding of TGFβ to its receptor. In this specification, TGFβ signaling pathway inhibitors include both inhibitors of the classical TGFβ signaling pathway mediated by Smad and inhibitors of other non-classical TGFβ signaling pathways, and any TGFβ signaling pathway inhibitor known in the art can be used. For example, classical TGFβ signaling pathway inhibitors can be suitably used.

[0215] The types of classical TGFβ signaling pathway inhibitors are not particularly limited, but examples include TGFβ receptor inhibitors, Smad phosphorylation inhibitors, Smad nuclear translocation inhibitors, and inhibitors of the activity or expression of one or more factors of the classical TGFβ signaling pathway. In the case of TGFβ receptor inhibitors, the type of receptor inhibited is not particularly limited. For example, inhibitors that show inhibitory activity against one or more of ALK1 to ALK7 (e.g., one or more of ALK4, ALK5, and ALK7) can be used. Specifically, examples include A8301, SB431542, SB505124, SB525334, D4476, LY2109761, LY2157299, LY364947, GW788388, RepSox, SD-208, TEW-7197, LDN-212854, or combinations thereof. For example, TGFβ receptor inhibitors such as A8301 can be suitably used as classical TGFβ signaling pathway inhibitors.

[0216] As non-classical TGFβ signaling pathway inhibitors, any of the following may be used: MAPK pathway inhibitors [MAPK / ERK pathway inhibitors (trametinib, U0126, etc.), p38 MAPK pathway inhibitors (SB202190, SB203580, etc.), MAPK / JNK pathway inhibitors (SP600125, JNK-IN-8, etc.)], PI3K / Akt pathway inhibitors (GDC-0941, etc.), Rho GTPase pathway inhibitors (TRIPa, etc.), etc.].

[0217] Multiple types of TGFβ signaling pathway inhibitors can be used in combination. The types of combinations are not particularly limited, but for example, multiple types of classical TGFβ signaling pathway inhibitors may be used in combination, or classical TGFβ signaling pathway inhibitors may be used in combination with non-classical TGFβ signaling pathway inhibitors.

[0218] The content of each component is not particularly limited, as long as it is in an effective amount. ROCK inhibitors can be used for a certain period, for example, at the start of culture. The concentration and duration should be in accordance with the description of ROCK inhibitors in the first embodiment.

[0219] The final concentration of anti-inflammatory agents (such as steroids like dexamethasone) is not particularly limited, but examples of lower limits include 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 45 nM, and 50 nM, while examples of upper limits include 10,000 nM, 5,000 nM, 1,000 nM, 500 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 90 nM, 70 nM, 60 nM, 55 nM, and 50 nM.

[0220] The final concentration of the JAK-STAT3 pathway activator (such as oncostatin M) is not particularly limited, but examples of lower limits include 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 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, 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.

[0221] The final concentration of classical TGFβ signaling pathway inhibitors (ALK inhibitors such as A8301) is not particularly limited, but examples of lower limits include 0.001 μM, 0.005 μM, 0.01 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, and 0.5 μM, and examples of upper limits include 100 μM, 50 μM, 10 μM, 5 μM, 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 0.9 μM, 0.7 μM, 0.6 μM, and 0.5 μM.

[0222] The culture period is not particularly limited. For example, it can be cultured for 10 hours to 30 days, 10 hours to 20 days, 10 hours to 15 days, 10 hours to 10 days, 10 hours to 5 days, 10 hours to 4 days, 10 hours to 3 days, 10 hours to 2 days, 20 hours to 30 days, 20 hours to 20 days, 20 hours to 15 days, 20 hours to 10 days, 20 hours to 5 days, 20 hours to 4 days, 20 hours to 3 days, 20 hours to 2 days, 1 day to 30 days, 1 day to 20 days, 1 day to 15 days, 1 day to 10 days, 1 day to 5 days, 1 day to 4 days, 1 day to 3 days, or 1 day to 2 days.

[0223] 1-3-5. Differentiation Induction Step The differentiation induction step is an essential step of the method according to this embodiment and is a step in which differentiation of raw material cells is induced. It can be performed simultaneously with or after the co-culture step.

[0224] This process can be carried out in accordance with the descriptions in the co-culture process and the tissue construction process. Typically, these processes induce differentiation of each raw material cell in the organoid.

[0225] 1-3-6. Tissue Construction Process The tissue construction process is an essential step of the method according to this embodiment, and is a process of constructing a tissue structure composed of multiple layers of cells. It can be performed simultaneously with or after the co-culture process.

[0226] The culture method for this process is not particularly limited, but for example, it is cultured by a gas-liquid phase culture method.

[0227] "Gas-liquid interface culture" refers to a culture method in which cells or a portion of a cell group in contact with a culture medium are cultured in a state where they are in contact with the gas phase. Typically, this is done by seeding cells on a mesh membrane and supplying the culture medium from below.

[0228] 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).

[0229] In this process, a tissue structure composed of multiple layers of cells is constructed. The tissue structure constructed is not particularly limited, but for example, vascular structures such as sinusoidal structures may be formed.

[0230] Therefore, preferably, at least a portion of the culture in this process is carried out in an environment that promotes the stacking of cells in the height direction.

[0231] Specifically, for example, by seeding the cell population after co-culturing into a mold that restricts horizontal diffusion, cell layering can be promoted.

[0232] The specific shape and material of the mold are not particularly limited. Examples of shapes include cylindrical, conical, and approximately spherical. The material is not particularly limited, but for example, the materials mentioned above can be used in relation to the culture vessel. The mold may be composed of multiple materials combined together, or it may be continuously composed of a single material. For example, commercially available products developed for culture inserts may be used. Examples of such commercially available products include Culture-Inserts 3 Well for self-insertion (Inbidi, 80369) and Culture-Inserts 4 Well for self-insertion (Inbidi, 80469), but are not particularly limited.

[0233] The mold may be used throughout the entire culture period or only temporarily. If used temporarily, the timing and duration are not particularly limited.

[0234] For example, it may be used at the start of a 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. Typically, once the organoid has adhered to the culture material, it can be cultured without using a mold.

[0235] While not specifically limited, the mold can be filled with culture medium during the period it is in use, and the organoids can be cultured in an environment where they do not come into contact with the gas phase.

[0236] The culture period in this process 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.

[0237] Seeding density is not particularly limited, for example, cell density is not particularly limited, for example, hepatoblasts are 10^2 cells / cm 2 ~10^8 cells / cm 2 , 10^3 cells / cm 2 ~10^7 cells / cm 2 , 10^5 cells / cm 2 ~10^7 cells / cm 2 , 10^6 cells / cm 2 ~10^7 cells / cm 2 , 2×10^6 cells / cm 2 ~8×10^6 cells / cm 2 , 4×10^6 cells / cm 2 ~6×10^6 cells / cm 2 Seeds can be sown to achieve a certain density.

[0238] The culture medium and other culture conditions used shall be in accordance with those described in the co-culture step. This step may be carried out using the same culture medium and conditions as those used in the co-culture step, or it may be carried out using different culture medium and conditions. For example, this step may be carried out using the same culture medium and conditions as those used in the co-culture step.

[0239] 1-4. Multilayer liver organoids The present invention also relates to multilayer liver organoids comprising hepatic parenchymal cells, hepatic stellate cells, hepatic sinusoidal endothelial cells, and Kupffer cells.

[0240] Preferably, the organoid of the present invention includes quiescent hepatic stellate cells. In this case, the quiescent hepatic stellate cells may be proliferative quiescent hepatic stellate cells, such as fetal-like quiescent hepatic stellate cells.

[0241] Furthermore, the organoids of the present invention preferably have polarity with two or more axes of directionality. The tissue structures included in the organoids of the present invention are not particularly limited, but preferably include vascular structures, and especially sinusoidal structures.

[0242] Other cells may be included as needed. The types of these other cells are not particularly limited.

[0243] Preferably, one or more cell types included in the organoid of the present invention—hepatic parenchymal cells, hepatic stellate cells, hepatic sinusoidal endothelial cells, and Kupffer cells—are derived from pluripotent stem cells such as iPS cells. The organoid of the present invention can be manufactured, for example, by the method of this embodiment.

[0244] 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 2The 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. Also, in terms of height, for example, 50μm to 1000μm, 50μm to 700μm, 50μm to 500μm, 50μm to 400μm, 50μm to 300μm, 70μm to 1000μm, 70μm to 700μm, 70μm to 500μm, 70μm to 400μm, 70μm to 300μm, 100μm to 1000μm, 100μm to 700μm, 100μm to 5 Any size range such as 00μm, 100μm-400μm, 100μm-300μm, 150μm-1000μm, 150μm-700μm, 150μm-500μm, 150μm-400μm, 150μm-300μm, 200μm-1000μm, 200μm-700μm, 200μm-500μm, 200μm-400μm, 200μm-300μm, etc. is acceptable.

[0245] 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 in experiments such as drug testing. Examples of in vitro experiments include preparing disease model organoids by subjecting them to arbitrary treatments.

[0246] The organoids of the present invention may be applied to healthy individuals or those suffering from some 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.

[0247] 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.

[0248] The organoids of the present invention possess well-developed blood vessels and form complex vascular structures. Furthermore, they can exhibit liver-specific functions such as albumin production, urea production, CYP3A4 production, and NH4 metabolism.

[0249] In particular, liver-specific functions such as albumin production, urea production, CYP3A4 production, and NH4 metabolism are significantly enhanced compared to cases where hepatic endodermal cells are used instead of hepatoblasts. Furthermore, the complex tissue structure and intercellular interactions characteristic of the liver, including the microenvironment containing Kupffer cells around the sinusoids, Disse space, and hepatic stellate cells, are reproduced by the organoids of the present invention. Moreover, hepatic parenchymal cells are continuously and uniformly arranged within the organoids of the present invention.

[0250] 1-5. Non-human animals The present invention also relates to non-human animals comprising the multilayer liver organoid of the present invention. Non-human animals can be produced by the method of the transplantation step of the second embodiment.

[0251] The species of non-human animals are not particularly limited. For example, the organisms exemplified as species from which pluripotent stem cells are derived (such as rodents like mice, as well as experimental animals and livestock) can be cited. 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.

[0252] The non-human animals of the present invention preferably have at least a portion of their endogenous liver destroyed. This destruction includes not only physical defects but also functional defects.

[0253] 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.

[0254] The use of non-human animals according to the present invention is not particularly limited. Because they carry liver organoids that faithfully reproduce the liver in vivo, non-human animals according to the present invention can be used, for example, to search for risk factors for liver disease or to verify pharmacokinetics, including side effects.

[0255] 1-6. Effects The organoid manufacturing method of this embodiment makes it possible to prepare organoids that highly reproduce the in vivo liver, containing various cell types characteristic of the liver, such as Kupffer cells, and having sinusoidal structures, which are vascular structures characteristic of the liver.

[0256] The use of organoids prepared by the methods described herein offers several advantages compared to organoids developed to date. Firstly, by possessing liver-specific immune cells and vascular structures, it is possible to construct disease models that precisely reproduce inflammatory symptoms characteristic of the liver. Secondly, due to their well-developed vascular structures, a high engraftment rate can be expected when transplanted into vivo. Thirdly, because the hepatic stellate cells contained in the organoids are in the quiescent state observed in a normal liver, they reproduce the properties of a normal liver with high accuracy, and do not promote inflammatory responses or liver fibrosis when transplanted. Furthermore, because the expression of cirrhosis-related genes is low, transplantation does not worsen the disease or condition. Moreover, because the organoids of the present invention possess high liver function, they are expected to exert a high improvement effect on the disease or condition when transplanted.

[0257] These characteristics provide the present invention with a new and promising option for exploring and realizing methods for treating liver failure.

[0258] 2. Method for Producing Model Organoids or Model Non-Human Animals for Fatty Liver Disease 2-1. Overview The second aspect of the present invention is a method for producing model organoids or model non-human animals for fatty liver disease. The method of this aspect includes a lipid addition step, a disease progression step, an organoid preparation step, and an implantation step. According to the method of this aspect, model organoids or model non-human animals for fatty liver disease can be easily produced.

[0259] 2-2. Process 2-2-1. Organoid preparation process The organoid process is an optional process of the method of this embodiment, and is a process for preparing a multilayer liver organoid.

[0260] In this process, the method for producing a multilayer liver organoid according to the first embodiment is carried out. The specific details are the same as those described in the first embodiment.

[0261] 2-2-2. Transplantation Process The transplantation process is an optional step in the method for producing non-human animals that are models of fatty liver disease, and involves transplanting multilayer liver organoids into the non-human animals. If the organoid preparation process is performed, it can be carried out afterward.

[0262] The method of organoid transplantation according to the present invention is not particularly limited, but may be performed by incising the target site and implanting the organoid, or by systemic or local administration. Local administration includes, for example, tissue administration and organ administration, while systemic parenteral administration includes intracirculatory administration (e.g., intravenous administration, intra-arterial administration, and intra-lymphatic administration). For example, in this process, transplantation is usually performed by incision and implantation. The amount applied should be an amount that is effective for the engraftment of the organoid. The effective amount is appropriately selected according to the information of the target, etc.

[0263] In this specification, "information about 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.

[0264] The organoids may be subjected to any treatment before application. For example, the organoids may be stored. There are no specific limitations on the storage method, storage period, or storage temperature.

[0265] The species from which the cells constituting the transplanted organoid originate is not particularly limited, but for example, each cell originates from a species or individual that is the target of the drug being tested in a non-human animal (such as mammals including primates such as humans, or pets, etc.). Preferably, the organoid transplanted in this embodiment originates from a different species or individual than the non-human animal in this embodiment. Preferably, the non-human animal in this embodiment carries a (complete or partial) human liver.

[0266] The non-human animal in this embodiment carries organoid-derived liver tissue. Whether or not the tissue is organoid-derived can be determined by any method. For example, it may be determined by distinguishing between transplanted cells and cells from the non-human animal, or by the presence or absence of phenomena detected after organoid transplantation.

[0267] When distinguishing between transplanted cells and non-human animal cells, the specific method is not particularly limited. For example, the distinction 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.

[0268] When judging based on the presence or absence of phenomena detected after organoid transplantation, the type of phenomenon detected is not particularly limited, as long as it reflects changes 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.

[0269] Phenomena based on structural changes in the liver are not particularly limited, but include, for example, changes in liver size, cell number, weight, and the ratio of fibrotic areas. Phenomena based on functional changes in the liver are not particularly limited, but include, for example, changes in drug metabolism function, albumin production function, and bilirubin 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 destroyed liver cells.

[0270] Whether or not a non-human animal carries organoid-derived liver tissue 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 bloodstream decreases, the accumulation of bilirubin and ammonia decreases, and the total protein amount, albumin amount, and albumin / globulin ratio increase after organoid administration, or if these values ​​are at the same level as those of a normal individual, it can be determined that the non-human animal carries organoid-derived liver tissue.

[0271] 2-2-3. Lipid Addition Step The lipid addition step is an essential step of the method according to this embodiment, and is a step of adding lipid molecules to a multilayer liver organoid containing hepatic parenchymal cells, hepatic stellate cells, hepatic sinusoidal endothelial cells, and Kupffer cells. If an organoid preparation step is to be performed, it can be performed afterward. In the case of a method for producing a non-human animal model of fatty liver disease, this step is carried out as a lipid administration step in which lipid molecules are administered to the non-human animal.

[0272] Except for the addition of lipid molecules, the basic culture conditions for this step are the same as those described in the tissue construction step of the first embodiment. When this step is performed following the preparation of multilayer liver organoids, the culture may be performed under the same conditions as the organoids used in the organoid preparation step, or under different conditions.

[0273] The lipid molecules used in this process are not particularly limited. For example, simple lipids, complex lipids, derived lipids, and steroid lipids may be used. For example, derived lipids such as free fatty acids can be suitably used. The specific free fatty acids used are not particularly limited, but examples include oleic acid, palmitic acid, stearic acid, linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, or combinations thereof.

[0274] The method of application is not particularly limited. For example, when performing gas-liquid interface culture, it may be added directly to the organoid from the gas phase, added to the liquid phase, or both. Preferably, it is added from both the gas and liquid phases.

[0275] The amount added is not particularly limited, as long as it is an excess amount. Specific dosages include, for example, lower limits of 0.1 nmol, 0.5 nmol, 1 nmol, 5 nmol, 10 nmol, 20 nmol, 30 nmol, 40 nmol, 45 nmol, and 50 nmol, and upper limits of 10000 nmol, 5000 nmol, 1000 nmol, 500 nmol, 300 nmol, 250 nmol, 200 nmol, 150 nmol, 100 nmol, 90 nmol, 70 nmol, 60 nmol, 55 nmol, and 50 nmol.

[0276] The addition may be carried out continuously, intermittently, or only once. Preferably, it is carried out at the frequency exemplified for medium exchange, for example. Specifically, for example, the addition may be carried out from the gas phase at the frequency exemplified for medium exchange, and the addition may be carried out continuously from the liquid phase.

[0277] In the manufacturing methods for non-human animals, lipid molecules can be administered orally or parenterally. Parenteral administration can be further subdivided into systemic administration and local administration. Local administration includes, for example, intrasplenic, subcutaneous, intradermal, intravenous, intramuscular, intrafocal, tissue, and organ administration. Systemic parenteral administration includes intracirculatory administration (e.g., intravenous, intra-arterial, and intra-lymphatic administration), intraperitoneal administration, etc. For example, the cell preparation of the present invention can be administered locally, in which case it can be directly administered to the target site by, for example, injection. Alternatively, it can be used by incorporating it into 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 effective amount for the lipid molecules to be effective. The effective amount is appropriately selected according to the information of the target, as described above.

[0278] When administered orally, it is given in solid, liquid, or a combination thereof. The specific method of administration in this case is not particularly limited. For example, methods commonly used for feeding high-fat diets can be used.

[0279] 2-2-4. Disease Progression Process The disease progression process is an essential step of the method according to this embodiment, and involves growing multilayer liver organoids to which lipid molecules have been added to promote disease progression. This process can be performed simultaneously with or after the lipid addition process.

[0280] In the case of organoid manufacturing methods, the disease state can be advanced by culturing for a certain period of time, and in the case of non-human animal manufacturing methods, the disease state can be advanced by rearing for a certain period of time.

[0281] The culture conditions shall be the same as those for the lipid addition step. If the lipid addition step is performed simultaneously with this step, lipid addition may be carried out throughout the entire duration of this step.

[0282] The duration of this process varies depending on the degree of progression of the target disease and is not particularly limited. For example, by performing the process over periods of 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, and 10 to 15 days, it is possible to induce progression to hepatitis such as MASH or NASH.

[0283] 2-3. Fatty Liver Disease Model Organoids The present invention also relates to fatty liver disease model organoids comprising hepatocytes, hepatic stellate cells, hepatic sinusoidal endothelial cells, and Kupffer cells that accumulate lipids within the cells.

[0284] Preferably, the model organoid of the present invention includes balloon-like degenerated hepatocytes. The model organoid of the present invention also includes, for example, Mallory bodies, proliferated activated hepatic stellate cells, etc.

[0285] Furthermore, compared to multilayer liver organoids that do not contain hepatocytes accumulating lipids, the model organoids of the present invention show a significant increase in one or more of the following: for example, the amount of ROS, the amount of IL6 secretion, the amount of activated hepatic stellate cells, and the expression level of inflammation-related genes. On the other hand, compared to multilayer liver organoids that do not contain hepatocytes accumulating lipids, the expression levels of one or more of the following of liver function-related genes, for example, ALB, OTC, G6CP, and CYP3A4, are significantly decreased.

[0286] The model organoid of the present invention may be subjected to additional processing as needed. The specific processing in such cases is not particularly limited.

[0287] The model organoid of the present invention can be manufactured, for example, by the method of this embodiment.

[0288] The use of the model organoid of the present invention is not particularly limited. It 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 screening for therapeutic effects by applying candidate drugs.

[0289] When the model organoid of the present invention is transplanted into a non-human animal, the non-human animal produced by the transplant can be used as a non-human animal model of fatty liver disease. This non-human animal model of fatty liver disease is produced by transplanting the model organoid of the present invention, prepared according to this embodiment, based on the description of the transplantation process in this embodiment. The model organoid of the present invention may be applied to healthy individuals or individuals suffering from some disease or condition.

[0290] The target diseases for the model organoids of the present invention may be any fatty liver disease, and are not particularly limited. For example, they may be used as model organoids for any of the various fatty liver diseases exemplified in the definition section. Specifically, they can be used as, for example, NAFLD model organoids, NASH model organoids, MASLD model organoids, MASH model organoids, etc.

[0291] 2-4. Non-human animal models of fatty liver disease The present invention also relates to non-human animal models of fatty liver disease, including fatty liver disease model organoids, which include hepatic parenchymal cells, hepatic stellate cells, hepatic sinusoidal endothelial cells, and Kupffer cells that accumulate lipids intracellularly.

[0292] The detailed contents of the model organoids are as described above. For non-human animals, the description is basically the same as that for non-human animals in the first aspect.

[0293] The model non-human animal of the present invention can be produced, for example, by the method of this embodiment or by transplanting the model organoid of the present invention.

[0294] 2-5. Effects According to the method of this embodiment, by adding lipid molecules, it is possible to construct a fatty liver disease model that can reproduce not only hepatitis-specific degenerations such as balloon-like degeneration and Mallory bodies, which could not be reproduced with conventional organoids, but also changes in cell composition and gene expression.

[0295] This provides a new and extremely useful option for screening therapeutic drugs and elucidating disease mechanisms.

[0296] Furthermore, since the serious condition of hepatitis is reproduced after a certain period of treatment, this provides a new option that can be used to elucidate the mechanism of disease progression and to screen for earlier-stage therapeutic drugs.

[0297] 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. All concentrations of additives mentioned in the examples represent the final concentrations. Furthermore, all cultivation was carried out at 37°C in a 5% CO2 environment.

[0298] <Example 1. Preparation of organoids using hepatoblasts, vascular endothelial progenitor cells, and mesenchymal cells as raw material cells and evaluation of their properties> (Objective) To prepare organoids using hepatoblasts, vascular endothelial progenitor cells, and mesenchymal cells as raw material cells and to investigate their properties.

[0299] (Methods) 1. iPS cells: M48 (CiRA Foundation) was used as the human iPS cell (hiPSC) line. The plate was iMatrix-511 TM (Nippi, 892011): Coat with iMatrix solution mixed with PBS in a 1:150 ratio, then apply StemFit (R) Cells were cultured in 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.

[0300] 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). hiPSCs were labeled with either GFP or Kusabira Orange gene knock-in.

[0301] 2. Preparation of raw material cells 2-1. Preparation of liver progenitor cells 2-1-1. Preparation of endoderm cells (DE) First, human iPS cells (hiPSCs) are placed on an iMatrix-511 coated dish in a 1.5 × 10⁻¹⁶ size. 5 cells / cm 2 Seeds were sown at the following density (Day 0). The culture medium was 1% B-27. TMCells were cultured for 7 days in RPMI1640 medium (Wako, 189-02025) supplemented with supplement (50×) (Gibco, 17504-001), 100 ng / mL of activin A (molecular weight: approximately 26 kDa; Ajinomoto, 18585-81), and 2 μM CHIR99021 (Cayman, 13122). 10 μM of Y-27632 was added to the medium only on day 0 of culture. 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. Differentiated cells obtained on day 7 of culture were endoderm cells (hiPSC-DE).

[0302] 2-1-2. Differentiation Induction to Hepatic Endoderm Cells (HE) Differentiation induction from induced hiPSC-DEs to hepatic endoderm cells was performed as follows. From day 7 of culture onward, the culture medium was changed to hepatic endoderm cell differentiation medium. StemFit Basic03 (Ajinomoto) was used as the hepatic endoderm cell differentiation medium, 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). The culture medium was changed daily. Differentiated cells obtained on day 10 of culture were hepatic endoderm cells (hiPSC-HE).

[0303] 2-1-3. Differentiation Induction to Hepatoblasts (HB) Differentiation induction from induced hiPSC-DEs to hepatoblasts was performed as follows. From day 7 of culture onward, the culture medium was changed to hepatoblast differentiation medium. As hepatoblast differentiation medium, serum-free differentiation medium (SFD medium) supplemented with 100 nM dexamethasone (DEX: Sigma, D2915), 10 mM nicotinamide (Sigma, N636), 0.5 mM L-ascorbic acid 2-phosphate (Sigma, A8960), 10 ng / mL oncostatin M (OSM; molecular weight: approx. 30 kDa; R&D Systems, 295-OM), 10 ng / mL fibroblast growth factor 2 (FGF2; molecular weight: approx. 17 kDa; Wako, 060-04543), and 20 ng / mL hepatic growth factor (HGF; molecular weight: approx. 79 kDa; REPROCELL, 03-0019) was used. The culture medium was changed daily.

[0304] 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. TM A 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% 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. The culture medium was changed daily. Differentiated cells obtained on day 13 of culture were hepatoblasts (hiPSC-HB).

[0305] 2-2. Preparation of Vascular Endothelial Progenitor Cells Differentiation induction of Kusabira Orange-labeled hiPSCs into vascular endothelial progenitor cells was performed according to previously reported procedures (Takebe et al., Cell Reports, 2017). In summary, human iPS cells (hiPSCs) were seeded on iMatrix-511 coated dishes and cultured for 24 hours in StemFit medium supplemented with 10 μM Y-27632. The following day, the medium was changed to DMEM / F12 medium supplemented with 1% Glutamax, 1% B27, 8 μM CHIR99021 (Cayman Chemical), and 25 ng / mL BMP4. After 3 days of culture, the medium was changed to StemPro-34 SFM medium supplemented with 200 ng / mL VEGF (Fujifilm Wako Pure Chemical Industries) and 2 μM forskolin (Cayman Chemical). Quality checks were performed on day 7 after the start of differentiation induction by confirming the expression of CD31 and CD144 using FACS. The obtained vascular endothelial progenitor cells were subcultured on fibronectin-coated culture dishes using StemPro-34 SFM medium supplemented with 50 ng / mL of VEGF, and then expanded into larger cultures.

[0306] 2-3. Preparation of Mesenchymal Cells (MCs) Differentiation induction from iPSCs to mesenchymal cells was performed according to previously reported methods (Takebe et al., Cell Reports, 2017). In summary, similar to liver progenitor cells, the cell density was 2 × 10^3 cells / cm². 2 ~8×10^3 cells / cm 2 iPSCs were seeded at a density and cultured for 4 to 6 days in StemFit medium supplemented with 10 μM Y-27632. The medium was then changed to DMEM / F12 medium supplemented with 1% Glutamax, 1% B27, 8 μM CHIR99021 (Cayman Chemical), and 25 ng / mL BMP4, and cultured for 3 days. After that, the medium was changed again to StemPro-34 SFM medium supplemented with 10 ng / mL FGF2 and 10 ng / mL PDGFBB (PeproTech) instead of CHIR99021 and BMP4. After 3 days, the medium was changed again to StemPro-34 SFM medium supplemented with 10 ng / mL FGF2 and 10 ng / mL PDGFBB, and cultured for another 3 days.

[0307] 3. Preparation of organoids: Prepared hepatoblasts (or hepatic endodermal cells), vascular endothelial progenitor cells, and mesenchymal cells in ultra-low adhesion 6-well micro-dimple Elplasia (R) Microliver organoids were co-cultured for 24 hours on Corning plates in organoid culture medium supplemented with 10 μM Y-27632. Co-culture was performed with a composition ratio of 1 × 10^6 cells of hepatoblasts (or hepatic endodermal cells), 2 × 10^5 cells of vascular endothelial progenitor cells, and 2 × 10^5 cells of mesenchymal cells per well to prepare microliver organoids (D1).

[0308] For organoid culture medium, we used a medium prepared by mixing equal volumes of DMEM (Fujifilm Wako Pure Chemical Industries) and KBM-VEC1 medium (Kojin Bio), to which we added 0.5% fetal bovine serum (FBS; Biowest), 50 nM dexamethasone (Merck), 10 ng / mL oncostatin M (R&D Systems), and 0.5 μM A8301 (TOCRIS).

[0309] Liver organoids were prepared by culturing the prepared microliver organoids using a gas-liquid phase culture method. First, a cell culture insert (Falcon, 353090) was placed in a 6-well flat-bottom plate, and a silicone chamber (Ibidi, 80369; culture area / well: 0.22 cm²) was placed in the center of the cell culture insert as a mold. 2 A microliver organoid was installed. The prepared microliver organoids were seeded in the silicon chamber of each well so that each well contained 1 × 10^6 hepatoblast cells (or hepatic endodermal cells), and cultured in 1.2 mL of organoid culture medium.

[0310] After 18 hours of culture, when the cells had attached to the cell culture insert, the mold was removed, the culture medium on the cell culture insert was removed (D2), and the cells were cultured for 8 days at a 5% CO2 concentration and 37°C (D10). The culture medium was changed every other day.

[0311] 4. Evaluation of organoid properties 4-1. qPCR analysis PowerMasher (R) After homogenizing the organoids with II (Nippi), use PureLinkTM Total 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.

[0312] 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 liver organoids using hepatic endodermal cells (HE).

[0313]

[0314] 4-2. Albumin Assay: 1.2 mL of culture medium was collected from the lower chamber of the cell culture insert during the cultivation of liver organoids and stored at -80°C. The assay was performed using a human albumin ELISA quantitative set (Bethyl Laboratories) with the thawed medium.

[0315] Absorbance was measured at a wavelength of 450 nm using a Promega GloMax Explorer. Protein secretion amounts were standardized by the volume of collected culture medium. The culture medium samples used were diluted 400-fold.

[0316] 4-3. Urea Production Assay The mesh of the cell culture insert surrounding the liver organoid was cut, and the liver organoid with the attached mesh was placed in a 6-well plate. The organoids were cultured for 24 hours at 37°C in 1 mL of RPMI1640 medium (Fujifilm Wako Pure Chemical Industries) supplemented with 1% B27 (Gibco) and 2 mM NH4CL. The culture medium after incubation was analyzed using QuantiChrom. TMUrea production was evaluated using the Urea Assay Kit (BioAssay Systems) according to the manufacturer's protocol. Fluorescence was measured by detecting fluorescence at a wavelength of 450 nm using a Promega GloMax Explorer (Promega). Measurements were standardized against the results from a control well that did not contain liver organoids.

[0317] 4-4. CYP3A4 Activity Assay: To perform the assay, after aspirating the culture medium, luciferin-IPA (Promega), a luminescent CYP substrate included with the CYP450-Glo CYP3A4 Assay Kit (Promega), was diluted in 1 mL of culture medium and added to the upper chamber of the cell culture insert for incubation for 60 minutes. After incubation, the culture medium was collected, and the luciferin reaction was performed using the CYP450-Glo CYP3A4 Assay Kit according to the manufacturer's protocol. CYP3A4 activity was measured by detecting the fluorescence of luciferin using a Promega GloMax Explorer (Promega). The measured values ​​were calculated as relative luminescence units (RLU) relative to the measured values ​​in an empty well.

[0318] 4-5. Ammonia Metabolism Assay The culture medium used for culturing for the urea production assay was diluted 100-fold with ddH2O, and ammonia metabolism was measured using Ammonia Test Kit II test strips (Arkray) and PocketChem BA Blood Ammonia Analyzer (Arkray) according to the manufacturer's protocol. The measured values ​​were standardized against the results from a control well that did not contain liver organoids.

[0319] 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. The Mann-Whitney U test was used for two-group comparisons.

[0320] (Results) The results are shown in Figures 2 and 3. Liver organoids containing hepatoblasts (HB) had a clearer vascular network and a significantly increased area of ​​hepatic parenchymal cells compared to liver organoids containing hepatic endodermal cells (HE). This was also supported by gene expression, as shown in Figure 2, in liver organoids containing HB (labeled "HB" in the figure), the expression of liver marker genes was significantly increased compared to liver organoids containing HE (labeled "HE" in the figure). Furthermore, as shown in Figure 3, liver organoids containing HB were significantly superior to those containing HE in various liver-specific functions such as albumin production (Figure 3A), urea production (Figure 3B), CYP3A4 activity (Figure 3C), and ammonia metabolism (Figure 3D).

[0321] This suggests that using hepatoblasts instead of hepatic endodermal cells significantly improves the properties of liver organoids, making it possible to prepare organoids that better reflect liver function.

[0322] <Example 2. Preparation of organoids using hepatic stellate cells and monocyte cells and evaluation of their properties> (Objective) To prepare organoids using hepatic stellate cells and monocyte cells as raw material cells, in addition to hepatoblasts, vascular endothelial progenitor cells and mesenchymal cells, and to investigate their properties.

[0323] (Methods) 1. Differentiation induction into hepatic stellate cells (HSCs) 1-1. Differentiation induction into septum transversum mesenchyme cells (STMs) iMatrix-511 TM GFP-labeled human iPS cells (hiPSCs) were 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 10 μM Y-27632 (day 1). Then, the medium was replaced with mesoderm cell induction medium and cultured for 3 days to induce mesoderm cells (day 4). The mesoderm cell induction medium consisted of 1% Glutamax and 1% B27 TMDMEM / F12 medium supplemented with 8 μM 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 PDGFBB (R&D Systems) (day 6). Furthermore, cells were cultured for 2 days in STM secondary induction medium in which activin A and PDGFBB 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).

[0324] 1-2. Differentiation induction of hepatic stellate cells: iPSC-STM on day 8 of culture is converted to iMatrix-511 TM Cells were seeded on plates coated with (Nippi, 892011) 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% fetal bovine serum (FBS; Biowest), 1 mM N-acetylcysteine ​​(NAC; Sigma-Aldrich), and 10 ng / mL FGF2. The medium was changed every other day.

[0325] 2. Preparation of monocyte cells using iMatrix-511 TM 300 hiPSC cells were seeded onto coated 12-well plates, and StemFit was used. (R) Expanded culture was performed in AKOKO2N (Ajinomoto) for 4 days. 10 μM Y-27632 was added to the culture medium only during the first 24 hours. On the 4th day of culture, the medium was converted to STEMdiff. TM The cells were replaced with a Hematopoietic Kit (StemCell Technologies) to induce erythro-myeloid progenitor cells (EMP).

[0326] EMP was collected on day 10 of culture and 0.5% Lipidure was added at a density of 2 × 10^5 cells per well. (R)Monocytes were seeded in 12-well plates coated with (NOF) and induced in serum-free differentiation medium (SFD) supplemented with 50 ng / mL of human M-CSF (Peprotech). The medium was changed every other day by centrifugation, and monocytes were harvested on day 6 of induction culture.

[0327] 3. Preparation of organoids: The procedure was the same as in Example 1, except that the organoids included hepatic stellate cells and monocyte cells.

[0328] Hepatic stellate cells were co-cultured with hepatoblasts at a ratio of 1 × 10^6 cells to 1 × 10^5 cells. Before subjecting the microliver organoids to gas-liquid phase culture, they were mixed with prepared monocyte cells at a cell ratio of 10:2 and suspended in organoid culture medium. The suspension was then subjected to gas-liquid phase culture as described in Example 1. Some liver organoids were prepared without the addition of monocyte cells.

[0329] 4. Evaluation of the properties of liver organoids The properties of liver organoids were evaluated using the various methods described in Example 1. These procedures and statistical analyses were carried out in accordance with the description in Example 1. In addition, the following methods were used for evaluation.

[0330] 4-1. Staining of Sections Liver organoids fixed in 4% paraformaldehyde were embedded in Tissue-Tek OCT compound (Sakura) and stored at -80°C.

[0331] Paraffin sections were prepared as follows: Fixed liver organoids were solidified in 0.2% agarose (Nacalai Tesque) and dehydrated with 30% ethanol and 50% ethanol. Subsequently, Tissue-Tek (R) The process was carried out overnight using VIP 5 Jr (Sakura), and Tissue-Tek (R) TEC TM Paraffin embedding was performed using 5 (Sakura). Both frozen sections and paraffin sections were prepared to a thickness of 6 μm.

[0332] HE staining of paraffin samples was performed by treating them with Mayer's Hematoxylin (Muto Pure Chemicals) for 7 minutes, followed by washing and then treating with Eosin (Muto Pure Chemicals) for 10 seconds.

[0333] Immunostaining was performed on paraffin sections or frozen sections depending on the affinity of the primary antibody. Blocking treatment was performed on frozen sections using Amersham ECL Prime. TM The procedure was performed by treating the sections with Blocking Agent (Cytiva) at room temperature for 1 hour. For paraffin samples, the procedure was performed by sequentially dehydrating with xylene and ethanol, followed by treatment with citrate buffer at 121°C for 20 minutes. After washing the sections, the primary antibody reaction was carried out overnight at 4°C. After washing, the secondary antibody reaction was carried out at room temperature for 1 hour, and the sections were mounted with a mounting medium containing DAPI (Dojindo).

[0334] The following antibodies were used: anti-CD31 antibody (Dako); anti-CK19 antibody (Dako); anti-FCGR2β antibody (Santa Cruz).

[0335] Images were acquired using a Leica THUNDER microscope and a Leica SP8 confocal microscope.

[0336] 4-2. Staining of whole-mount organoids Liver organoids fixed with 4% paraformaldehyde were immersed in ice-cold organoid washing buffer (PBS supplemented with 0.2% BSA and 0.1% Triton X-100) and incubated at 4°C for 1 hour. Primary antibody reactions were carried out overnight at 4°C using anti-αSMA antibody (Agilent) and anti-GFP antibody (Abcam) as primary antibodies. After washing, secondary antibody reactions were also carried out overnight at 4°C. After washing the samples three times with buffer, they were incubated at room temperature for 30 minutes in tissue clearing reagent CUBIC-R+(M) (Tokyo Chemical Industry) diluted with an equal volume of PBS. The cleared organoids were transferred to a dish, PBS containing DAPI (Dojindo) was added, and the dish was covered with a coverslip.

[0337] The following antibodies were used: anti-αSMA antibody (Agilent); anti-GFP antibody (Abcam).

[0338] Images were acquired using a Leica SP8 confocal microscope and analyzed using Leica LAS-X and Imaris 9 software.

[0339] 4-3. Bile Acid Assay: Total bile acid levels were measured using the Total Bile Acid Assay Kit (fluorometric) (Cell Biolabs). First, liver organoids were subjected to PowerMasher. (R) After homogenization using II, sonication was performed, and measurements were carried out according to the manufacturer's protocol. The measurement solution was diluted 20-fold, and fluorescence at an excitation wavelength of 520 nm was measured using a Promega GloMax Explorer.

[0340] 4-4. Liver organoids were recovered from cell culture inserts for transmission electron microscopy observation. They were pre-fixed at 4°C in 0.1M phosphate buffer containing 4% paraformaldehyde and 4% glutaraldehyde, and then post-fixed overnight at 4°C in 0.1M phosphate buffer containing 2% glutaraldehyde. The fixed samples were cut into 1mm × 1mm blocks and incubated at 4°C for 1 hour in equal volumes of 4% paraformaldehyde and 4% glutaraldehyde solution, followed by overnight incubation at 4°C in a 2% glutaraldehyde solution using 0.1M phosphate buffer as the solvent. The fixed samples were post-fixed with 2% tetrooxide osmium, dehydrated using a series of ethyl alcohols, and embedded in fresh 100% resin.

[0341] 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 (JEOL) transmission electron microscope at an acceleration voltage of 100 kV. A CCD camera EM-14830RUBY2 (JEOL) was used to acquire digital images.

[0342] 4-5. Vascular endothelial cells selected using Kusabira Orange as an indicator by scanning electron microscopy and flow cytometry were seeded into 48-well plates coated with collagen-I and incubated for 6 hours in organoid culture medium supplemented with 10 μM Y-27632.

[0343] After incubation, the samples were pre-fixed at 4°C in 0.1M cacodylate buffer containing 2% paraformaldehyde and 2% glutaraldehyde, and then incubated overnight at 4°C in 0.1M phosphate buffer containing 2% glutaraldehyde. Furthermore, the samples were incubated at 4°C for 1 hour in 0.1M cacodylate buffer containing 1% tannic acid, and then washed with 0.1M cacodylate buffer. Post-fixation was performed by incubation at 4°C for 1 hour in 0.1M cacodylate buffer containing 2% osmium tetrooxide.

[0344] Dehydration of the samples was performed by incubation at RT for 1 hour in a 5:5 solution of ethanol and tert-butanol, and by immersion in a stepwise dilution series of ethanol. After dehydration, the samples were immersed in tert-butanol, followed by freezing and drying under reduced pressure at 4°C. The samples were coated with a 30 nm thick osmium film using an osmium plasma coater (Nippon Laser; NL-OPC80NS).

[0345] The images were taken using a field emission scanning electron microscope (JEOL; JSM-7500F) with an acceleration voltage of 3.0 kV.

[0346] 4-6. RNA Sequencing Analysis Some RNA sequencing analyses used cell populations that had been classified by flow cytometry.

[0347] Liver organoids were harvested from the cell culture insert, washed, and shredded with a razor, then dispersed in 5 mL of dispersion buffer at 37°C for 60 minutes. After dispersion, the buffer was removed, TrypLE (Gibco) was added, and the cells were incubated at room temperature for 10 minutes. After incubation, the cells were separated into single cells by pipetting, collected in washing buffer (PBS containing 5% FBS), centrifuged at 200 × G for 4 minutes, and resuspended in washing buffer.

[0348] As a dispersion buffer, HBSS (-Ca, -Mg, -phenol red) (Gibco) containing 3 mg / mL collagenase-IV (Gibco), 1% HEPES (Gibco), 0.5% CaCl2, and 0.5% MgCl2 was used.

[0349] Single-cell isolated cells 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 APC Mouse Anti-Human CD14 antibody on ice for 30 minutes. The samples were then washed and resuspended in PE buffer. Flow cytometry was performed using BD FACSAria. (R) The analysis was performed using the III Cell Sorter (BD Biosciences) and the collected data using FlowJo 10.7.1 (BD Biosciences) software. Cells were classified into Kusabira Orange-positive cells, GFP-positive cells, and CD14-positive cells.

[0350] From a mixed or already classified cell population, PureLink (R) RNA Micro Kit (Invitrogen) and PureLink (R)RNA extraction was performed using DNase / Carrier RNA (Invitrogen). RNA-seq library preparation and sequencing were performed using the Ion AmpliSeq Transcriptome Human Gene Expression Kit. Transcriptome data analysis of the obtained RNA quantities was performed using GeneSpring GX 14 (Agilent) software, and gene ontology analysis was performed using the DAVID bioinformatics database. Gene set enrichment analysis was performed using GSEA v4.3.2 (Broad Institute).

[0351] (Results) The results are shown in Figures 4-8. Live imaging confirmed the engraftment of hepatic stellate cells and monocytes within the liver organoids. As shown in Figure 4, the hepatic stellate cells contained in the liver organoids were αSMA-negative, indicating that the quiescent state of hepatic stellate cells was maintained.

[0352] The vascular density and vascular diameter within the liver organoids were significantly increased, suggesting active angiogenesis. Furthermore, as shown in Figure 5, CD31-positive cells within the liver organoids (Figure 5C) were confirmed to be co-positive for FCGR2β (Figure 5B), suggesting that these liver organoids contained sinusoidal cells. This was supported by genetic analysis, which confirmed an increase in the expression levels of hepatic sinusoidal endothelial cell-specific genes (LYVE-1, FVIII, FCGR2β, CD36, etc.) due to the inclusion of monocyte cells in the liver organoids. In addition, when CD31-positive cells within the liver organoids were isolated by flow cytometry and their cell membrane structure was examined using a scanning electron microscope, the presence of a cribriform plate structure characteristic of hepatic sinusoidal endothelial cells was confirmed on the cell membrane (Figure 6).

[0353] This suggests that the inclusion of monocytes promotes the differentiation of functional hepatic sinusoidal endothelial cells within liver organoids.

[0354] When the expression of human Kupffer cell-related genes (CD68, ADORA3, C1QA, TIMD4, MARCO, CD5L, etc.) was examined in monocyte cells, it was confirmed that all of them were significantly elevated compared to monocyte cells before co-culture. The top genes that were upregulated in primary Kupffer cells were all confirmed to be highly expressed in monocyte cells derived from liver organoids (Figure 7).

[0355] This suggests that the liver organoids of the present invention contain a large number of Kupffer cells.

[0356] Furthermore, urea production, CYP3A4 activity, ammonia metabolism, and bile acid levels were checked in the same manner as in Example 1, and all were found to be favorable. Gene set enrichment analysis confirmed that the expression of these related genes was significantly increased compared to the microorganoids.

[0357] Detailed observation of the ultrastructure of liver organoids revealed hepatocytes ("Hep" in Figure 8), sinusoidal lumen ("Lu" in Figure 8A), and bile canaliculi ("BC" in Figure 8B), as shown in Figure 8. Furthermore, the sinusoidal lumen was lined with hepatic sinusoidal endothelial cells ("*" in Figure 8A), and Kupffer cells ("†" in Figure 8A) were observed on both the luminal and parenchymal sides. On the parenchymal side, Disse's space ("SD" in Figure 8A) containing hepatic stellate cells was observed, along with hepatic stellate cells ("\" in Figure 8A). This confirmed that a diverse range of cells similar to those found in vivo were observed around the sinusoidal lumen, indicating the construction of a complex tissue. Tight junctions ("TJ" in Figure 8B) were also observed between hepatocytes.

[0358] From the above, it has been shown that the liver organoid of the present invention comprises hepatic parenchymal cells and non-parenchymal cells, has a tissue structure and ultrastructure similar to that of the liver in vivo, and is a liver organoid that has liver-specific metabolic functions.

[0359] <Example 3. Preparation of fatty liver disease model organoids and evaluation of their properties> (Objective) To induce a fatty liver disease model from the liver organoids of the present invention and evaluate its properties.

[0360] (Methods) 1. Preparation of fatty liver disease model organoids In the D10 liver organoids prepared in Example 2, 10 μL of organoid culture medium containing 5 mM oleic acid (Sigma-Aldrich) was added to the top of the organoid on a cell culture insert. Subsequent culture was carried out under conditions where organoid culture medium was present in the lower chamber of the cell culture insert. During this time, the medium and oleic acid were changed every other day. As a control, organoids cultured for the same period without the addition of oleic acid were used. Hepatic stellate cell proliferation was monitored by live cell imaging using GFP as an indicator.

[0361] 2. Evaluation of the properties of fatty liver disease model organoids The properties of fatty liver disease model organoids were evaluated using the various methods described in Examples 1 and 2. These procedures and statistical analyses were carried out in accordance with the descriptions in Examples 1 and 2. In addition, the following methods were used for evaluation.

[0362] To visualize the presence of reactive oxygen species, 2 μM CellROX TM Orange Reagent (Invitrogen) was added to the organoids and incubated at 37°C for 30 minutes. After incubation, the culture medium was removed, the cells were washed, and then observed and quantified using a fluorescence microscope.

[0363] To visualize lipid accumulation, first, in the case of frozen sections, use 5 μM BODIPY TM The sections were incubated with 493 / 503 (Invitrogen) at 37°C for 30 minutes. After incubation, the sections were washed and mounted with a mounting medium containing DAPI (Dojindo). The stained sections were observed under a Leica THUNDER microscope.

[0364] When targeting the entire organoid, fix the organoid with 4% paraformaldehyde and then use HCS LipidTOX. TM The samples were stained by incubation with a 200-fold diluted solution of Deep Red Neutral Lipid Stain (Invitrogen) at 37°C for 30 minutes, and observed using a Leica SP8 confocal microscope, as in the whole-mount staining method described above.

[0365] 3. Image Analysis: Staining data for hepatic stellate cell proliferation, lipid accumulation, and balloon-like degeneration of p62-positive hepatocytes were analyzed using ImageJ software.

[0366] (Results) The results are shown in Figures 9 to 15.

[0367] Since the accumulation of free fatty acids is a contributing factor to the development of fatty liver disease, liver organoids were loaded with free fatty acids (FFA) for 10 days. As shown in Figure 9, compared to liver organoids that were not loaded with FFA (Figure 9A), the liver organoids loaded with FFA (Figure 9B) showed a significant increase in signals indicating lipid accumulation, suggesting that a large amount of lipid accumulation occurred.

[0368] Furthermore, as shown in Figures 10 and 11, balloon-like degeneration (Figure 10A), a lesion characteristic of fatty liver disease, was observed in liver organoids that underwent FFA loading (Figures 10C and D), but was not observed in liver organoids that did not undergo FFA loading (Figures 10A and B). In addition, when the fluorescence signal of p62, a Mallory body marker, was examined, it was not observed in liver organoids that did not undergo FFA loading (Figure 11A), but was significantly observed in liver organoids that underwent FFA loading (Figure 11B).

[0369] Quantitative results also confirmed the pathological features characteristic of fatty liver disease. Compared to liver organoids that did not undergo FFA loading, liver organoids that underwent FFA loading showed a significant increase in p62 signaling (Figure 12A) and a significant increase in reactive oxygen species (ROS) (Figure 12B).

[0370] Furthermore, liver organoids subjected to FFA loading showed a significant increase in the expression of inflammation-related genes compared to liver organoids not subjected to FFA loading (Figure 13A), and increased IL-6 secretion suggested an enhanced inflammatory response (Figure 13B). Hepatic stellate cells, which were in a quiescent state before FFA addition, also showed synchronized changes; as shown in Figure 14, FFA loading increased the number of αSMA-positive cells, an activation marker. In addition, as shown in Figure 15, the amount of hepatic stellate cells increased with the number of days since the start of FFA loading, suggesting that the inflammatory response progressed with the passage of time.

[0371] On the other hand, as shown in Figure 16, in liver organoids subjected to FFA loading, albumin secretion (Figure 16A), urea production (Figure 16B), and CYP3A4 activity (Figure 16C) were decreased. This suggests that FFA causes impaired liver function. These results were also supported by gene expression analysis. In liver organoids subjected to FFA loading, the expression of liver function-related genes such as ALB, OTC, G6CP, and CYP3A4 was significantly reduced compared to liver organoids that were not subjected to FFA loading. Furthermore, GO analysis of the top distinguishing genes with and without FFA loading revealed that inflammation-related genes such as cellular responses to IL1, inflammatory responses, neutrophil chemotaxis, cellular responses to TNF, chemokine-mediated pathways, cellular responses to LPS, monocyte chemotaxis, and lymphocyte chemotaxis were among the factors whose expression was upregulated by FFA loading. Conversely, factors whose expression was decreased by FFA loading included liver function-related genes such as arachidonic acid metabolism, GPCR signaling pathways, cholesterol efflux, triglyceride homeostasis, cholesterol biosynthesis pathways, cholesterol homeostasis, responses to heterologous stimuli, and linoleic acid metabolism pathways.

[0372] The results above demonstrate that, with the liver organoid of the present invention, spontaneous inflammatory responses are induced solely by the addition of FFA, liver function is impaired, and characteristic disorders and lesions of fatty liver disease are reproduced, such as the generation of reactive oxygen species, the appearance of balloon-like degeneration, the formation of Mallory bodies, and the proliferation and activation of hepatic stellate cells, thereby constructing a fatty liver disease model similar to that of fatty liver in vivo.

[0373] <Example 4. Evaluation of drugs using fatty liver disease model organoids> (Objective) To evaluate fatty liver disease treatment drugs using fatty liver disease model organoids prepared in Example 3.

[0374] (Methods) The preparation of fatty liver disease model organoids was carried out in the same manner as in Example 3, except that during the culture period in which oleic acid was added, organoid culture medium containing Resmetirom (Selleck), Niclosamide (Selleck), Pirfenidone (Selleck), Cenicriviroc (Selleck), Tipelukast (Selleck), and Lanifibranor (Selleck) at effective concentrations as therapeutic agents for fatty liver disease was placed in the lower chamber of the cell culture insert and cultured. As a control, fatty liver disease model organoids prepared without the addition of therapeutic agents were used.

[0375] (Results) The results are shown in Figures 17-19. As shown in Figure 17, lipid accumulation was significantly suppressed by all the drugs tested, including Resmetirom and Lanifibranor. Similarly, the amount of p62, a Mallory body marker, was also significantly suppressed by all the drugs tested (Figure 18). On the other hand, Lanifibranor and Cenicriviroc did not show a significant inhibitory effect on the proliferation rate of hepatic stellate cells (HSCs), which reflect the severity of inflammation. In addition, results for albumin production and the inflammatory markers CCL2, IL6, and IL8 suggested that Resmetirom and Niclosamide, in particular, had high therapeutic effects. For example, a paper on the therapeutic effect of Lanifibranor (Francque, et al, 2021; doi:10.1056 / NEJMoa2036205) pointed out that while Lanifibranor showed significant improvement in lipid-related items, it did not show a significant effect in some inflammation-related items, suggesting that its effect may be limited. Furthermore, for example, a paper on clinical trials of Resmetirom (Harrison, et al, 2024; doi:10.1056 / NEJMoa2309000) reported that Resmetirom significantly improved cholesterol-related parameters and also showed remarkable improvements in inflammation-related parameters. Thus, the results shown in this embodiment based on the model organoid of the present invention are consistent with the therapeutic effects suggested for each drug.

[0376] Based on the above, it is suggested that the fatty liver disease model organoid of the present invention is highly reproducible as a model of actual fatty liver in vivo and is useful for drug screening and evaluation.

[0377] <Example 5. Evaluation of the cellular composition of organoids using hepatic stellate cells and monocyte cells> (Objective) To evaluate the cellular composition of organoids using hepatic stellate cells and monocyte cells as raw material cells, in addition to hepatoblasts, vascular endothelial progenitor cells, and mesenchymal cells, based on a comparison with human liver.

[0378] (Methods) Organoid preparation and immunohistochemical staining were performed in the same manner as in Example 2. Normal human liver samples were obtained from LifeNet Health LifeSciences. The dispersion of normal human liver samples was performed using gentleMACS. TM The study was conducted by Dissociators (Miltenyi Biotec).

[0379] The anti-SOX9 antibody (Abcam) was used as the SOX9 antibody. Single-cell RNA sequencing analysis was performed by Novogene Japan. The procedure outline is as follows.

[0380] Using a Chromium controller (10x Genomics), single cells derived from the sample were captured and lysed. Single-cell RNA was then contained within each oil droplet, and each droplet was tagged with a bead containing a unique molecular identifier. The single-cell RNA in the droplets was reverse transcribed and amplified. Subsequently, library preparation was performed using the Chromium Single Cell 3' v3 reagent kit (10x Genomics) according to the manufacturer's instructions.

[0381] Sequencing was performed using the NovaSeq X Plus platform (Illumina). The sequencing results were aligned to the human genome reference sequence (GRCh38) and pre-processed using CellRanger 10x Genomics software (10x Genomics) before use in subsequent analysis. Only genes expressed in three or more cells were used for subsequent analysis.

[0382] UMAP analysis and violin plot matrix creation based on the results of single-cell RNA sequencing were performed using RStudio. Here, cell types were distinguished based on specific markers of each cell.

[0383] The cell type-specific markers used for classifying each cell type are as follows: Hepatocytes: APOA1, APOC3, ALB; Hepatic sinusoidal endothelial cells: LYVE-1, STAB1, STAB2, KDR; Vascular endothelial cells: VWF, CD34; Kupffer cells: CD5L, MARCO, VCAM1; Monocytes: LYZ, CD300E, VCAN; Macrophages: ITGAX, CIITA; T cells: CD3D, TRAC, TRBC1, TRBC2, CD4, CD8A; Natural killer cells: KLRD1, NKG7, GNLY, GZMB, PRF1, KLRF1, KLRG1; B cells: CD19, CD79A, MS4A1, IGHM, IGHD; Hepatic stellate cells: HAND2, RSPO3, HGF, DCN; Vascular smooth muscle cells: MYH11, ITGA7, NOTCH3; Fibroblasts: LAMC3, PODN, AEBP1, ELN; Biliary epithelial cells: KRT7, AQP1, SPP1.

[0384] (Results) The results are shown in Figures 20-25. UMAP analysis was performed to analyze the clusters of cells constituting normal human liver samples and liver organoids. The results confirmed that liver organoids (Figure 20A) contain hepatocytes, hepatic stellate cells, as well as vascular cells such as bile duct epithelial cells, hepatic sinusoidal endothelial cells, and vascular smooth muscle cells, and immune system cells such as macrophages and Kupffer cells, similar to normal human liver samples (Figure 20B).

[0385] Furthermore, immunohistochemical staining revealed ALB and CYP3A4 signals in many cells of the liver organoids (Figure 22), confirming that the hepatocytes contained in the liver organoids were performing hepatic functions such as albumin production and CYP3A4 production. It was also confirmed that the liver organoids contained biliary tract cells (Figure 23) and sinusoidal cells (Figure 24), and that Kupffer cells were located near hepatic sinusoidal endothelial cells (Figure 25).

[0386] <Example 6. Evaluation of changes in cellular composition and gene expression associated with disease progression> (Objective) To evaluate the changes in cellular composition and genes of fatty liver disease model organoids associated with disease progression, based on comparison with human liver exhibiting MASH pathology.

[0387] (Methods) Organoid preparation and RNA sequencing analysis were performed in the same manner as in Example 2. Furthermore, fatty liver disease model organoids were prepared in the same manner as in Example 3.

[0388] Human liver samples were obtained from LifeNet Health LifeSciences. The human liver samples used included those from normal human individuals ("NAS0"), those from MASH individuals classified as category 2 by NAS ("NAS2"), those from MASH individuals classified as category 4 by NAS ("NAS4"), and those from MASH individuals classified as category 6 by NAS ("NAS6"). The variance of normal human liver samples was determined using gentleMACS. TM This was conducted at Dissociators.

[0389] As the SOX9 antibody, the anti-SOX9 antibody (Abcam) was used. In the gene expression fluctuation analysis, analysis was performed for each cell type using genes whose expression level increased by logFC (Fold Change) > 0.5 compared to the gene expression level in control samples that did not exhibit fatty liver disease.

[0390] In this study, we performed gene ontology analysis on genes whose expression increased in NAS6 compared to NAS0, and whose expression also increased upon induction of disease from liver organoids to fatty liver disease model organoids.

[0391] (Results) The results are shown in Figures 26-35. UMAP analysis was performed to analyze the clusters of cells constituting normal human liver samples and liver organoids. The results confirmed that the decrease in hepatocytes and vascular cells such as LSECs, and the increase in immune cells such as macrophages and monocytes (Figures 26 and 27) observed in human liver samples as MASH progresses, were reproduced in the induction from liver organoids (Figure 28A) to fatty liver disease model organoids (Figure 28B). This was also confirmed quantitatively, and as shown in Figure 29, the trends of increase and decrease in various cells were consistent between the organoids and human liver samples (Figure 29).

[0392] Furthermore, when we examined the commonality between human liver samples and organoids of genes whose expression levels increased with the progression of the disease, we found that a considerable number of genes commonly showed increased expression levels. This included increased expression levels of cytotoxicity-related and oncogenesis-related genes in liver cells (Figure 30), increased expression levels of inflammatory response-related genes in Kupffer cells (Figure 31), macrophages (Figure 32), and monocytes (Figure 33), and increased expression levels of fibrosis-related genes in hepatic stellate cells (Figure 34) and hepatic fibroblasts (Figure 35). These increases in the expression of important genes related to MASH pathology were also reproduced.

[0393] This demonstrates that the organoids of the present invention not only accurately reproduce normal human liver tissue, but can also be easily applied to accurately reproduce the MASH pathology, including not only macroscopic pathological manifestations but also changes in cell composition and gene expression.

[0394] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A method for producing a multilayer liver organoid, comprising: a co-culture step of co-culturing raw material cells; a differentiation induction step of inducing differentiation of the raw material cells; and a tissue construction step of constructing a tissue structure composed of multiple layers of cells, wherein the raw material cells include hepatocytes, vascular endothelial progenitor cells, and mesenchymal cells.

2. The method according to claim 1, wherein the raw material cells further comprise hepatic stellate cells.

3. The method according to claim 2, wherein the hepatic stellate cells are quiescent hepatic stellate cells.

4. The method according to claim 1, wherein the raw material cells further comprise monocyte cells.

5. The method according to claim 1, further comprising a growth culture step of growing and culturing the raw material cells.

6. The method according to claim 1, further comprising a raw material cell preparation step of preparing the raw material cells from pluripotent stem cells.

7. A multilayer liver organoid manufactured by the method described in claim 1.

8. Multilayer liver organoids containing hepatic parenchymal cells, hepatic stellate cells, hepatic sinusoidal endothelial cells, and Kupffer cells.

9. A method for producing a fatty liver disease model organoid, comprising: a lipid addition step of adding lipid molecules to a multilayer liver organoid containing hepatic parenchymal cells, hepatic stellate cells, hepatic sinusoidal endothelial cells and Kupffer cells; and a disease progression step of culturing the multilayer liver organoid to which lipid molecules have been added to induce disease progression.

10. The method according to claim 9, wherein the lipid molecule includes a free fatty acid.

11. The method according to claim 9, wherein the multilayer liver organoid is the organoid described in claim 7.

12. A fatty liver disease model organoid manufactured by the method described in any one of claims 9 to 11.

13. A fatty liver disease model organoid containing hepatocytes, hepatic stellate cells, hepatic sinusoidal endothelial cells, and Kupffer cells that have accumulated lipids within the cells.

14. A non-human animal comprising the organoid described in claim 7.

15. A method for producing a non-human animal model of fatty liver disease, comprising a lipid administration step of administering a lipid molecule to the non-human animal described in claim 14.

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