Methods of hepatoblast proliferation and methods of liver disease treatment using hepatoblasts

Abstract

Provided are a method for producing hepatoblasts in high purity and in large quantities, and a method for producing liver lineage cells that achieve high survival rates in vivo. Provided are a hepatoblast proliferation promoter and a culture medium comprising a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor, and a proliferation culture method using the same.

Description

Technical Field

[0001] This invention relates to a method for proliferating hepatoblasts, a method for manufacturing hepatoblast populations, a pharmaceutical composition containing hepatoblasts for treating liver diseases, and a treatment method for liver diseases using hepatoblasts. Background Technology

[0002] The liver is a highly regenerative organ capable of recovering from injury. This is because hepatocytes, which make up about 70% of liver tissue, are plastic. However, when liver damage exceeds its regenerative capacity, or when the regenerative capacity itself is reduced or impaired due to factors such as hepatocyte aging, liver function significantly declines, leading to liver failure. Liver failure is a major cause of high mortality.

[0003] Liver transplantation is known as one of the treatments for liver failure. While liver transplantation has long been recognized as an effective cure for liver failure, the shortage of available donor livers has prevented patients from receiving treatment at the necessary time. Cell replacement therapy, a simpler treatment option that does not require extensive surgery, has been explored. However, cell replacement therapy involves administering donor liver-derived hepatocytes containing hepatocytes and fetal hepatocytes / hepatoblasts, thus also relying on the availability of donor livers. Furthermore, these methods, due to the donor-recipient combination, even with suppression of the recipient's immune system to reduce rejection, still suffer from low survival rates one year after transplantation and limited long-term therapeutic effects.

[0004] Among these methods, cell replacement therapy and liver transplantation using human pluripotent stem cells are promising options for treatment that can be performed without relying on the quantity of donor livers. In particular, iPS cells derived from autologous cells and pluripotent stem cells that have undergone genome editing have low immunogenicity and are expected to be less prone to recipient rejection. Due to these advantages, various studies have been conducted to date, achieving the generation of hepatocytes and liver-like organoids using human pluripotent stem cells. However, when these cells and organoids are applied in vivo, their reproliferative capacity is limited, indicating that the proportion of transplanted cells in the recipient's liver is less than 1%. Typically, to achieve a therapeutic effect for patients with liver failure, the transplanted cells must occupy more than 10% of the hepatocytes in the recipient's liver (Non-Patent Literature 1).

[0005] To date, methods for inducing differentiation of hepatocytes from pluripotent stem cells as hepatocyte precursor cells have been reported (Patent Document 1). Hepatocytes are highly proliferative hepatic precursor cells present in the fetal liver, indicating their potential as a cell supply source for cell replacement therapy in liver diseases. In mouse allograft experiments, the reproliferative capacity of hepatocytes derived from the fetal liver was confirmed in livers with liver injury. However, compared to transplantation of hepatocytes derived from the mature liver, a significantly lower proportion of transplanted hepatocytes was observed in the recipient liver (Non-Patent Document 2). Hepatocytes are a cell type that is typically found only in fetuses, making it difficult to ensure a sufficient number of cells for cell transplantation. Therefore, cell replacement therapy using hepatocytes has not yet reached practical application.

[0006] For these reasons, cell replacement therapy using hepatocytes derived from pluripotent stem cells and liver transplantation, although having many advantages, have not yet been put into practical use. It is hoped that a method for preparing liver lineage cells with high survival rates in vivo can be established.

[0007] Existing technical documents Patent documents Patent Document 1: WO2021 / 060380 Non-patent literature Non-patent literature 1: Puppi, J., et al., Cell Transplant 21, 1-10 (2012) Non-patent literature 2: Dhivya H. et al., The American Journal of Pathology 175(4), 1483-1492 (2009) Summary of the Invention The problem that the invention aims to solve The inventors have explored the establishment of a method for cell transplantation that achieves high viability in vivo. To test the usefulness of hepatoblasts derived from pluripotent stem cells in cell transplantation, it is necessary to prepare large quantities of these hepatoblasts with high purity. However, it is known from conventional culture methods that, with prolonged culture, hepatoblasts differentiate into hepatocytes and bile duct cells, making it difficult to prepare high-purity and large quantities of hepatoblasts while maintaining their undifferentiated state. Therefore, the objective of this invention is to provide a method for preparing high-purity and large quantities of hepatoblasts, and further, to provide a method for preparing liver lineage cells with high viability in vivo.

[0008] Methods for solving problems The inventors conducted in-depth research and discovered that by culturing hepatoblasts in a culture medium composed of specific components, the hepatoblasts could stably and continuously proliferate in an undifferentiated state for more than 15 generations. Furthermore, they found that when these hepatoblasts were transplanted into the livers of mice with liver failure, the transplanted hepatoblasts could stably survive in vivo, replenishing liver tissue and restoring liver function.

[0009] That is, the present invention is based on the new insights and the like, and provides the following [1] to

[38] .

[0010] [1] A method for proliferating hepatocytes, comprising: a proliferation culture step, wherein hepatocytes are proliferated and cultured in a hepatocyte proliferation culture medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator and growth factors.

[0011] [2] A method for manufacturing a population of hepatocytes, comprising: a proliferation culture step, wherein hepatocytes are proliferated and cultured in a hepatocyte proliferation culture medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator and growth factors.

[0012] [3] The method according to [1] or [2], wherein the growth factor is one or more proteins selected from the group consisting of FGF2, EGF, IGF, VEGF and PDGF.

[0013] [4] The method according to any one of [1] to [3], wherein the ROCK inhibitor is Y-27632, Rho Kinase Inhibitor IV or thiazolinone.

[0014] [5] The method according to [4], wherein the ROCK inhibitor is Y-27632.

[0015] [6] The method according to any one of [1] to [3], wherein the TGFβ signaling pathway inhibitor is one or more inhibitors selected from the group consisting of A8301, SB431542, SB505124, LY364947, LY2157299 and RepSox.

[0016] [7] According to the method described in [6], the TGFβ signaling pathway inhibitor is A8301.

[0017] [8] The method according to any one of [1] to [3], wherein the Wnt signal transduction pathway activator is one or more substances selected from the group consisting of WNT3A, CHIR99021, SB216763, BIO, TWS119, SB216763, and Recombinant R-Spondin 1.

[0018] [9] According to the method described in [8], the Wnt signal transduction pathway activator is WNT3A.

[0019]

[10] The method according to any one of [1] to [9], wherein the culture medium for hepatoblast proliferation further comprises a steroid agent.

[0020]

[11] According to the method described in

[10] , the steroid is a glucocorticoid or a derivative thereof.

[0021]

[12] According to the method described in

[11] , wherein the glucocorticoid or a derivative thereof is dexamethasone.

[0022]

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

[12] , wherein the hepatoblasts express IL1RAPL1, NRP1, SOX9, ONECUT1, NID1 and LYST.

[0023]

[14] According to the method described in

[13] , wherein the hepatoblasts further express one or more genes selected from the group consisting of SLC4A4, TNFSF4, PLEKHA2 and RNF207.

[0024]

[15] A method for manufacturing a cell preparation for treating liver disease, comprising the method described in any one of [1] to

[14] .

[0025]

[16] A method for manufacturing a cell preparation for treating liver disease, comprising: a first step of inducing pluripotent stem cells to differentiate into fixed-type endoderm cells; a second step of inducing the fixed-type endoderm cells to differentiate into hepatocytes; and a third step of proliferating and culturing hepatocytes to create a hepatocyte population. The first step includes the following steps: culturing in a serum-free medium containing Y-27632 during the early stage of culture; and culturing in a medium containing B-27 supplement, activin A, and WNT signaling pathway activator after the middle stage of culture. The second step includes the following steps: culturing in a serum-free medium containing FGF2, HGF, oncogene M, dexamethasone, and nicotinamide. The third step includes a proliferation culture process, in which hepatocytes are proliferated and cultured in a hepatocyte proliferation culture medium containing ROCK inhibitors, TGFβ signaling pathway inhibitors, Wnt signaling pathway activators and growth factors.

[0026] [16-1] The method for manufacturing the cell preparation according to

[16] , wherein the serum-free culture medium contains 10 μM of Y-27632.

[0027]

[17] The method for manufacturing the cell preparation according to

[16] or [16-1] further includes, after the third step: a maturation promotion step, adding oncogene M to the proliferated hepatocytes and culturing them.

[0028]

[18] A method for treating liver disease includes the following steps: administering a cell preparation to a subject, the cell preparation comprising hepatocytes that have been proliferated and cultured in a hepatocyte proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator and growth factors.

[0029]

[19] The treatment method according to

[18] , wherein the cell preparation comprises further addition of oncogene M to proliferating and cultured hepatoblasts to promote cell maturation.

[0030]

[20] A method for manufacturing hepatocytes, comprising: a proliferation culture step, wherein hepatocytes are proliferated and cultured in a hepatocyte proliferation culture medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator and growth factors; and a hepatocyte induction step, wherein differentiation from the proliferated and cultured hepatocytes into hepatocytes is induced.

[0031]

[21] A method for manufacturing bile duct cells, comprising: a proliferation culture step, wherein hepatocytes are proliferated and cultured in a hepatocyte proliferation culture medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator and growth factors; and a bile duct cell induction step, wherein differentiation from the proliferated and cultured hepatocytes into bile duct cells is induced.

[0032]

[22] A cell preparation for treating liver disease comprising cells expressing IL1RAPL1, NRP1, SOX9, ONECUT1, NID1 and LYST.

[0033]

[23] According to the cell preparation described in

[22] , wherein the cells are cells that further express one or more genes selected from the group consisting of SLC4A4, TNFSF4, PLEKHA2, RNF207, REG3A, SAA1, SAA2, HP, SPINK1, LBP, KCNG2, ANXA10, ITIH3, ITIH4, MTUS1, CRP, CEACAM5, C4B, ATP10B, NNMT, LCN2, LOC145837, ORM1, ORM2, PC, SERPINA7, SOCS3, C4A, SERPINA3, C8A, CD38, TMEM45B, CDA and UGT2B4.

[0034]

[24] A treatment for liver disease includes administering cells expressing IL1RAPL1, NRP1, SOX9, ONECUT1, NID1 and LYST to a subject.

[0035]

[25] According to the treatment method described in

[24] , the cells are cells that further express one or more genes selected from the group consisting of SLC4A4, TNFSF4, PLEKHA2, RNF207, REG3A, SAA1, SAA2, HP, SPINK1, LBP, KCNG2, ANXA10, ITIH3, ITIH4, MTUS1, CRP, CEACAM5, C4B, ATP10B, NNMT, LCN2, LOC145837, ORM1, ORM2, PC, SERPINA7, SOCS3, C4A, SERPINA3, C8A, CD38, TMEM45B, CDA and UGT2B4.

[0036]

[26] A cell for the treatment of liver disease, characterized in that it expresses IL1RAPL1, NRP1, SOX9, ONECUT1, NID1 and LYST.

[0037]

[27] The cells according to

[26] further express one or more genes selected from the group consisting of SLC4A4, TNFSF4, PLEKHA2, RNF207, REG3A, SAA1, SAA2, HP, SPINK1, LBP, KCNG2, ANXA10, ITIH3, ITIH4, MTUS1, CRP, CEACAM5, C4B, ATP10B, NNMT, LCN2, LOC145837, ORM1, ORM2, PC, SERPINA7, SOCS3, C4A, SERPINA3, C8A, CD38, TMEM45B, CDA and UGT2B4.

[0038]

[28] A method for proliferating hepatocytes, comprising: a proliferation culture step, wherein hepatocytes are proliferated and cultured in a hepatocyte proliferation culture medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor or a Wnt signaling pathway activator, and growth factors.

[0039]

[29] According to the method of

[28] , wherein the growth factor is one or more proteins selected from the group consisting of FGF2, EGF, IGF, VEGF and PDGF.

[0040]

[30] The method according to

[28] or

[29] , wherein the ROCK inhibitor is Y-27632, Rho Kinase Inhibitor IV or thiazolinone.

[0041]

[31] The method according to any one of

[28] to

[30] , wherein the TGFβ signaling pathway inhibitor is one or more inhibitors selected from the group consisting of A8301, SB431542, SB505124, LY364947, LY2157299 and RepSox.

[0042]

[32] The method according to any one of

[28] to

[30] , wherein the Wnt signal transduction pathway activator is one or more substances selected from the group consisting of WNT3A, CHIR99021, BIO, TWS119, SB216763, and Recombinant R-Spondin 1.

[0043]

[33] The method according to any one of

[28] to

[32] , wherein the culture medium for hepatoblast proliferation further comprises glucocorticoids or derivatives thereof.

[0044]

[34] A hepatoblast proliferation promoter comprising a ROCK inhibitor, a TGFβ signaling pathway inhibitor and / or a Wnt signaling pathway activator, and growth factors.

[0045]

[35] The hepatoblast proliferation promoter according to

[34] , wherein the growth factor comprises one or more selected from the group consisting of FGF, EGF, IGF, VEGF and PDGF.

[0046]

[36] The hepatoblast proliferation promoters described in

[34] or

[35] further comprise glucocorticoids or derivatives thereof.

[0047]

[37] A kit for promoting hepatoblast proliferation, comprising a ROCK inhibitor, a TGFβ signaling pathway inhibitor and / or a Wnt signaling pathway activator, and growth factors.

[0048]

[38] A culture medium for hepatoblast proliferation, comprising any one of

[34] to

[36] hepatoblast proliferation promoters.

[0049] This specification contains the disclosures of Japanese Patent Applications No. 2024-059423 and No. 2025-013968, which form the basis of the priority of this application.

[0050] Invention Effects The hepatoblast proliferation method according to the present invention enables the proliferation and culture of hepatoblasts while maintaining an undifferentiated state. Furthermore, the method for manufacturing a hepatoblast population according to the present invention enables the proliferation and culture of hepatoblasts while maintaining an undifferentiated state, thereby creating a hepatoblast cell population. Moreover, the hepatoblast proliferation method of the present invention provides cell preparations containing hepatoblasts for treating liver diseases and treatment methods for liver diseases utilizing hepatoblasts.

[0051] The present invention also provides a hepatocyte proliferation promoter, a hepatocyte proliferation promoter kit, and a hepatocyte proliferation culture medium for proliferating and culturing hepatocytes while maintaining their undifferentiated state. Attached Figure Description

[0052] Figure 1 This diagram schematically illustrates the process of inducing differentiation into hepatoblasts and the regeneration of liver tissue by hepatoblasts according to the present invention. Figure 1 A shows the differentiation of human iPS cells into hepatocytes and hepatocytes via the induction of type-specific endoderm cells. Figure 1 B illustrates liver tissue regeneration through transplantation of proliferated hepatoblasts.

[0053] Figure 2 This is a graph showing the results of the qPCR analysis in Example 1. In the graph, "hiPSC" represents the results from human iPS cells, "hiPSC-DE" represents the results from definitive endoderm cells induced by human iPS cells, and "hiPSC-HB" represents the results from hepatocytes induced by human iPS cells. In the graph, "fold change" represents the fold change calculated as a relative value to the expression level obtained from hiPSC, and the horizontal dashed line represents the fold change level of 1. In the graph, the circles on each bar represent the data points.

[0054] Figure 3This is a graph showing the results of the transcriptome analysis in Example 1. Each box uses color to indicate the expression level of each gene (row) in each cell (column); generally, darker colors indicate higher expression levels. Row labels indicate the names of characteristic genes from the hepatocytes used as the target cells, and column labels indicate the cell types used. In the figure, "hiPSC-HE" represents the results from hepatic endoderm induced from human iPS cells, "hiPSC-Hep" represents the results from hepatocytes induced from human iPS cells, and "PHH" represents the results from primary hepatocytes.

[0055] Figure 4 This is a graph showing the results of flow cytometry in Example 1. Figure 4 Figure A shows an exemplary plot illustrating the expression levels of CD13 and EpCAM in hepatoblasts induced with differentiation using WNT3A or CHIR99021. Figure 4 In diagram A, each point represents data from one cell. Data enclosed by solid-line boxes represents cells with high EpCAM expression (EpCAM-high), and data enclosed by dashed-line boxes represents cells with low EpCAM expression (EpCAM-low). Figure 4 In A, the data shown in the lower left corner of the solid and dashed boxes represents the data obtained when isotype control antibodies were used. Figure 4 B is a graph showing the proportion of EpCAM-low in CD13-positive cells. Figure 4 C is a graph showing the proportion of EpCAM-high in CD13-positive cells. Figure 4 In B and 4C, each circle represents the data from each experiment, and the error bars represent the standard deviation.

[0056] Figure 5 This is a graph showing the results of flow cytometry in Example 1. Figure 5 A shows the relationship with Figure 4 The same diagram as the one on the left of A. Figure 5 B shows the results of comparing the distribution of CD90 expression levels in EpCAM-low cells with the distribution when an isotype control antibody was used. Figure 5 C shows the results in EpCAM-high cells.

[0057] Figure 6-1This is a graph showing the results of the qPCR analysis at the end of the initial passage in Example 2. In the graph, "YAC" represents the candidate culture medium for proliferation containing Y-27632, A8301, and CHIR99021, and "YAW" represents the candidate culture medium for proliferation containing Y-27632, A8301, and WNT3A. "Fold change" represents the fold change calculated as a relative value to the expression level (hiPSC-HB) obtained in hiPSC-induced hepatocytes. In the graph, the names of each series indicate the maintenance candidate culture medium used. In the graph, the circles on each bar represent the data points, and the error bars represent the standard deviation.

[0058] Figure 6-2 This is a graph showing the cell proliferation rate results and qPCR analysis results at the end of the initial passage in Example 2. In the graph, "fold change" represents the change in expression relative to the expression levels in hepatocytes cultured using proliferation medium containing Y27632, A8301, WNT3A, EGF, and DEX. The horizontal dashed line represents the fold change level of 1. In the graph, the names of each series indicate the maintenance candidate medium used. In the graph, the error bars represent the standard deviation.

[0059] Figure 6-3 This is a graph showing the results of the qPCR analysis at the end of the initial passage in Example 2. In the graph, "Related Expression" represents the fold change calculated as a relative value of the expression levels in hepatocytes cultured using proliferation medium containing Y27632, A8301, WNT3A, EGF, and DEX. The series names in the graph indicate the maintenance candidate medium used. In the graph, "WNT3A-50" represents the result when WNT3A was used at a concentration of 50 ng / mL, and "CHIR-0.5", "CHIR-1", "CHIR-2", and "CHIR-3" represent the results when CHIR99021 was used at concentrations of 0.5 μM, 1 μM, 2 μM, or 3 μM, respectively. Error bars represent standard deviations.

[0060] Figure 6-4 This graph shows the results of investigating the proliferation capacity of hiPSC-HB in the sixth generation using various ROCK inhibitors. In the graph, "Proliferation Rate" represents the proliferation rate when the cell number on Day 1 is set to 1, and the horizontal axis represents the name and concentration of the added substance. In the graph, "Rho" indicates the results when Rho Kinase Inhibitor IV was used. The error bars represent the standard deviation.

[0061] Figure 6-5This is a graph showing the results of investigating the proliferation capacity of hiPSC-HB in the sixth generation using various TGFβ / Smad inhibitors. In the graph, "Proliferation Rate" represents the proliferation rate when the cell number on Day 1 is set to 1, and the horizontal axis represents the name and concentration of the added substance. Error bars represent the standard deviation.

[0062] Figure 6-6 This graph shows the results of investigating the proliferation capacity of hiPSC-HB in the sixth generation using various WNT3 signaling activators. In the graph, "Proliferation Rate" represents the proliferation rate when the cell number on Day 1 is set to 1, and the horizontal axis represents the name and concentration of the added substance. In the graph, "R-Spondin" represents the results when Recombinant R-Spondin1 was used. The error bars represent the standard deviation.

[0063] Figure 7 This is a graph showing the relationship between passage number and cumulative cell proliferation rate in Example 2. The labels for each data series indicate the name of the iPS cell line used in the differentiation induction of the hepatoblasts used. In the figure, the numbers shown with "#" indicate different batches, and "cryopreserved" indicates that cells thawed from cryopreservation were used.

[0064] Figure 8 This is a graph showing the relationship between the number of passages and the expression level of EpCAM in Example 3. The results are shown after 1 passage (A), 5 passages (B), 10 passages (C), and 15 passages (D). In the graph, the proportion shown below the solid box represents the proportion of EpCAM-high in CD13-positive cells.

[0065] Figure 9 This is a graph showing the results of transcriptome analysis in Example 3. Each box uses color to represent the expression level of each gene (row) in each cell (column); generally, darker colors indicate higher expression levels. Row labels indicate the names of characteristic genes in the hepatocytes used as the target cells, and column labels indicate the number of passages of the target cells. In the graph, "hiPSC" represents the results from human iPS cells, "DE" represents the results from induced endoderm cells from human iPS cells, "HE" represents the results from induced liver endoderm cells from human iPS cells, "HB P0" represents the results from hepatocytes before passage, "HB P1" represents the results after one passage, "HB P5" represents the results after five passages, "HB P10" represents the results after ten passages, "HB P15" represents the results after fifteen passages, and "MH" represents the results from mature hepatocytes.

[0066] Figure 10This is a graph showing the results of the main component analysis in Example 3. In the graph, the ratio of the vertical axis to the horizontal axis represents the contribution rate of each component.

[0067] Figure 11 This is a graph showing the results of the qPCR analysis in Example 4. In the graph, "fold change" represents the fold change calculated as a relative value to the expression level obtained from hiPSC-induced hepatoblasts, and the horizontal dashed line represents the fold change level of 1. In the graph, "HB" represents the results from hiPSC-induced hepatoblasts, "HB-Hep" represents the results from hepatocytes differentiated from HB, and "HB-cholangioblasts" represents the results from cholangioblasts differentiated from HB. In the graph, the circles on each bar represent the data points, and the error bars represent the standard deviation.

[0068] Figure 12-1 The figure shows the results of serum ALB levels in humans after transplantation of hepatoblasts (hiPSC-HB) into TK-NOG mice in Example 5. In the figure, the circles on the curves represent data points, and the error bars represent standard deviations.

[0069] Figure 12-2 The graph shows the results of the qPCR analysis in Example 5. In the graph, "ploidy change" represents the ploidy change calculated as a relative value to the expression level obtained from hiPSC-induced hepatocytes. In the graph, "hiPSC-HB" represents the results from hiPSC-induced hepatocytes, "hiPSC-HB engraftment" represents the results from liver tissue reconstructed from human cells after hiPSC-HB transplantation, and "PHH" represents the results from primary human hepatocytes. In the graph, the circles on each bar represent the data points, and the error bars represent the standard deviation.

[0070] Figure 13 This diagram shows an overview (A) of the transplantation of hepatoblasts (hiPSC-HB) into mice in Example 6 and the results of serum ALB level measurements (B). Figure 13 In A, "TK-NOG" hOSM "GCV" indicates TK-NOG mice inoculated with AAV8-hOSM, and "GCV" indicates the timing of ganciclovir administration. Figure 13 In B, "hiPSC-HB-Hep" indicates the result of hepatocyte differentiation from hepatoblasts induced by hiPSC.

[0071] Figure 14The survival curves (A) and serum levels (B) of ALT, AST, total bilirubin (T-Bil), and direct bilirubin (D-Bil) in mice transplanted with hepatoblasts in Example 6 are shown. In the figure, "Sham" represents the results of mice with liver failure without hepatoblast transplantation, "hiPSC-HB" represents the results of mice with liver failure after hepatoblast transplantation, "PHH" represents the results of mice with liver failure after primary hepatocyte transplantation, and "Normal" represents the results of mice without induced liver failure. In the figure, the circles on each bar represent the data points, and the error bars represent the standard deviation.

[0072] Figure 15 The results of transmission electron microscopy observations in Example 6 are shown. In the figures, "BC" represents bile capillaries, "ER" represents the endoplasmic reticulum, and "M" represents mitochondria. The scale bars in each figure represent 10 μm (A), 2 μm (B), and 1 μm (C and D), respectively.

[0073] Figure 16-1 This diagram illustrates an outline of in vitro and / or in vivo human OSM (hOSM) stimulation treatments during hepatoblast transplantation in Example 6. The figure shows “TK-NOG”. hOSM "AAV8 hOSM" indicates TK-NOG mice that have been introduced with AAV8-hOSM. "hOSM" indicates the administration of OSM in vitro, and "AAV8 hOSM" indicates the in vivo production of AAV8 using hOSM.

[0074] Figure 16-2 The results of serum ALB level determination in humans in Example 6 are shown. Series are represented as follows. Figure 16-1 The results for each hOSM stimulation treatment group are shown in the figure. The error bars in the figure represent the standard deviation. Detailed Implementation

[0075] <Definition> The following defines the terms used in this specification.

[0076] "Pluripotent stem cells" refer to cells possessing the ability to differentiate into all types of cells that make up an organism (pluripotency). Typically, pluripotent stem cells, when cultured in vitro under appropriate conditions, can proliferate indefinitely while maintaining pluripotency. Here, "pluripotency" refers to the ability to differentiate into cells from all types of germ layers (ectoderm, mesoderm, and endoderm in vertebrates) that make up an individual. In this specification, both Naive and Primed types of pluripotent stem cells are included.

[0077] "iPS cells" are cells created from human somatic cells such as skin and blood through culture methods including the introduction of pluripotency-inducing factors and treatment with low-molecular-weight compounds. They are "artificial pluripotent stem cells" that have the ability to proliferate almost indefinitely and differentiate into cells of various tissues and organs.

[0078] Hepatic endoderm cells (hereafter sometimes referred to as "HE") are precursor cells that form early in liver development. They are cell types induced from the endoderm during embryonic development. Hepatic endoderm cells receive specific signals (such as FGF and BMP) and differentiate into hepatoblasts, which then develop into mature hepatocytes and bile duct cells.

[0079] Hepatoblasts (hereinafter sometimes referred to as "HBs") are tissue stem cells derived from the foregut endoderm and present in the fetus. They are cells capable of differentiating into both bile duct epithelial cells and mature hepatocytes. Typically, hepatoblasts are not observed in adults; they are a cell type observed only during fetal liver development. Typically, in addition to expressing alpha-fetoprotein (AFP) and delta-like Notch ligand (DLK), they are known to express, depending on the situation, markers such as albumin (ALB), T-box transcription factor 3 (TBX3), hepatocyte growth factor receptor (C-MET), and epithelial cell adhesion molecule (EpCAM). They do not express markers such as OCT4 and CD90, except for keratin 19 (KRT19).

[0080] "Hepatocyte" refers to the 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 hepatic progenitor cells and other cells that define all stages of differentiation towards hepatocytes. Mature hepatocytes are terminally differentiated cells that perform various functions specific to the liver, such as protein and cholesterol synthesis, metabolism of biological molecules like proteins, carbohydrates, and lipids, and drug detoxification. Typically, in addition to expressing ALB, they are known to express, depending on the situation, markers such as α-1 antitrypsin (A1AT), carbamoyl phosphate synthase 1 (CPS1), cytochrome P450 3A4 (CYP3A4), and glucose-6-phosphatase catalytic subunit (G6PC). They do not express AFP or DLK, and depending on the situation, they may not express markers such as KRT19, EpCAM, or CD90.

[0081] "Cholangiocytes" are used synonymously with bile duct epithelial cells and are the epithelial cells that make up the bile ducts. Cholangiocytes play a role in regulating the flow and properties (including ion concentration and pH) of bile secreted by hepatocytes. Typically, in addition to expressing KRT19, they are known to express, depending on the situation, markers such as gamma-glutamyl transferase 1 (GGT1), aquaporin 1 (AQP1), somatostatin receptor 2 (SSTR2), and EpCAM. They do not express AFP or DLK, and depending on the situation, they also do not express markers such as A1AT.

[0082] "Proliferation" refers to increasing the number of cells, and "proliferation culture" refers to increasing the number of cells while maintaining their original properties without differentiation. "Proliferation culture process" refers to the process of proliferating and culturing hepatocytes in a culture medium for hepatocyte proliferation, and may also include a passage step.

[0083] "Cell population" refers to a homogeneous cell population obtained through proliferation culture.

[0084] "Differentiation" refers to the specialization and increased specificity of a cell's morphology and / or function. In this specification, differentiation specifically refers to a restricted differentiation fate of the cell, resulting in a reduction in the types of cells that may arise from it.

[0085] "ROCK inhibitor" refers to an inhibitor of the ROCK (Rho-associated coiled-coil forming kinase)-mediated signaling pathway. The ROCK inhibitors described in this specification include either drugs that directly inhibit ROCK function or drugs that inhibit its signaling pathway without directly inhibiting ROCK function. Any ROCK inhibitor known in the art can be used.

[0086] "TGFβ signaling pathway inhibitor" refers to an inhibitor of a signaling pathway based on the binding of TGFβ (transforming growth factor β) to its receptor. In addition to inhibitors of the Smad-mediated classical TGFβ signaling pathway, the TGFβ signaling pathway inhibitors in this specification also include any inhibitor of non-classical TGFβ signaling pathways, and any TGFβ signaling pathway inhibitor known in the art can be used.

[0087] "Wnt signaling pathway" refers to the classical Wnt signaling pathway, as well as other non-classical Wnt signaling pathways, which mediate the inhibition and stabilization of β-linkin degradation through the binding of Wnt to Wnt receptors such as Frizzled (e.g., Fizzled, Celsr, Vangl, etc.). In this specification, "Wnt signaling pathway activator" refers to a drug capable of activating any one or both of the classical and non-classical Wnt signaling pathways.

[0088] "Growth factor" refers to molecules that promote cell proliferation and differentiation in living organisms. It encompasses any of the following: 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 in living organisms.

[0089] "Steroids" are drugs that have a steroid skeleton in their chemical structure.

[0090] The “hepatoblast induction process” refers to the process of inducing differentiation from cells that are the source of hepatoblasts (such as pluripotent stem cells) into hepatoblasts.

[0091] 1. Proliferation culture of hepatocytes and creation of hepatocyte populations This invention relates to a method for proliferating hepatocytes or a method for manufacturing a hepatocyte population. The culture medium used in the method for proliferating hepatocytes or a method for manufacturing a hepatocyte population of this invention is a culture medium to which a ROCK inhibitor, a TGFβ signaling pathway inhibitor, and / or a Wnt signaling pathway activator, and growth factors are further added. By culturing with this culture medium, hepatocyte proliferation can be promoted while maintaining the properties of hepatocytes. During proliferation, when the cell confluence reaches a certain level, appropriate passage is performed to further increase the cell number. Therefore, the proliferation culture in this invention also includes the concept of a passage step. In one embodiment, the method for proliferating hepatocytes of this invention includes a proliferation culture step of proliferating and culturing hepatocytes in a hepatocyte proliferation culture medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and growth factors. In another embodiment, the method for proliferating hepatocytes of this invention includes a proliferation culture step of proliferating and culturing hepatocytes in a hepatocyte proliferation culture medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, and growth factors. In one embodiment, the method for proliferating hepatoblasts according to the present invention includes a proliferation culture step of proliferating and culturing hepatoblasts in a hepatoblast proliferation culture medium containing a ROCK inhibitor, a Wnt signaling pathway activator, and growth factors. In one embodiment, the method for manufacturing a hepatoblast population according to the present invention includes a proliferation culture step of proliferating and culturing hepatoblasts in a hepatoblast proliferation culture medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and growth factors. In one embodiment, the method for manufacturing a hepatoblast population according to the present invention includes a proliferation culture step of proliferating and culturing hepatoblasts in a hepatoblast proliferation culture medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, and growth factors. In one embodiment, the method for manufacturing a hepatoblast population according to the present invention includes a proliferation culture step of proliferating and culturing hepatoblasts in a hepatoblast proliferation culture medium containing a ROCK inhibitor, a Wnt signaling pathway activator, and growth factors.

[0092] Hepatoblasts Hepatoblasts are foregut endoderm-derived stem cells present in the fetal stage. They are capable of differentiating into both bile duct epithelial cells and mature hepatocytes. Typically, in addition to expressing alpha-fetoprotein (AFP) and delta-like Notch ligand (DLK), they are known to express, depending on the situation, albumin (ALB), T-box transcription factor 3 (TBX3), hepatocyte growth factor receptor (C-MET), and epithelial cell adhesion molecule (EpCAM). Furthermore, besides not expressing keratin 19 (KRT19), they may not express, depending on the situation, markers such as OCT4 and CD90.

[0093] The source of hepatoblasts used in this invention is not particularly limited. For example, they can be hepatoblasts isolated from animal tissues, organs or individuals, hepatoblasts induced to differentiate from pluripotent stem cells, or mixtures thereof.

[0094] When hepatoblasts are derived from pluripotent stem cells, the source of the pluripotent stem cells is not particularly limited. For example, they can be derived from pluripotent stem cells from all vertebrates (e.g., mammals, primates, etc.), including humans. The source animal can be healthy or diseased. For example, cells derived from mammals are preferred, particularly cells derived from primates such as humans. In one embodiment, the hepatoblasts used in this invention are derived from pluripotent stem cells from humans or other vertebrates. In another embodiment, the hepatoblasts used in this invention are derived from human-derived pluripotent stem cells.

[0095] In the case of inducing differentiation of hepatocytes from pluripotent stem cells, any pluripotent stem cells known in the art can be appropriately selected, obtained, or prepared and used according to the purpose, and the type is not particularly limited. Examples include induced pluripotent stem cells (iPS cells) of somatic cell origin, embryonic stem cells (ES cells) of early embryonic origin, embryonic germ cells (EG cells) of primordial germ cells, germ cell lineage stem cells (GS cells) of testis, Muse cells of mesenchymal tissue origin, adult stem cells (mesenchymal stem cells derived from bone marrow, adipose tissue, dental pulp, placenta, oocyte membrane, umbilical cord blood, amnion, chorion, etc., neural stem cells, 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) are preferred. The method for preparing the pluripotent stem cells used can be appropriately selected according to the type of stem cells used, and is not particularly limited. In one embodiment, the hepatocytes used in the hepatocyte proliferation method and the hepatocyte population manufacturing method of the present invention are derived from iPS cells. In one embodiment, the hepatoblasts used in the hepatoblast proliferation method and the hepatoblast population manufacturing method of the present invention are derived from human iPS cells or vertebrate iPS cells. In another embodiment, the hepatoblasts used in the hepatoblast proliferation method and the hepatoblast population manufacturing method of the present invention are derived from human iPS cells.

[0096] For example, when using hepatocytes proliferated using the hepatocyte proliferation method and hepatocyte population manufacturing method of the present invention for the treatment of a specific individual, hepatocytes with a reduced risk of rejection due to transplantation can be used. In this case, for example, hepatocytes induced to differentiate from iPS cells derived from the somatic cells of the applying individual, hepatocytes derived from individuals whose major histocompatibility complex (MHC) genotype (human leukocyte antigen (HLA) genotype in the case of a human) is the same as or substantially the same as the applying individual, or hepatocytes induced to differentiate from pluripotent stem cells derived from such individuals can be used.

[0097] In addition to expressing the typical markers mentioned above (AFP, DLK, ALB, TBX3, C-MET, CER1, EpCAM, etc.), the hepatocytes used in the method for manufacturing the hepatocyte population of the present invention may also express one or more genes or proteins selected from the group consisting of SLC4A4 (Solute Carrier Family 4 Member 4), TNFSF4 (TNF Superfamily Member 4), IL1RAPL1 (Interleukin 1 Receptor Accessory Protein Like 1), NRP1 (Neuropilin 1), SOX9 (SRY-Box Transcription Factor 9), ONECUT1 (One Cut Homeobox 1), PLEKHA2 (Pleckstrin Homology Domain Containing A2), RNF207 (Ring Finger Protein 207), NID1 (Nidogen 1), and LYST (Lysosomal Trafficking Regulator). In one embodiment, the hepatocytes used in the method for manufacturing a hepatocyte population of the present invention are characterized by expressing SLC4A4, TNFSF4, IL1RAPL1, NRP1, SOX9, ONECUT1, PLEKHA2, RNF207, NID1, and LYST.

[0098] <Culture medium> The culture medium used in the hepatoblast proliferation method of the present invention (also referred to herein as "hepatoblast proliferation culture medium") further comprises, as a basic culture medium (hereinafter referred to as "basal culture medium"), a ROCK inhibitor, a TGFβ signaling pathway inhibitor and / or a Wnt signaling pathway activator, and growth factors as essential components, and contains steroids as optional components. Preferably, the culture medium used in the hepatoblast proliferation method of the present invention contains both a TGFβ signaling pathway inhibitor and a Wnt signaling pathway activator; however, the hepatoblast proliferation method of the present invention can also be used even if the culture medium contains only either a TGFβ signaling pathway inhibitor or a Wnt signaling pathway activator. The components of each culture medium will be specifically described below.

[0099] As a basal culture medium, any vertebrate cell culture medium known in the art, such as a mammalian cell culture medium, can be used alone or in combination, with the addition of culture medium additives as needed. For example, Eagle Minimum Essential Medium (MEM) and any modified Eagle medium can be used as a basal culture medium. For example, specific culture media for mammalian cell culture include Dulbecco's Modified Eagle's Medium (DMEM), Advanced DMEM, Iscove's Modified Dulbecco's Medium (IMDM), Ham's F-12 medium or its modified media (e.g., Ham's F-12 K medium), Eagle's Minimum Essential Medium α-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., DMEM / F-12 medium), etc. The basal medium can also be a combination of multiple culture media. There are no particular limitations on the volume ratio when combining multiple culture media. For example, two or more culture media can be mixed in equal volumes, or a certain culture medium can be mixed in larger quantities than others. When the volumes of the mixed culture media differ, there are no particular limitations on the content and mixing ratio of the master medium (the medium with the largest volume) and other culture media. For example, the master medium can be more than 1 / 4, 1 / 3, 1 / 2, 2 / 3, or 3 / 4 of the total volume. Furthermore, for example, the master medium can be mixed at a ratio of more than 1, 1.5, 2, 2.5, or 3 times the volume of other culture media. There are no particular limitations on the type of master medium. For example, Iscove modified Duroc medium is preferably used as the master medium.

[0100] Additives such as proteins and compounds may be further added to the basal medium. When the additive is a protein, it may be a protein derived from a specific organism, a recombinant, or an artificially synthesized protein. When the protein is derived from a specific organism, the biological species is not particularly limited, and for example, a protein derived from a biological species that is the same as or closely related to the biological species from which the hepatoblasts are derived can be used. For example, proteins derived from mammals are preferably used, and in particular, proteins derived from primates such as humans can be used. The medium used preferably contains an organic buffer, L-glutamine or a derivative thereof, and sodium pyruvate. When a plurality of media are used in combination, it is sufficient that these components are contained after the combination. For example, a medium obtained by combining IMDM, which contains HEPES as an organic buffer and does not contain L-glutamine, and Ham's F-12 or a modified medium thereof (such as Ham's F-12 K medium) which contains L-glutamine and does not contain HEPES can be preferably used. Further, when the above components are not contained in the mixed medium or the above components are insufficient, they may be added from the outside. As the organic buffer, any organic compound having a pH buffering effect can be used. Preferred organic buffers include, for example, Good's buffers, and specific examples thereof include HEPES, MOPS, TES, Bis-Tris, PIPES, MES and the like, without particular limitation. Any inorganic buffer such as sodium bicarbonate may be contained as needed.

[0101] The basal medium used in the present invention may be a serum-containing medium or a serum-free medium. When a serum-free medium is used, the entire medium of the present embodiment may be a serum-free medium, or serum may be added to make it a serum-containing medium. As one embodiment, the basal medium used in the present invention is a serum-free medium.

[0102] The types of the ROCK inhibitor, the TGFβ signaling pathway inhibitor, the Wnt signaling pathway activator, the growth factor, the steroid agent, and the basal medium used in the medium for proliferating hepatoblasts of the present specification are not particularly limited as long as they are types commonly used by those skilled in the art.

[0103] <ROCK Inhibitor> The specific type of ROCK inhibitor used in this invention is not particularly limited; for example, any type of ROCK1 inhibitor or ROCK2 inhibitor can be used. Furthermore, the ROCK1 inhibitor here does not need to selectively inhibit ROCK1; it broadly includes drugs that exhibit inhibitory effects on ROCK1. Specifically, in addition to examples such as Thiazovivin, Ripasudil, Y-39983 (4-[(1R)-1-aminoethyl]-N-1H-pyrrolo[2,3-b]pyridin-4-ylbenzamide dihydrochloride), AR-13324, Wf-536 ((+)-(R)-4-(1-aminoethyl)-N-(4-pyridyl)benzamide monohydrochloride), AZD-5363, K-155, AR-13503, compounds such as Y-27632, which can inhibit ROCK1, are also included. ]-N-pyridin-4-ylcyclohexane-1-carboxamide or its salts (e.g., dihydrochloride), hydroxyfasudil hydrochloride, RKI-1447, GSK-429286A, AT13148, GSK-269962A hydrochloride, BAY-549, Seman 1, etc.; and compounds that specifically inhibit ROCK2, such as Fasudil (1-(5-isoquinolinesulfonyl)piperazine) or its salts (e.g., dihydrochloride), H-1152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-hexahydro-1H-1,4-diazazepine) or its salts (e.g., dihydrochloride), etc. Expression inhibitors that inhibit the expression of ROCK or its downstream factors may also be used as ROCK inhibitors in this specification. Examples of expression inhibitors used in this specification include, for example, antisense nucleic acids, RNA interference-inducible nucleic acids (e.g., siRNA), dominant-negative mutants, and their expression vectors. For example, ROCK1 inhibitors, and more specifically Y-27632 or its salts, may be preferred as ROCK inhibitors.

[0104] Multiple ROCK inhibitors can be used in combination. There are no particular limitations on the types of combinations; for example, multiple ROCK function inhibitors can be used in combination, or ROCK function inhibitors and ROCK expression inhibitors can be used in combination.

[0105] The content ratio of the ROCK inhibitor is not particularly limited as long as it is a concentration that allows the drug used to exhibit activity. Examples of the lower limit of the final concentration of the ROCK inhibitor (for example, a ROCK1 inhibitor such as Y-27632) include 0.001 μM, 0.01 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM and the like, and examples of the upper limit thereof include 200 μM, 100 μM, 50 μM, 20 μM and the like. In one embodiment, the ROCK inhibitor used in the present invention is Y-27632 or thiazovivin. In one embodiment, the ROCK inhibitor used in the present invention is Y-27632. In one embodiment, the concentration of Y-27632 used in the present invention is 0.01 μM to 500 μM. In one embodiment, the concentration of Y-27632 used in the present invention is 0.1 μM to 200 μM. In one embodiment, the concentration of Y-27632 used in the present invention is 1 μM to 100 μM.

[0106] <TGFβ signaling pathway inhibitor> The type of TGFβ signaling pathway inhibitor used in this invention is not particularly limited; any type of classical or non-classical TGFβ signaling pathway inhibitor can be used. Examples of classical TGFβ signaling pathway inhibitors include TGFβ receptor inhibitors, Smad phosphorylation inhibitors, Smad nuclear translocation inhibitors, and inhibitors of the activity or expression of one or more factors in the TGFβ signaling pathway. Examples of non-classical TGFβ signaling pathway inhibitors include 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.), and Rho GTPase pathway inhibitors (TRIPa, etc.). In the case of TGFβ receptor inhibitors, the type of receptor inhibited is not particularly limited. For example, substances that exhibit inhibitory effects on 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. In one embodiment, the TGFβ signaling pathway inhibitor used in this invention is selected from one or more inhibitors selected from the group consisting of A8301, SB431542, SB505124, LY364947, and RepSox. In one embodiment, the TGFβ signaling pathway inhibitor used in this invention is an inhibitor selected from the group consisting of A8301, SB431542, SB505124, LY364947, and RepSox.

[0107] The content ratio of the TGFβ signaling pathway inhibitor is not particularly limited as long as it is at a concentration that allows the drug used to exhibit activity. Examples of the lower limit of the final concentration of the TGFβ signaling pathway inhibitor (such as TGFβ receptor inhibitors including A8301) include 0.001 μM, 0.005 μM, 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, etc., and examples of the upper limit thereof include 100 μM, 50 μM, 10 μM, 5 μM, 1 μM, etc. In one embodiment, the TGFβ signaling pathway inhibitor used in the present invention is A8301. In one embodiment, the concentration of A8301 used in the present invention is 0.005 μM to 50 μM. In one embodiment, the concentration of A8301 used in the present invention is 0.01 μM to 10 μM. In one embodiment, the concentration of A8301 used in the present invention is 0.05 μM to 5 μM.

[0108] <Wnt signaling pathway activator> The Wnt signaling pathway activator used in the present invention is not particularly limited, and for example, Planar Cell Polarity (PCP) pathway activators, Wnt / Ca 2+ pathway activators, activators that promote the binding of Wnt to receptors, Wnt (such as WNT3A) and substitutes thereof, Notum pectin acetylesterase inhibitors (LP-922056, Notum pectin acetylesterase-1), Notum inhibitors (ARUK3001185, 8BTC, etc.), JNK activators (R-spondin1), Wnt expression promoters (Wnt expression vectors, γ-Glutamylvaline, etc.), GSK3β inhibitors, β-catenin stabilizers (Axin-LRP6 complex formation promoters (Lycorine and derivatives thereof, etc.), Axin-β-catenin complex formation promoters (SKL2001, etc.), etc.), phosphodiesterase 5 inhibitors (tadalafil, vardenafil, mirodenafil and derivatives thereof, etc.), other drugs (BML-284, SM-04554, Dalosirvat, methyl vanillate, etc.), expression promoters of molecules in the Wnt signaling pathway, and the like can be used.

[0109] Examples of GSK3β inhibitors include CHIR99021 (Laduviglusib) and its hydrochloride, CHIR98014, Tideglusib, 6-bromoindorubin-3-oxime (BIO), 6-bromoindorubin-3-acetyloxime (BIO acetyloxime), SB415286, SB216763, TWS119, Tideglusib, A1070722, LY2090314, AZD1080, 1-Azakenpaullone, AR-A014418, IM-12, Indirubin, TDZD-8, and other low-molecular-weight inhibitors, as well as GSK3β expression inhibitors and combinations thereof, without particular limitation.

[0110] Wnt or its analogues may preferably be used as activators of the Wnt signaling pathway. In this case, the substance may be derived from a specific organism (e.g., human) or a recombinant. When using Wnt receptor agonists such as WNT3A as activators of the Wnt signaling pathway, the agonist is included in concentrations of 0.001 nM or more, 0.01 nM or more, 0.05 nM or more, 0.1 nM or more, 0.133 nM or more, 0.5 nM or more, 1 nM or more, 1.33 nM or more, 2 nM or more, and 5 nM or more, and in concentrations of 10 μM or less, 1 μM or less, 500 nM or less, 100 nM or less, 50 nM or less, and 10 nM or less. When using, for example, a GSK3β inhibitor as an activator of the Wnt signaling pathway, the inhibitor is included in concentrations of 0.001 nM or higher, 0.01 nM or higher, 0.05 nM or higher, 0.1 nM or higher, 0.133 nM or higher, 0.5 nM or higher, 1 nM or higher, 1.33 nM or higher, 2 nM or higher, and 5 nM or higher, and in concentrations of 10 μM or lower, 1 μM or lower, 500 nM or lower, 100 nM or lower, 50 nM or lower, and 10 nM or lower. In one embodiment, the Wnt signaling pathway activator used in this invention is one or more activators selected from the group consisting of Wnt3A, CHIR99021, and SB216763, or a combination thereof. In one embodiment, the Wnt signaling pathway activator used in this invention is Wnt3A. In one embodiment, the concentration of Wnt3A used in this invention is 0.5 ng / mL to 5 μg / mL. In one embodiment, the concentration of Wnt3A used in this invention is 5 ng / mL to 500 ng / mL.

[0111] <Growth Factors> As the growth factor used in this invention, any growth factor known in the art can be used. Specifically, examples include fibroblast growth factor (FGF: FGF1~FGF23, etc., containing basic FGF (bFGF, FGF2),) epidermal growth factor (EGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), its substitutes, or combinations thereof, without particular limitation. The growth factor can be derived from a specific organism (e.g., human) or a recombinant. For example, the growth factor in the hepatoblast proliferation promoter of this method may preferably contain EGF or its substitutes.

[0112] The content ratio of growth factors is not particularly limited, as long as it is at a concentration that makes the drug used exhibit activity. For example, growth factors (e.g., EGF) can be included at molar concentrations of 0.1 times, 0.2 times, 0.4 times, 0.5 times, 1 times, 1.1 times, 1.2 times, 1.3 times, or 1.35 times that of the Wnt signaling pathway activator. Alternatively, growth factors (e.g., EGF) can be included at molar concentrations of less than 10 times, 5 times, 3 times, 2 times, 1.5 times, or 1.4 times that of the Wnt signaling pathway activator. Examples of lower limits for the final concentration of growth factors (e.g., EGF) include 0.01 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.5 nM, 0.6 nM, 1 nM, 1.2 nM, 1.4 nM, 1.5 nM, and 1.6 nM, while examples of upper limits include 10 nM, 5 nM, 2.5 nM, 2.2 nM, 2.1 nM, 2 nM, 1.9 nM, 1.8 nM, 1.7 nM, 1.65 nM, and 1.6 nM. In one embodiment, the growth factor used in this invention is one or more proteins selected from the group consisting of FGF2, EGF, IGF, VEGF, and PDGF. In one embodiment, the growth factor used in this invention is FGF2. In one embodiment, the concentration of FGF2 used in this invention is 0.01 μM to 10 μM. In one embodiment, the concentration of FGF2 used in this invention is 10 ng / mL.

[0113] <steroids> In the method for proliferating hepatoblasts or the method for manufacturing a population of hepatoblasts according to the present invention, the culture medium for hepatoblast proliferation may further contain a steroid agent. Examples of steroid agents that can be used in the present invention include, for example, glucocorticoids and their analogues, derivatives, etc. For example, glucocorticoids or their analogues, derivatives, or substitutes are preferably used as steroid agents. The specific types of glucocorticoids or their analogues, derivatives, etc. used are not particularly limited; examples include long-acting steroids with a biological half-life of 36 hours or more (dexamethasone (DEX), betamethasone, peramisone, 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, etc. For example, the steroid in the hepatoblast proliferation promoter of this method may preferably include a long-acting steroid such as dexamethasone (DEX). Furthermore, multiple steroids can be used in combination as the steroid in this invention. In this case, multiple glucocorticoids can be used in combination, or glucocorticoids can be used in combination with other steroids.

[0114] The concentration of the steroid agent is not particularly limited as long as it is the concentration at which the drug exhibits activity. Examples of lower limits for the final concentration of the steroid agent (e.g., glucocorticoids such as DEX) include 0.001 μM, 0.005 μM, 0.01 μM, 0.05 μM, and 0.1 μM, while examples of upper limits include 10 μM, 5 μM, 1 μM, 0.5 μM, 0.3 μM, 0.2 μM, and 0.1 μM. In one embodiment, the steroid agent used in this invention is a glucocorticoid or a derivative thereof. In one embodiment, the glucocorticoid or a derivative thereof used in this invention is dexamethasone. In one embodiment, the concentration of dexamethasone used in this invention is 0.1 μM.

[0115] <Other Ingredients> The hepatocyte proliferation culture medium used in the hepatocyte proliferation method or the hepatocyte population manufacturing method of the present invention may also contain any other components. Other components may include, for example, antioxidants, serum or its substitutes, vitamins, albumin, amino acids, antibiotics, supplements, sugars, and other substances (additives) commonly used by those skilled in the art.

[0116] When the culture medium for hepatocyte proliferation contains antioxidants, it may contain any molecule with antioxidant activity known in the art. There are no particular limitations on the specific types; examples include glutathione and its precursors, L-ascorbic acid and its derivatives, 2-mercaptoethanol or its substitutes (monothioglycerol), etc. Specific precursors to glutathione include, for example, L-cysteine ​​or its derivatives (N-acetyl-L-cysteine, etc.), γ-glutamylcysteine ​​or its derivatives, etc., without particular limitation. For example, other components of the drug of this manner may preferably include L-cysteine ​​or its derivatives (N-acetyl-L-cysteine, etc.).

[0117] When the culture medium for hepatocyte proliferation contains serum or serum substitutes, it may contain any serum or serum substitute known in the art. Examples of serum include, for example, fetal bovine serum (FBS), human serum, sheep serum, or mixtures thereof; examples of serum substitutes include, for example, Knockout serum. TM Serum replacement (KSR), XF212 XerumFree, CDM-HD serum replacement, StemSure serum replacement, Nu-Serum TM etc., without any specific restrictions.

[0118] When the culture medium for hepatoblast proliferation contains antibiotics, such as penicillin, streptomycin, penicillin-streptomycin, sulfonamides, phenoxyethyl penicillin, chlortetracycline, oxytetracycline, tetracycline, desmethylcycline, doxycycline, methacycline, and minocycline can be used, but are not limited to these. There are no particular limitations on the amount of antibiotic added; for example, it can be used at the concentration recommended by the manufacturer.

[0119] When the culture medium for hepatocyte proliferation contains supplements, such as B27, it can be used. TM supplement, N2 supplement, etc.

[0120] When the culture medium for hepatocyte proliferation contains sugars, the following sugars can be used: monosaccharides such as glucose, galactose, mannose, and fructose; disaccharides such as sucrose, maltose, and lactose; and water-soluble or water-insoluble polysaccharides such as hyaluronic acid, gellan gum, chitin, and chitosan.

[0121] In addition, substances commonly used by those skilled in the art include, for example, vitamins, albumin, amino acids, inorganic salts, stabilizers, and buffers. These substances can be those commonly chosen by those skilled in the art and can be used according to methods and concentrations commonly used by those skilled in the art.

[0122] <Proliferation and Culture Process> The proliferation culture process is the process of proliferating and culturing hepatocytes in a culture medium for hepatocyte proliferation, and may also include a passage step.

[0123] Typically, the proliferation culture process refers to culturing hepatocytes seeded at any cell density on a culture plate in a hepatocyte proliferation medium at appropriate temperature, humidity and CO2 concentration.

[0124] As a cultivation method, any cultivation method known in this art can be used without particular limitation. For example, static cultivation using culture plates, static cultivation using Cell Factory or CellSTACK, static cultivation using culture bags, etc., and cultivation using bioreactors of any type (hollow fiber type, corrugated type, vertical waterwheel stirring type, reciprocating motion type, stirred layer type, etc.) can be used. In addition, surface coating can be performed during cultivation, and microcarriers can also be used.

[0125] There is no particular limit to the cell density in the proliferation culture process; for example, 10 can be listed. 2 cells / cm 2 ~10 6 cells / cm 2 10 3 cells / cm 2 ~10 5 cells / cm 2 Density, etc.

[0126] The culture plate can be coated as needed. There are no particular limitations on the extracellular matrix used for coating. Examples include laminin-1~12, collagen, fibronectin, hyalin, matrix gel, Geltrex, iMatrix-511, etc.

[0127] As for suitable temperature, cultivation can generally be carried out in the range of about 30°C to 40°C, for example, at 37°C. As for humidity, cultivation can generally be carried out in the range of about 70% to 100%, for example, in the range of about 95% to 100%. As for CO2 concentration, cultivation can generally be carried out in the range of about 1% to 10%, for example, in the range of about 5%. In addition, there is no particular limitation on O2 concentration, and cultivation can be carried out at normal oxygen concentration (18% to 22%) or low oxygen concentration (0% to 10%).

[0128] During the culture period, cells can be cultured until they proliferate to the desired number or degree of confluence, without any particular limitation. For example, they can be cultured for 2–15 days, 3–15 days, 7–15 days, 7–14 days, or 7–12 days.

[0129] The culture medium can be changed as needed. There is no particular limit to the frequency of medium replacement. For example, it can be done regularly or irregularly. If done regularly, it can be done every 1-5 days, every 1-4 days, every 1-3 days, every 1-2 days, or every 2 days. If done irregularly, the timing of medium replacement can be determined based on changes in pH, turbidity, etc. The composition of the culture medium can be the same as or different from the medium used before replacement; preferably, the composition of the medium should remain unchanged when replacing it with fresh medium.

[0130] The timing of subculturing can be determined appropriately based 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 specific limitation on the exact degree of confluence at the time of subculturing. 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, or 95% or higher.

[0131] There are no particular limitations on the specific methods of cell passage. Typically, cells are detached from the culture dish, dispersed in the culture medium, and re-seeded into new culture dishes. Cell detachment and dispersion can be achieved through mechanical stimulation such as pipetting, or through chemical stimulation such as the action of enzymes like 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, dispersase, trypsin, trypsin / EDTA, trypsin / collagenase, ReLeSR TM The cell density at inoculation and the coating of the culture dish are not particularly limited. The cell density at inoculation and the coating of the culture dish can be performed according to the descriptions related to the proliferation culture procedure. Furthermore, the composition of the culture medium can be the same as or different from the culture medium used before subculture; preferably, the composition of the culture medium before and after subculture is the same. The culture conditions after subculture can be performed according to the descriptions in the proliferation culture procedure. The conditions at inoculation and / or the culture conditions after subculture can be the same as or different from the conditions used in the proliferation culture procedure. The subculture step can be performed multiple times. The conditions used in each subculture step can be the same or different.

[0132] <Preparation of pluripotent stem cells> The pluripotent stem cells used in this invention can be prepared from cells isolated from individuals, tissues, etc., and the preparation method can be appropriately selected according to the type of stem cells used, without any particular limitation.

[0133] Pluripotent stem cells other than iPS cells are usually prepared by culturing cells isolated from individuals, tissues, etc., under culture conditions that can maintain their ability to differentiate into multiple types.

[0134] Methods for preparing iPS cells have been extensively studied by those skilled in the art, and any of these methods can be used. Typically, iPS cells are induced by introducing specific reprogramming factors (DNA or proteins) into somatic cells, including skin cells, bone marrow cells, digestive tract cells, hepatocytes, adult stem cells, etc. 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 mutant), N-Myc, L-Myc, etc.), Nanog family genes (Nanog, etc.), Lin family genes (Lin28, Lin28b, etc.), and other well-known genes (Fbx15, ERas, ECAT15-2, Tcl1, β-linkin, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, p53shRNA, Glis1, etc.). Combinations of specific reprogramming factors, for example, are known to include: 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, etc.

[0135] Pluripotent stem cells can be cultured using well-known methods without particular limitations. For example, they can be cultured together with feeder cells that produce the factor sets necessary for maintaining pluripotency and self-replication in a culture vessel coated with an extracellular matrix. Whether coating is necessary depends on the culture vessel and method used, and they can be cultured without coating. The extracellular matrix used for coating may include molecules described later related to the proliferation culture process of this method. As for the culture method, adhesion culture or suspension culture may be used, and the appropriate method can be selected depending on the purpose. For example, in cases where spheroids are formed, suspension culture can be used. Culture can be static or by any mechanical stimulation (rotation culture, spiral culture, etc.).

[0136] The culture medium used is not particularly limited, and any culture medium commonly used by those skilled in the art can be used. In this invention, a culture medium provided as particularly suitable for pluripotent stem cells can be used. For example, commercially available culture media for ES cells and iPS cells include 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 AK02N (Ajinomoto), etc.

[0137] Whether feeder cells are needed depends on the culture medium and additives used. For example, feeder cells are not needed when using a culture medium without a feeder layer. 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.

[0138] Other culture conditions are not particularly limited, and additional arbitrary treatments can be performed during culture. In particular, when using feeder cells, treatments to inhibit cell proliferation (mitomycin C treatment, radiation irradiation, etc.) can be applied.

[0139] <Hepatoblast Induction Process> The hepatoblast induction process involves inducing the culture of pluripotent stem cells and their differentiation into hepatoblasts. It includes: a first step, inducing the differentiation of pluripotent stem cells into defined endoderm cells; and a second step, inducing the differentiation of defined endoderm cells into hepatoblasts. A proliferation culture step can be performed after the second step of the hepatoblast induction process.

[0140] The first step may include the following steps: culturing in serum-free medium containing 10 μM Y-27632 during the early stage of culture; and culturing in serum-free medium containing B-27 supplement, activator A, and WNT signaling pathway activator after the middle stage of culture. The second step may include the following steps: culturing in serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide. In this specification, "early stage of culture" refers to days 0, 1, 2, 3, and 4 of the culture start date. In this specification, "middle stage of culture" refers to days 1, 2, 3, 4, and 5 of the culture start date. In one embodiment, the first step may include the following steps: culturing in serum-free medium containing 10 μM Y-27632 on day 0 of the culture; and culturing in serum-free medium containing B-27 supplement, activator A, and WNT signaling pathway activator after day 1 of the culture. The second step may include the following steps: culturing in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide. In one embodiment, the first step may include the following steps: culturing on day 0 in a serum-free medium containing 10 μM Y-27632; and culturing thereafter on day 2 in a serum-free medium containing B-27 supplement, activator A, and a WNT signaling pathway activator. The second step may include the following steps: culturing in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide. In one embodiment, the first step may include the following steps: culturing on day 0 in a serum-free medium containing 10 μM Y-27632; and culturing thereafter on day 3 in a serum-free medium containing B-27 supplement, activator A, and a WNT signaling pathway activator. The second step may include the following steps: culturing in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide. There are no particular limitations on the method for inducing differentiation of hepatocytes from pluripotent stem cells, and any method known in the art can be used. Specifically, for example, pluripotent stem cells can be first induced to differentiate into morphological endoderm cells, and the induced morphological endoderm cells can be further differentiated into hepatocytes, but there are no particular limitations. 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.

[0141] The pluripotent stem cells used in this process can be newly prepared pluripotent stem cells derived from healthy individuals or patients, or they can be obtained from biobanks, etc., and there are no particular limitations on their type. The method for newly preparing pluripotent stem cells can follow methods commonly used by those skilled in the art. In the case of obtaining them from biobanks, for example, if they are iPS cells or ES cells, they can be obtained from RIKEN Bioresource Center Cell Materials Development Laboratory, ATCC (American Type Culture Collection), iPS Academia Japan Co., Ltd., etc. Commonly used cell lines include, for example, human ES cells such as WA01(H1), WA09(H9), KhES-1, KhES-2, and KhES-3, and human iPS cells such as M48, QHJI, 201B7, 253G1, 409B2, 454E2, 585A1, 606A1, 610B1, 648A1, 1201C1, 1231A3, 1383D2, 1383D6, HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2, but are not limited to these. Pluripotent stem cells can be used in one type or in combination. Pluripotent stem cells can also be used after long-term preservation. For example, cryopreserved pluripotent stem cells can be thawed and used. In this case, the preservation temperature and duration are not particularly limited. For example, specific temperatures can be used for storage below 0℃, -20℃, -30℃, -40℃, -50℃, -79℃, -80℃, -100℃, -130℃, -150℃, -180℃, and -196℃. The temperature can remain constant during storage, vary naturally, or be manually changed. There are no specific limitations on the storage period; for example, it can be set to more than 1 day, more than 1 week, more than 2 weeks, more than 3 weeks, more than 4 weeks, more than 30 days, more than 1 month, more than 2 months, or more than 3 months.

[0142] The serum-free culture medium or other components, or culture conditions used, are not particularly limited and can be referenced from culture media and conditions commonly used by those skilled in the art, or the basal culture medium described in the section on "Proliferation Culture of Hepatoblasts and Production of Hepatoblast Populations". Furthermore, any additional arbitrary treatments may be performed during culture.

[0143] The steps for inducing pluripotent stem cells to differentiate into morphological endoderm cells (first step) and the steps for inducing morphological endoderm cells to differentiate into hepatocytes (second step) are described illustratively.

[0144] <Induction of Differentiation from Pluripotent Stem Cells to Defined Endoderm Cells> In this step, differentiation from pluripotent stem cells into defined endoderm cells is induced.

[0145] The specific method for differentiation induction in this step is not particularly limited. For example, differentiation from pluripotent stem cells to mature endoderm cells can be induced by culturing pluripotent stem cells in a medium containing Wnt signaling pathway activators and / or TGFβ family proteins.

[0146] Regarding the types and concentrations of Wnt signaling pathway activators and TGFβ family proteins used in this step, please refer to the foregoing description. For example, CHIR99021 or WNT3A can be used as Wnt signaling pathway activators, and activin and / or BMP4 can be used as TGFβ family proteins.

[0147] Growth factors can also be added to the culture medium used for differentiation induction. Regarding growth factors and their concentrations, refer to the description in the section on <Growth Factors>. For example, FGF2 and VEGF are preferred growth factors.

[0148] The basal culture medium and other components should be prepared according to the instructions in the proliferation culture procedure. For example, B-27 can be used. TM As a supplement, RPMI 1640 medium and / or serum-free medium can be preferably used as the basal medium. Additionally, additives such as sodium butyrate can be used. There are no specific limitations on the culture period; for example, it can be set for more than 1 day, 3 days, 4 days, 5 days, 6 days, or 7 days, or for example, 1 day to 150 days, 1 day to 90 days, 1 day to 50 days, 1 day to 30 days, 1 day to 20 days, 3 days to 10 days, etc. Furthermore, a ROCK inhibitor (e.g., Y-27632) can be added only on day 0 of the culture. During this step, the composition of the medium can be changed more than once. For example, as the number of culture days increases, a medium containing multiple TGFβ family proteins and multiple growth factors can be used. Furthermore, there are no specific limitations on the oxygen concentration in this step. For example, it can be a low oxygen concentration, a normal oxygen concentration, or a combination thereof.

[0149] Differentiation into morphological endoderm cells can be determined based on the presence or absence of any properties characteristic of morphological endoderm cells and / or the loss of properties characteristic of pluripotent stem cells. For example, this can be determined by the expression of morphological endoderm cell markers and / or pluripotent stem cell markers. Any marker known in the art can be used as a morphological endoderm cell marker or pluripotent stem cell marker, without particular limitation. Specific examples of morphological endoderm cell markers include, for example, FOXA2, SOX17, CXCR4 (CD184), GATA4, GATA6, CD117, and CER1. Specific examples of pluripotent stem cell markers include, for example, OCT4, NANOG, SOX2, CD9, DNMT3B, GABRB3, GAL, GDF3, IFITM1, PODXL, TDGF1, and ZFP42.

[0150] In one embodiment, the hepatoblast induction process includes a first step of inducing pluripotent stem cells to differentiate into defined endoderm cells, the first step comprising the following steps: culturing on day 0 in serum-free medium containing 10 μM Y-27632; and culturing after day 1 in serum-free medium containing B-27 supplement, activin A and WNT signaling pathway activator.

[0151] In one embodiment, the hepatoblast induction process includes a first step of inducing iPS cells to differentiate into defined endoderm cells, the first step comprising the following steps: culturing on day 0 in serum-free medium containing 10 μM Y-27632; and culturing after day 1 in serum-free medium containing B-27 supplement, activin A and WNT signaling pathway activator.

[0152] <Induction of Differentiation from Determined Endoderm Cells to Hepatocytes> In this step, differentiation from defined endoderm cells into hepatocytes is induced. This step is usually performed after the defined endoderm cell induction step, but if defined endoderm cells are obtained, this step can be performed without the above steps. Furthermore, in this step, for example, hepatocytes can also be induced to differentiate from defined endoderm cells into other cell types (e.g., hepatic endoderm cells, foregut progenitor cells).

[0153] The basal culture medium and culture conditions used are not particularly limited, and can be referred to the culture media and culture conditions commonly used by those skilled in the art or those described in the section on "Proliferation Culture of Hepatoblasts and Production of Hepatoblast Populations". In addition, any additional arbitrary treatments can be performed during culture.

[0154] The specific method for differentiation induction in this step is not particularly limited. For example, differentiation into hepatocytes can be induced by culturing well-formed endoderm cells in a serum-free medium containing one or more of the following groups: steroids, TGFβ family proteins, dimethyl sulfoxide, inflammatory cytokines, and growth factors. For example, the medium may include dimethyl sulfoxide, growth factors, TGFβ family proteins and growth factors, dimethyl sulfoxide and growth factors, or steroids, inflammatory cytokines, and growth factors.

[0155] Regarding steroids and growth factors, please refer to the description in the section on "Proliferation Culture of Hepatoblasts and Production of Hepatoblast Populations". Regarding serum-free culture medium and the concentrations of steroids and growth factors, please refer to the description in the section on "Proliferation Culture of Hepatoblasts and Production of Hepatoblast Populations". For example, as steroids, glucocorticoids such as DEX are preferred; as growth factors, FGF (FGF1, FGF2, FGF4, FGF10, etc.), VEGF, and / or HGF are preferred. As TGFβ family proteins, activin and / or BMP4 are preferred, for example.

[0156] There are no particular limitations on the inflammatory cytokines used. Specific examples of inflammatory cytokines include thrombopoietin (TPO), leukemia inhibitory factor (LIF), FLT-3 ligand, TNF-α, IFN-γ, IL-1, IL-6 family proteins, and IL-17a. Examples of IL-6 family proteins include oncogenes M (OSM), IL-6, IL-11, IL-27, IL-35, IL-39, LIF, CT-1, CNTF, CLCF1, and combinations thereof. For example, oncogenes M may be preferred.

[0157] The concentration of inflammatory cytokines (such as OSM and other IL-6 family proteins) is not particularly limited as long as it is an effective amount. Lower limits for their concentrations include, for example, 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 upper limits include, for example, 10 nM, 5 nM, 1 nM, 0.6 nM, 0.5 nM, 0.45 nM, 0.4 nM, and 0.35 nM.

[0158] The cultivation conditions shall be in accordance with the description of the proliferation and cultivation procedures in this method. For example, as a culture medium additive, vitamin B such as nicotinamide or its derivatives, and vitamin C such as L-ascorbic acid or its derivatives may be preferred. There are no particular limitations on the cultivation period; for example, it may be set to more than 1 day, more than 3 days, more than 5 days, more than 6 days, more than 7 days, or for example, it may be set to 1 day to 150 days, 1 day to 90 days, 1 day to 50 days, 1 day to 30 days, 1 day to 20 days, 3 days to 10 days, etc.

[0159] During this step, the composition of the culture medium can be changed more than once. Furthermore, the oxygen concentration in this step is not particularly limited. For example, it can be a low oxygen concentration, a normal oxygen concentration, or a combination thereof.

[0160] Induction of differentiation into hepatocytes can be determined based on the presence or absence of any properties characteristic of hepatocytes and / or the loss of properties characteristic of morphological endoderm cells. For example, this can be determined by the expression of hepatocyte markers and / or morphological endoderm cell markers. Any marker known in the art can be used as a hepatocyte marker without particular limitation, such as 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, and CD133. As a marker for defining endoderm cells, any of the aforementioned markers for defining endoderm cells in this method may be used, for example.

[0161] In one embodiment, the hepatoblast induction process includes a second step of inducing shaped endoderm cells to differentiate into hepatoblasts, the second step including culturing them in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide.

[0162] <Maturation Acceleration Process> This step is optional and involves treatment to promote the maturation of hepatocytes. This step can be performed simultaneously with or after the proliferation and culture step. Furthermore, this step can be performed as a pretreatment, particularly when transplanting hepatocytes proliferated and cultured using the method of this invention into a biological individual.

[0163] In this process, hepatoblasts are exposed to an expression promoter of genes expressed in the later stages of liver development from the biological species from which the hepatoblasts originate. The types of genes expressed in the later stages of liver development are not particularly limited, as long as they are genes related to liver maturation. For example, proteins from the IL-6 family, tumor necrosis factor (TNF) superfamily proteins such as TNF-α, and JAK-STAT3 pathway activators are preferred. Examples of JAK-STAT3 pathway activators include FGF19, the aforementioned inflammatory cytokines used in the hepatoblast induction process of this method, and overexpression vectors for JAK-STAT3 pathway-related genes. Furthermore, one or more of the following can be used: growth factors (FGF2, FGF10, etc., HGF, etc.), steroids (glucocorticoids such as dexamethasone, etc.), and Wnt signaling pathway activators (Wnt signaling pathway activators such as GSK3β inhibitors, etc.). Regarding the components and their concentrations, the above description applies. In one embodiment, the substance used in the hepatoblast maturation promotion process is oncogene M.

[0164] During this step, the composition of the culture medium can be changed more than once. Furthermore, the oxygen concentration in this step is not particularly limited. For example, it can be a low oxygen concentration, a normal oxygen concentration, or a combination thereof.

[0165] In the case of human hepatoblasts (including hepatoblasts differentiated from human pluripotent stem cells), the hepatoblasts are exposed to human-derived proteins. The human-derived protein can be a protein produced in other organisms or a chemically synthesized protein, provided its amino acid sequence is identical to that of human proteins. There are no particular limitations on the exposure method; examples include adding the target protein to the culture medium, co-culturing with cells expressing the target protein, or combinations thereof. There are also no particular limitations on the exposure period; for example, exposure can continue until STAT3 activity increases (e.g., until it increases significantly), or until the expression levels of mature hepatocyte markers such as ALB, CPS1, CYP3A4, and G6PC increase. Specifically, exposure can be for more than 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. For example, exposed cells can also express proteins selected from SLC4A4, TNFSF4, PLEKHA2, RNF207, REG3A (Regenerating Family Member 3 Alpha), SAA1 (Serum Amyloid A1), SAA2 (Serum Amyloid A2), HP (Haptoglobin), SPINK1 (Serine Peptidase Inhibitor Kazal Type 1), LBP (Lipopolysaccharide Binding Protein), KCNG2 (Potassium Voltage-Gated Channel Modifier Subfamily G Member 2), ANXA10 (Annexin A10), ITIH3 (Inter-Alpha-Trypsin Inhibitor Heavy Chain 3), ITIH4 (Inter-Alpha-Trypsin Inhibitor Heavy Chain 4), MTUS1 (Microtubule Associated Scaffold Protein 1), CRP (C-Reactive Protein), CEACAM5 (CEA Cell Adhesion Molecule 5), and C4B (Complement Protein). C4B), ATP10B (ATPase PhospholipidTransporting 10B), NNMT (Nicotinamide N-Methyltransferase), LCN2 (Lipocalin 2), LOC145837, ORM1 (Orosomucoid 1), ORM2 (Orosomucoid 2), PC (PyruvateOne or more genes from the group consisting of Carboxylase, SERPINA7 (Serpin Family A Member 7), SOCS3 (Suppressor Of Cytokine Signaling 3), C4A (Complement C4A), SERPINA3 (Serpin Family A Member 3), C8A (Complement C8 Alpha Chain), CD38, TMEM45B (Transmembrane Protein 45B), CDA (Cytidine Deaminase), and UGT2B4 (UDP Glucuronosyltransferase Family 2 Member B4).

[0166] <Effect> The hepatoblast cell proliferation method according to the present invention does not induce overexpression of oncogenes and achieves cell proliferation while maintaining the undifferentiated state of hepatoblast cells. Furthermore, the hepatoblast cell manufacturing method according to the present invention can produce a homogeneous cell population containing hepatoblast cells with high purity based on stable cell proliferation. Typically, more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and 99.5% of the cells in the manufactured cell population are hepatoblast cells. In one embodiment, the purity of the hepatoblast cell population manufactured by the hepatoblast cell manufacturing method of the present invention is more than 85%, 90%, 95%, 96%, 97%, 98%, 99%, and 99.5%. In one embodiment, the purity of the hepatoblast cell population manufactured by the hepatoblast cell manufacturing method of the present invention is more than 85%. In one embodiment, the purity of the hepatoblast cell population manufactured by the hepatoblast cell manufacturing method of the present invention is more than 90%. In one embodiment, the hepatoblast population manufactured by the hepatoblast manufacturing method of the present invention has a purity of 95% or higher. In one embodiment, the hepatoblast population manufactured by the hepatoblast manufacturing method of the present invention has a purity of 97% or higher. In one embodiment, the hepatoblast population manufactured by the hepatoblast manufacturing method of the present invention has a purity of 99% or higher. The manufactured hepatoblast population can survive in vivo for a long time after being given to an individual and exert the tissue repair capacity of liver tissue in the organism.

[0167] 2. Methods for manufacturing hepatocytes or bile duct cells The present invention also provides a method for manufacturing hepatocytes or bile duct cells. According to the method for manufacturing hepatocytes or bile duct cells of the present invention, a large number of hepatocytes or bile duct cells can be produced from hepatocytes that have undergone proliferation culture.

[0168] The method for manufacturing hepatocytes or bile duct cells according to the present invention may include a pluripotent stem cell preparation step, a hepatoblast induction step, a proliferation culture step, and a differentiation induction step. The hepatoblast induction step is the step of inducing the culture of pluripotent stem cells and inducing their differentiation into hepatoblasts, and includes: a first step of inducing the differentiation of pluripotent stem cells into defined endoderm cells; and a second step of inducing the differentiation of defined endoderm cells into hepatoblasts. The pluripotent stem cell preparation step and the hepatoblast induction step are arbitrary steps in this method, while the proliferation culture step and the differentiation induction step are essential steps.

[0169] <Methods for manufacturing liver cells> Hepatocytes can be induced to differentiate into hepatocytes by culturing them in a medium for hepatocyte induction. There are no particular limitations; for example, 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.

[0170] The hepatocyte induction culture medium used can be any culture medium known in the art for use in inducing differentiation into hepatocytes. For example, a culture medium containing one or more of the group consisting of inflammatory cytokines, steroids, and growth factors can be used. As inflammatory cytokines, for example, any inflammatory cytokine described in the section on "Induction of Differentiation from Determined Endoderm Cells to Hepatocytes" can be used; as steroids and growth factors, any steroids and growth factors described in the section on "Proliferation Culture of Hepatocytes and Production of Hepatocyte Populations" can be used. For example, as inflammatory cytokines, IL-6 family proteins such as OSM are preferred; as steroids, glucocorticoids such as DEX are preferred; and as growth factors, HGF is preferred. The concentrations of inflammatory cytokines, steroids, and growth factors are not particularly limited and can be used at concentrations commonly used by those skilled in the art.

[0171] There are no particular limitations on the culture conditions. For example, the proliferation culture procedure described in Method I can be followed. There are no particular limitations on the culture period; culture can be carried out until differentiation into hepatocytes is induced. For example, it can be set to more than 1 day, more than 3 days, more than 5 days, more than 7 days, more than 9 days, or more than 10 days. Specifically, for example, the culture period can be set to 1 day to 150 days, 1 day to 90 days, 5 days to 50 days, 8 days to 30 days, 9 days to 20 days, or 9 days to 15 days.

[0172] Induction of differentiation into hepatocytes can be determined based on whether the cell possesses any properties characteristic of hepatocytes, whether it has lost properties characteristic of hepatoblasts, and / or whether it lacks any properties characteristic of cholangioblasts. For example, this can be determined by the expression of hepatocyte markers, hepatoblast markers, and / or cholangioblast markers. As hepatocyte markers, any marker known in the art can be used without particular limitation. Specifically, examples include A1AT, ALB, CPS1, CYP3A4, SERPINA1, AHSG, APOA4, ASGR1, KRT8, FABP1, FGG, G6PC, PCK2, RBP4, TAT, TF, TDO2, HHEX, and HNF1A. As hepatoblast markers, any of the aforementioned hepatoblast markers in the first approach can be used, and as cholangioblast markers, any hepatocyte markers described later in this approach can be used.

[0173] <Methods for Manufacturing Bile Duct Cells> Hepatocytes can be induced to differentiate into bile duct cells by culturing them in a medium for bile duct cell induction. There are no particular limitations; for example, methods described in Sampaziotis, et al., Nat. Protoc. (2017) (doi:10.1038 / nprot.2017.011) and Ogawa et al., Nat. Biotechnol. (2015) (doi:10.1038 / nbt.3294) can be used.

[0174] The culture medium used for inducing cholangioblasts can be any culture medium known in the art for inducing differentiation into cholangioblasts. For example, a culture medium containing a Wnt signaling pathway activator, a ROCK inhibitor, TGFβ family proteins (activin, TGFβ, etc.), adenylate cyclase activator, and / or growth factor can be used. As the Wnt signaling pathway activator, ROCK inhibitor, and growth factor, the ROCK inhibitor and growth factor described in <Hepatocyte Proliferation Culture and Hepatocyte Population Production> can be used respectively. For example, as the Wnt signaling pathway activator, any one or more of Wnt, such as WNT3A, its analogues, and JNK activators such as R-spondin 1 can be used. As the ROCK inhibitor, a ROCK1 inhibitor such as Y-27632 can be used. As the growth factor, any one or more of EGF, HGF, and FGF (such as FGF10) can be used. The concentration of Wnt is not particularly limited, and can be set to, for example, 0.1nM~5nM, 0.5nM~3nM, 1nM~2nM, 1nM~1.5nM, etc. The concentration of JNK activator is not particularly limited, and can be set to, for example, 0.1nM~50nM, 1nM~20nM, 1nM~10nM, 5nM~10nM, 6nM~8nM, etc. The concentrations of ROCK inhibitor and growth factor are not particularly limited, and can be, for example, the final concentrations described in the first method.

[0175] There are no particular limitations on the adenylate cyclase activator; any known drug that enhances the activity of adenylate cyclase can be used. Specific examples of adenylate cyclase activators include: trichodin, trichodin analogues (such as Colforsin daropate); cholera toxin, carbacycline, isoproterenol, their analogues or salts thereof; biogenic amines such as octopamine; steroid hormones (glucagon, prostaglandins, vasopressin, etc.); peptides such as pituitary adenylate cyclase activating polypeptide (PACAP) and parathyroid hormone; nucleotide derivatives such as 8-Bromo-cAMP or combinations thereof. The concentration of adenylate cyclase activators such as trichodin is not particularly limited as long as it is an effective amount; for example, it can be set to 0.1 μM~100 μM, 1 μM~50 μM, 1 μM~20 μM, 5 μM~15 μM, etc.

[0176] As the basal culture medium, any culture medium described in the first method can be used, such as DMEM, which is preferably used. Furthermore, other components, additives, and culture medium additives described in the first method may be appropriately included. For example, vitamin A derivatives such as retinoic acid may be preferably additionally included.

[0177] There are no particular limitations on the culture conditions. For example, the proliferation culture procedure described in Method I can be followed. There are no particular limitations on the culture period; culture can be carried out until differentiation into bile duct cells is induced. For example, it can be set to more than 1 day, more than 3 days, more than 5 days, more than 7 days, more than 9 days, or more than 10 days. Specifically, for example, the culture period can be set to 1 day to 150 days, 1 day to 90 days, 5 days to 50 days, 8 days to 30 days, 9 days to 20 days, or 9 days to 15 days.

[0178] The induction of differentiation into bile duct cells can be determined based on whether the cells possess any of the properties of bile duct cells, whether they have lost the properties of hepatoblasts, and / or whether they lack any of the properties of hepatocytes. For example, this can be determined by the expression of bile duct cell markers, hepatoblast markers, and / or hepatocyte markers, without particular limitation. As bile duct cell markers, any marker known in the art can be used. Specifically, examples include KRT19, GGT1, KRT7, AQP1, HES1, and SSTR2. As hepatoblast markers, for example, any of the aforementioned hepatoblast markers in the first embodiment can be used; as hepatocyte markers, for example, any of the aforementioned hepatocyte markers in this embodiment can be used.

[0179] 3. Hepatoblast proliferation promoters and kits for promoting hepatoblast proliferation The present invention also provides a hepatoblast proliferation promoter and a kit for promoting hepatoblast proliferation.

[0180] The hepatoblast proliferation promoter of the present invention comprises a ROCK inhibitor, a TGFβ signaling pathway inhibitor and / or a Wnt signaling pathway activator, and a growth factor as essential components, and includes a steroid as an optional component. Regarding the ROCK inhibitor, TGFβ signaling pathway inhibitor, Wnt signaling pathway activator, growth factor, or steroid contained therein, it may include the substances and concentrations described in the section "<Hepatoblast proliferation culture and hepatoblast population creation>". In one embodiment, the hepatoblast proliferation promoter of the present invention comprises a ROCK inhibitor, a TGFβ signaling pathway inhibitor, and a growth factor as essential components. In one embodiment, the hepatoblast proliferation promoter of the present invention comprises a ROCK inhibitor, a Wnt signaling pathway activator, and a growth factor as essential components.

[0181] The hepatoblast cell proliferation promotion kit of the present invention, in addition to comprising a hepatoblast cell proliferation promoter containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor and / or a Wnt signaling pathway activator, and growth factors as essential components, and containing steroids and other components as optional components, may further include culture medium, culture medium additives, etc. In one embodiment, the hepatoblast cell proliferation promotion kit of the present invention contains a ROCK inhibitor, a TGFβ signaling pathway inhibitor, and growth factors as essential components. In another embodiment, the hepatoblast cell proliferation promotion kit of the present invention contains a ROCK inhibitor, a Wnt signaling pathway activator, and growth factors as essential components.

[0182] The kit of the present invention may further include a container. The container is not limited to any 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.

[0183] In the kit of the present invention, each active ingredient may be contained in a container. In the kit of the present invention, each active ingredient may be contained in one container or separately in multiple containers. When contained separately in multiple containers, the containers may be divided according to the ingredients, or according to other criteria (such as the amount used at one time), or according to their combination.

[0184] The kit of the present invention may include, for example, tools for adding culture medium (dropper, micropipette, syringe, needle, sprayer, etc.). Furthermore, the kit of the present invention may include instructions for use as needed.

[0185] The kit of the present invention has the advantage of being able to easily perform hepatocyte proliferation culture.

[0186] 4. Cell-based preparations containing hepatoblasts or treatments for liver diseases. This invention also provides a cell preparation containing hepatoblasts for treating liver disease or a method for treating liver disease. The cell preparation containing hepatoblasts for treating liver disease of this invention contains hepatoblasts as an essential component and, if necessary, includes a solvent and a carrier. The cell preparation containing hepatoblasts for treating liver disease according to this invention is capable of treating individuals with liver disease.

[0187] <Cellular preparations containing hepatoblasts for the treatment of liver disease> The cell preparation containing hepatoblasts for treating liver diseases of the present invention (hereinafter referred to as "the cell preparation of the present invention") comprises a population of hepatoblasts manufactured by the hepatoblast manufacturing method of the present invention.

[0188] The source of the hepatoblasts included in the cell preparations of the present invention is not particularly limited. For example, the hepatoblasts may be derived from the biological species or individual of the subject of administration or treatment. In one embodiment, the biological species from which the hepatoblasts included in the pharmaceutical composition of the present invention are derived is human.

[0189] The pharmaceutical composition of the present invention is characterized in that, in addition to expressing typical genes expressed in hepatoblasts (e.g., AFP, DLK, ALB, TBX3, C-MET, EpCAM, KRT19), it further expresses one or more genes selected from the group consisting of SLC4A4, TNFSF4, IL1RAPL1, NRP1, SOX9, ONECUT1, PLEKHA2, RNF207, NID1, and LYST. In one embodiment, the pharmaceutical composition of the present invention expresses one or more genes selected from the group consisting of SLC4A4, TNFSF4, IL1RAPL1, NRP1, SOX9, ONECUT1, PLEKHA2, RNF207, NID1, and LYST. In one embodiment, the pharmaceutical composition of the present invention expresses the genes or proteins of IL1RAPL1, NRP1, SOX9, ONECUT1, NID1, and LYST. In one embodiment, the pharmaceutical composition of the present invention further expresses the genes or proteins of SLC4A4, TNFSF4, PLEKHA2, and RNF207. In one embodiment, the cell preparation of the present invention, in addition to expressing typical genes expressed in hepatocytes (e.g., AFP, DLK, ALB, TBX3, C-MET, EpCAM, KRT19), further expresses one or more genes selected from the group consisting of SLC4A4, TNFSF4, IL1RAPL1, NRP1, SOX9, ONECUT1, PLEKHA2, RNF207, and LYST. In another embodiment, the cell preparation of the present invention, in addition to expressing typical genes expressed in hepatocytes (e.g., AFP, DLK, ALB, TBX3, C-MET, EpCAM, KRT19), further expresses IL1RAPL1, NRP1, SOX9, ONECUT1, NID1, and LYST. In one embodiment, the cell preparation of the present invention expresses typical genes expressed in hepatocytes (e.g., AFP, DLK, ALB, TBX3, C-MET, EpCAM, KRT19), as well as IL1RAPL1, NRP1, SOX9, ONECUT1, NID1, and LYST, and further expresses one or more genes or proteins selected from the group consisting of SLC4A4, TNFSF4, PLEKHA2, and RNF207.

[0190] The cell preparation of the present invention can be manufactured by a manufacturing method comprising the following steps: a hepatocyte induction step, which includes a first step of inducing pluripotent stem cells (e.g., iPS cells) to differentiate into morphological endoderm cells and a second step of inducing morphological endoderm cells to differentiate into hepatocytes; and a third step of proliferating and culturing hepatocytes to create a hepatocyte population. In the treatment of human liver diseases, cell preparations containing cells obtained from the steps of the first to the third step can also be used, but by using a hepatocyte population further cultured in a serum-free medium containing oncogene M in a maturation promotion step, it is expected to improve treatment outcomes.

[0191] The method for manufacturing the cell preparation of the present invention may further include: a maturation promotion step, in which oncogene M is added to culture a hepatocyte population. In one embodiment, the method for manufacturing the cell preparation of the present invention for treating liver disease includes: a first step of inducing pluripotent stem cells to differentiate into defined endoderm cells; a second step of inducing defined endoderm cells to differentiate into hepatocytes; a third step of proliferating and culturing hepatocytes to create a hepatocyte population; and a maturation promotion step of culturing the hepatocyte population in a serum-free culture medium containing oncogene M.

[0192] The first step may include the following steps: culturing in a serum-free medium containing Y-27632 (e.g., at a concentration of 10 μM) during the initial culture stage; and culturing in a serum-free medium containing B-27 supplement, activator A, and a WNT signaling pathway activator after the middle culture stage. The second step may include the following steps: culturing in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide. The third step may include a proliferation culture step, proliferating hepatocytes in a hepatocyte proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and growth factors.

[0193] In one embodiment, a method for manufacturing a cell preparation for treating liver disease includes: a first step of inducing pluripotent stem cells to differentiate into standardized endoderm cells; a second step of inducing standardized endoderm cells to differentiate into hepatocytes; and a third step of proliferating and culturing hepatocytes to create a hepatocyte population. The first step includes the following steps: culturing in a serum-free medium containing 10 μM Y-27632 during the early stage of culture; and culturing in a serum-free medium containing B-27 supplement, activin A, and a WNT signaling pathway activator after the middle stage of culture; the second step includes the following steps: culturing in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide; and the third step includes a proliferation culture step of proliferating and culturing hepatocytes in a hepatocyte proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and growth factors.

[0194] In one embodiment, the method for manufacturing the cell preparation for treating liver disease of the present invention includes: a first step of inducing iPS cells to differentiate into fixed endoderm cells; a second step of inducing fixed endoderm cells to differentiate into hepatocytes; a third step of proliferating and culturing hepatocytes to create a hepatocyte population; and a maturation promotion step of culturing the hepatocyte population in a serum-free medium containing oncogene M. The first step includes the following steps: culturing on day 0 of culture in a serum-free medium containing 10 μM Y-27632; and culturing after day 1 of culture in a serum-free medium containing B-27 supplement, activin A, and a WNT signaling pathway activator. The second step includes the following steps: culturing in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide. The third step includes: a proliferation culture step of proliferating and culturing hepatocytes in a hepatocyte proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and growth factors.

[0195] The cells used in this cell preparation can undergo any treatment before storage. Specific treatments are not particularly limited; for example, screening, transformation, gene manipulation, or combinations thereof can be performed. In one embodiment, the cells used in this cell preparation can undergo further gene manipulation after proliferation culture. In another embodiment, the cells used in this cell preparation can undergo further transformation after proliferation culture.

[0196] The cells in this cell preparation can be in a single-cell state or in cell clumps such as spheroids. A single-cell state is preferred. In this specification, "single-cell state" refers to a state in which cells exist as monomers and are not aggregated. The proportion of single-cell states among all cells in the cell preparation is, for example, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%. The proportion of single-cell states in the cell preparation can be determined using any method. For example, cells can be dispersed in a buffer solution (e.g., PBS), and a randomly selected number of cells can be observed under a microscope to investigate whether aggregation occurs, thereby determining the proportion.

[0197] Cells in cell preparations can be suspended or in contact with the inner wall of the container. Suspension is preferred. In this specification, "suspension" means that the cells are not fixed to the inner wall of the container containing the cell preparation by adhesives or other means.

[0198] The number of cells per unit dose contained in the cell preparation of the present invention is not particularly limited. Generally, the number of cells varies depending on the type of cells, route of administration, purpose of administration, and the type of carrier used as other constituent components (described later). Therefore, it can be appropriately determined considering the respective conditions. For example, it is sufficient for a single application dose of the cell preparation to contain a sufficient number of cells. Specifically, the number of hepatocytes per unit dose is not particularly limited; for example, it may contain 10... 3 More than 10 cells / mL 4 More than 10 cells / mL 5 More than 10 cells / mL 6 More than 10 cells / mL 7 More than 10 cells / mL 8 More than 10 cells / mL 9 Hepatocytes per mL or higher are acceptable. In the case of multiple administrations, it is sufficient that the total dose contains a sufficient number of cells. Furthermore, when administering the cell preparation of the present invention after dilution, the diluted preparation need only contain a sufficient number of cells to achieve the desired effect.

[0199] In addition to the hepatoblast cell population, any additional cells may be included. There are no particular limitations on the types of additional cells. Preferably, they should be cells from the same biological species or from the same individual source as the hepatoblast cells.

[0200] Solvent Cell formulations of this method may contain pharmaceutically acceptable solvents as needed. "Pharmaceutically acceptable solvents" refers to solvents commonly used in the field of formulation technology. Examples include water and aqueous solutions. Aqueous solutions may include, for example, physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer. Adjuvants may include, for example, inorganic salts such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride; organic acid salts such as citrate, gluconate, and succinate; and other low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.

[0201] The pH of the solution only needs to be sufficient to allow the preserved cells to survive; there are no specific limitations. Specific pH values ​​include, for example, 3.5–8.5, 4–8, 4.5–7.5, or 5–7.5.

[0202] The osmotic pressure of a solution is only required to ensure cell survival and is not particularly limited. For example, it can be a hypotonic solution (less than 250 mOsm / L), an isotonic solution (250 mOsm / L to 380 mOsm / L), or a hypertonic solution (greater than 380 mOsm / L). Osmotic pressure can be expressed as the ratio of osmotic pressure to that of physiological saline (e.g., 306 mOsm / L).

[0203] The solution of the present invention can be a self-prepared solution or a commercially available solution. For example, preferred examples of solutions include Ringer's solution (lactated Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, etc.), Ringer's base solution, and any other solution used for infusion.

[0204] The viscosity of the solution is not particularly limited as long as it allows the preserved cells to survive. For example, it is preferably 8 mPas or higher, and more preferably 18 mPas or lower, at the temperature during cell suspension and / or preservation. The viscosity of the non-cryopreservation solution can be measured, for example, using a TV-20 viscometer (Toki Sangyo Co., Ltd.) at a rotation speed of 10 rpm.

[0205] <Carrier> The cell formulations of this invention may include pharmaceutically acceptable carriers as needed. "Pharmaceutically acceptable carriers" refers to additives commonly used in the field of formulation technology. Examples include excipients, binders, disintegrants, emulsifiers, flow modifiers, lubricants, etc.

[0206] Excipients include, for example, monosaccharides, disaccharides, cyclodextrins and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, and pulmonol. （R） Kaolin, silica, or combinations thereof.

[0207] Adhesives may include, for example, starch pastes made from plant starches, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone, or combinations thereof.

[0208] Examples of disintegrants include, for example, the starch, lactose, carboxymethyl starch, croscarmellose, agar, kelp powder, sodium bicarbonate, calcium carbonate, alginate or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, glyceryl monostearate or its salts.

[0209] Examples of emulsifiers include sorbitan fatty acid esters, glycerol fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0210] Examples of flow modifiers and lubricants include silicates, talc, stearates, and polyethylene glycol.

[0211] In addition to the above, it may also include, as needed, commonly used solubilizers, suspending agents, diluents, dispersants, surfactants, analgesics, stabilizers, absorption enhancers, expanders, humectants, moisturizers, humectants, adsorbents, flavoring and odor-correcting agents, disintegration inhibitors, coating agents, coloring agents, preservatives, antiseptics, antioxidants, buffers, pH adjusters, isotonic agents, etc.

[0212] In addition to preventing or inhibiting the decomposition of the active ingredient in the body caused by enzymes, the carrier is also used to facilitate formulation, make administration easier, maintain dosage form and efficacy, and can be used as needed.

[0213] The cell preparation of the present invention is preferably sterile. Methods known in the art for achieving sterility can be used, and there are no particular limitations.

[0214] <Dosage Form> The cell preparation of the present invention is a liquid formulation. The liquid formulation contains any fluid-type preparation. Specifically, examples include injections, suspensions, and gels. Regarding the specific volume, etc., it is not particularly limited as long as it is within the range known in the art for each dosage form. Regarding the manufacturing method of the cell preparation of the present invention, formulation can be carried out according to conventional methods in the art.

[0215] <Treatment of liver diseases using cell preparations containing hepatoblasts> The cell preparation of the present invention can be used to treat various liver diseases.

[0216] <Administration Method> The method of administration of the cell preparation of the present invention is parenteral administration. Parenteral administration can be further subdivided into systemic administration and local administration. Local administration includes, for example, portal venous route, tissue administration, lesion-level route, organ administration, spleen-level route, subcutaneous route, intradermal route, intravenous route, and intramuscular route. Systemic administration of parenteral administration includes administration into the circulatory system (e.g., intravenous administration, intra-arterial administration, 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 administered directly to the target site, for example, by injection. In addition, for example, it can be used in a gel, suspension, or any other suitable substance. In the case of systemic administration, administration into the circulatory system can be performed, for example, by intravenous injection. The dosage is any amount that is effective in achieving cell efficacy. The effective amount is appropriately selected according to the information of the subject, as described above.

[0217] The application method and dosage of the cell preparation of the present invention can also be varied according to the information of the subject. In this specification, "information of the subject" refers to various information related to the characteristics and state of the subject. For example, in the case of an individual, information such as age, weight, sex, overall health status, presence or absence of disease, disease progression, severity, drug sensitivity, presence or absence of concurrent medications, and tolerance to treatment can be listed.

[0218] The cell preparations of this invention can be stored before application. Specific storage methods, storage periods, and storage temperatures are not particularly limited.

[0219] Furthermore, the cell preparation of the present invention can be applied directly or after any additional treatment. Specific examples of additional treatment include application through transplantation of a reservoir containing the cell preparation of the present invention, or application through further culturing the cell preparation after transplantation to form tissues, organs, or organoids, but are not limited thereto. Further culturing can be performed for the purpose of increasing cell number, cell differentiation, transformation, or gene introduction into cells.

[0220] Furthermore, the cell preparation of the present invention can also be used in combination with one or more known cell preparations.

[0221] <Application Objects> In this specification, "applicable object" refers to an object given or treated with the cell preparation of the present invention. The applicable object of the cell preparation of the present invention is not particularly limited; it can be a healthy person or a person suffering from a certain disease or in a certain state. The diseases and states in this case are not particularly limited; for example, diseases and states related to liver injury can be listed. Specifically, for example, the cell preparation of the present invention can be used as a cell preparation to improve, treat, or prevent one or more types of liver injury selected from the group consisting of: end-stage liver disease, cirrhosis, diabetes, obesity, acute hepatitis, chronic hepatitis, fatty liver, liver fibrosis, portal hypertension, aplastic anemia, hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis, autoimmune hepatitis, liver dysfunction, hepatic blood flow obstruction, acute liver failure, liver fibrosis, liver cancer, liver metabolic diseases, traumatic liver injury, drug-induced liver injury, and liver injury caused by liver failure.

[0222] Furthermore, there are no particular limitations on the intended use. For example, it can be used for purposes such as the improvement, treatment, and prevention of diseases and conditions.

[0223] <Effect> By administering the cell preparations in this manner, the introduced hepatoblasts can survive stably in vivo for a long period, and at least a portion of them can function in the same way as endogenous cells. Therefore, the cell preparations in this manner can be used in cell replacement therapy.

[0224] For example, the introduced hepatoblasts survived stably in the organism for more than 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, or 10 months.

[0225] Furthermore, the proportion of introduced cells in the recipient's liver is typically above 10% at a time point more than 2 months post-transplantation. Additionally, for example, the proportion of introduced cells in the recipient's liver is above 10%, 15%, 20%, 25%, and 27% at time points more than 14 weeks post-transplantation.

[0226] Moreover, the cell preparations produced in this manner will not form transplant-induced tumors, for example, during periods of more than 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, or 10 months, thus providing a treatment with a lower risk of tumor formation.

[0227] Hepatoblasts prepared using the methods described in this specification offer several advantages even compared to cell replacement therapy using primary cultured mature hepatocytes. First, pluripotent stem cell-derived hepatoblasts can be stably produced without donor dependence, thus ensuring a stable supply when treatment is needed. Second, the introduced hepatoblasts generate new mature hepatocytes in the recipient body, thus promising long-term therapeutic effects compared to mature hepatocytes with limited replication cycles. Third, hepatoblasts are considered to have greater tolerance to liver injury than mature hepatocytes, resulting in greater robustness of therapeutic effects after introduction. Fourth, the smaller cell size of hepatoblasts reduces the risk of immediate blood-mediated inflammatory response (BIMIR) in the portal venous region, which is commonly seen immediately after mature hepatocyte transplantation.

[0228] Based on these characteristics, the present invention provides a new and promising option for the treatment of liver failure.

[0229] To further understand the present invention, specific embodiments are provided below for reference, but these embodiments are for illustrative purposes only and do not limit the present invention.

[0230] Example <Example 1. Differentiation induction to hepatocytes and evaluation of hepatocyte properties> (Purpose) Inducing differentiation from iPS cells into hepatoblasts and investigating the properties of the induced hepatoblasts.

[0231] (method) 1. iPS cells Human iPS cell lines were used, including M48 (CiRA Foundation), QHJI (CiRA Foundation), and 1383D6 (Riken). Human iPS cell lines were cultured in StemFit AK02N medium (Ajinomoto, RCAK02N) at 37°C in the presence of 5% CO2 using plates coated with iMatrix-511 (Nippi, 892011) mixed with PBS at a ratio of 1:150. Cells were dispersed every 7 days using Accutase (Innovative Cell Technologies, AT104-500) and passaged as single cells. M48 cells were used after being cryopreserved at -196°C for 30 days in cryopreservation medium (Cellbanker1) and then thawed.

[0232] Cultured human iPS cells were seeded into StemFit AK02N medium (day 0) supplemented with 10 μM Y-27632 (Wako, 030-24026). The medium was replaced with fresh StemFit AK02N medium on day 1 of culture, and thereafter every 2 days.

[0233] 2. Induction of differentiation into hepatoblasts 2-1. Induction of differentiation into definitive endoderm cells 1. Similarly, on culture dishes coated with iMatrix-511, at a concentration of 1.5 × 10⁻⁶... 5 cells / cm 2 Human iPS cells (hiPSCs) were seeded at a density of [missing information] (day 0). Cells were cultured for 7 days in RPMI 1640 medium (Wako, 189-02025) supplemented with 1% B-27 supplement (50×) (Gibco, 17504-001), 100 ng / mL activin A (molecular weight: approx. 26 kDa; Ajinomoto, 18585-81), and 2 μM CHIR99021 (Cayman, 13122) or 50 ng / mL WNT3A (molecular weight: approx. 42 kDa; R&D Systems, 5036-WN-500). 10 μM Y-27632 was added to the medium only on day 0. Additionally, 500 μM sodium butyrate (NaB: Wako, 193-01522) was added to the medium from day 1 to day 3. The medium was changed daily. The differentiated cells obtained on day 7 of culture were defined endoderm cells (hiPSC-DE).

[0234] 2-2. Induction of differentiation into hepatoblasts After day 7 of culture, the culture medium was changed to hepatocyte differentiation medium. As the hepatocyte 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 oncogene 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 liver growth factor (HGF; molecular weight: approx. 79 kDa; REPROCELL, 03-0019) was used. The medium was changed daily.

[0235] As a serum-free differentiation medium (SFD medium), it was used in a medium containing 125 mL of Ham's F-12 K medium (Life Technologies), 5 mL of B27 supplement (Life Technologies), 2.5 mL of N2 supplement (Life Technologies) mixed with 375 mL of Iscove modified Duchenne broth (Life Technologies), and supplemented with 0.05% bovine serum albumin (Sigma-Aldrich), 2 mM L-glutamine (Life Technologies), 1% penicillin-streptomycin (Life Technologies), 0.45 mM monothioglycerol solution (Wako Pure Chemical Industries), and 0.5 mM L-ascorbic acid (Sigma-Aldrich).

[0236] Hepatoblasts differentiated on day 13 of culture (iPSC-HB) were recovered for evaluation of the properties of hepatoblasts as described in section 4.

[0237] 3. Preparation of other cells Hepatic endoderm cells and hepatocytes derived from human iPS cells were prepared using the following method. Additionally, human adherent hepatocytes (Bioreclamation IVT, IVT-F00995-P or IVT-M00995-P) were used as primary hepatocytes (PHH).

[0238] 3-1. Preparation of liver endoderm cells After 6 days of culture, the induced differentiated endoderm cells were recovered and cultured for 5 days in liver endoderm cell differentiation medium to prepare differentiated liver endoderm cells (hiPSC-HE). The liver endoderm cell differentiation medium used was DMEM medium supplemented with 10% KnockOut Serum Replacement (Gibco), 1% MEM Non-Essential Amino Acids Solution (Gibco), DMSO (Nacalai), 1 mM L-glutamine (Gibco), 10 μM 2-mercaptoethanol (Gibco), and 1% penicillin-streptomycin.

[0239] 3-2. Preparation of hepatocytes Hepatocytes (hiPSC-Hep) were cultured in hepatocyte differentiation medium for 10 days to prepare differentiated hepatocytes. DMED medium containing 10 ng / mL oncogene M (OSM: R&D Systems, 295-OM), 100 nM dexamethasone (DEX: Sigma, D2915), and 5% FBS was used as the hepatocyte differentiation medium.

[0240] 4. Evaluation of the properties of hepatoblasts 4-1. qPCR analysis Total RNA was isolated from hiPSC, hiPSC-DE, and hiPSC-HB cells using the PureLink RNA Mini Kit (Thermo Fisher Scientific, 12183025). Single-stranded cDNA was synthesized using the isolated RNA (<2 μg) as a template and a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, 4368814) according to the manufacturer's protocol. qPCR was performed using a CFX96 Real-Time System machine (BioRad) and TB Green Premix ExTaq II (Takara, RR820D) according to the manufacturer's protocol.

[0241] Primers used to detect gene expression are shown in Table 1. Gene expression levels were calculated using the ΔΔCT method with ACTB as the housekeeping gene, and fold change was calculated as a relative value to the expression level obtained from hiPSC.

[0242] For hiPSC, hiPSC-DE, and hiPSC-HB, the number of samples for each is n=8.

[0243] [Table 1] Table 1: Primers used in the qPCR analysis of Example 1

[0244] 4-2. Flow cytometry The isolated cells were dispersed with 0.05% trypsin / EDTA (Gibco), washed with DMEM containing 10% fetal bovine serum (FBS), and then washed with PBS. Next, the cells were stained with labeled antibodies on ice for 30 minutes. Afterward, the samples were washed and resuspended in FACS buffer. Flow cytometry was performed using a BD FACS Celesta Cell Analyzer or Aria III (BD Biosciences), and the collected data were analyzed using FlowJo 10.7.1 (BD Biosciences) software. Additionally, Biolgend or BD Science standards were used as isotype control antibodies.

[0245] The antibodies used are as follows: FITC mouse anti-human EpCAM (BioLegend, 324203) APC / CY7 anti-human CD13 (BioLegend, 301709) BV421 mouse anti-human CD90 (BD Bioscience, 562556).

[0246] 4-3. Transcriptome Analysis In the total RNA obtained as described above, RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies), and RNA samples with an RNA Integrity Number (RIN) greater than 9.0 were used for RNA sequencing (RNA-Seq) analysis. RNA-Seq libraries were prepared from 100 ng of total RNA using the Ion AmpliSeq Transcriptome Human Gene Expression kit (Thermo Fisher Scientific, A26326) according to the manufacturer's protocol. Library sequencing was performed using the Ion PI Hi-Q Sequencing 200 kit and Ion PI Chip v3 (Thermo Fisher Scientific, A26772) on the Ion Proton system, with sequencing reads mapped to hg19_AmpliSeq_Transcriptome_ERCC_v1 using the Torrent Mapping Alignment Program. Next, the QC metrics and normalized read counts for each gene were obtained using the AmpliSeqRNA plugin v5.2.0.3 and Torrent Suite Software v5.2.2 (Thermo Fisher Scientific). Data analysis was performed using GeneSpring (Agilent Technologies).

[0247] 5. Statistical Processing GraphPad Prism 8.0 (GraphPad Software) was used for statistical analysis. No data were excluded from the data analysis. The significance level was set at p < 0.05. Tests relating to the results of flow cytometry were performed using the Mann-Whitney t-test (two-sided test), and tests relating to the qPCR analysis in this example were performed using Dunnett's multiple comparison test.

[0248] (result) The results are shown in Figures 2 to 5 .

[0249] like Figure 2As shown, the gene expression analysis performed by qPCR revealed that in hiPSC-HB, the expression of typical hepatoblast-related genes such as alpha-fetoprotein (AFP), albumin (ALB), T-box transcription factor 3 (TBX3), delta-like Notch ligand 1 (DLK1), and hepatocyte growth factor receptor (C-MET) was significantly increased compared to predifferentiated iPS cells (hiPSC) and post-first-stage differentiated endoderm cells (hiPSC-DE). This indicates that the hiPSC-HB used in this study exhibits a hepatoblast-like gene expression profile.

[0250] Moreover, such as Figure 3 As shown, comprehensive transcriptomic analysis confirmed that hiPSC-HB strongly expresses all characteristic genes of hepatoblasts, indicating that hiPSC-HB is a hepatoblast-like cell. Furthermore, a comparison of the expression profiles of developmental stages and hepatoblast-specific markers in human liver organogenesis revealed that the gene expression profile of hiPSC-HB is similar to that of human embryonic liver cells at 5 post-conception weeks (data not shown).

[0251] Furthermore, during the differentiation induction process from hiPSC to hiPSC-HB, the induction efficiency of hiPSC-HB was compared between the case where WNT3A was used in the differentiation induction from hiPSC to hiPSC-DE and the case where CHIR99021, as a GSK3β inhibitor, was used. Figure 4 ).

[0252] CD13-positive cells, a marker of fetal liver stem cells, were extracted using flow cytometry, and the expression level of epithelial cell adhesion molecule (EpCAM), a marker of hepatocytes, was analyzed. The results showed that... Figure 4 As shown, compared with the case using WNT3A (6.5±1.6%), the proportion of cells with low EpCAM expression (EpCAM-low) was significantly reduced with the case using CHIR99021 (0.3±0.2%). Figure 4 A and B). In other words, it was found that by using CHIR99021, a cell population with high EpCAM expression (EpCAM-high) of 99.7 ± 0.2% and extremely high homogeneity could be obtained. Figure 4 (A and C).

[0253] To clarify the cell characteristics of the EpCAM-low and EpCAM-high cell populations, the expression of CD90, a marker of mesenchymal cells, was investigated in these populations. The results showed that... Figure 5As shown, CD90 expression is generally higher in the EpCAM-low cell population compared to the case where an isotype control antibody was used but no CD90 antibody was used. The EpCAM-low cell population consists of mesenchymal cells that express CD90. Figure 5 B). On the other hand, CD90 expression was low in the EpCAM-high cell population, comparable to the case using an isotype control antibody, indicating that the EpCAM-high cell population is a cell population that does not express CD90. Figure 5 C). In other words, this indicates that the EpCAM-low cell population is not a cell population containing hepatoblasts but rather a large number of mesenchymal stem cell lines, while the EpCAM-high cell population is a hepatoblast-like cell population. This result demonstrates that by using CHIR99021 to induce differentiation from hiPSCs to hiPSC-DE, a hepatoblast-like EpCAM-high cell population can be obtained with high purity.

[0254] <Example 2. Study and Evaluation of Hepatocyte Proliferation and Culture Methods> (Purpose) To utilize hepatoblasts differentiated from iPS cells (hiPSC-HB) for the treatment of liver injury in humans, a large quantity of hiPSC-HB needs to be prepared. Therefore, the culture conditions for proliferation of hiPSC-HB while maintaining its undifferentiated state were investigated.

[0255] (method) 1. Research on proliferation culture The hiPSC-HB differentiated from hiPSCs using the method described in Example 1, obtained by using CHIR99021, was dispersed in 0.05% trypsin / EDTA (Gibco) at a concentration of 1×10⁻⁶. 4 cells / cm 2 The culture medium was inoculated into new culture dishes at a density of [insert density here] and cultured using candidate culture medium for proliferation. As candidate culture medium for proliferation, a medium supplemented with the components listed in Table 2 was used, which was an SFD medium containing 10 μM Y-27632, 0.5 μM A8301 (TGFβ / Smad inhibitor: R&D Systems, 2939 / 10), 1 mM N-acetyl-L-cysteine ​​(Sigma, A7250), and 1% FBS (Biomedicals, 2916754). The culture medium was changed every 2 days.

[0256] [Table 2] Table 2: Additional components added to each of the maintenance candidate culture media used in Example 2

[0257] 2. Subculturing in propagation culture Cells were passaged when the confluence of hiPSC-HB cells reached approximately 90%. TrypLE Express Enzyme (Gibco, 12604013) was used for cell dissociation and dispersion. Cells were passaged at a rate of 1 × 10⁻⁶. 4 cells / cm 2 Cells were seeded at a density in new culture dishes using a maintenance supplement medium with the same composition as the pre-passage culture medium. The number of seeded cells and the total number of cells before the next passage were counted at each passage, and the proliferation rate was calculated by dividing the total number of cells by the number of seeded cells. The cumulative cell proliferation rate was calculated by setting the pre-passage hepatocyte count to 1 and cumulatively multiplying it by the proliferation rate at each passage.

[0258] 3. Evaluation of cell properties qPCR analysis, flow cytometry, transcriptome analysis, and statistical analysis were performed using the same methods as in Example 1. In the qPCR analysis, the fold change was calculated as a relative value to the expression level in hiPSC-HB before maintenance culture.

[0259] 4. Statistical Analysis Statistical analysis was performed using the methods described in Example 1. Tests relating to the results of the qPCR analysis were performed using Dunnett's multiple comparison test.

[0260] (result) The results are shown in Figures 6-1 to 6-3 and Figure 7 .

[0261] In the candidate culture medium for proliferation, in addition to Y-27632 and A8301 as a TGFβ / Smad inhibitor, CHIR99021 as a WNT3 signaling activator was also used, and EGF and FGF2 were further added. The expression levels of hepatoblast-related genes (AFP, ALB, DLK1, C-MET) in the cultured cells were significantly different from those in the original hiPSC-HB cells before proliferation culture. Figure 6-1 "YAC+EGF+FGF2"). On the other hand, when WNT3A is used in addition to Y-27632 and A8301, and EGF and / or FGF2 as growth factors are added, as well as DEX ( Figure 6-1The gene expression profiles of cells cultured in “YAW+EGF+FGF2”, “YAW+EGF”, and “YAW+EGF+DEX” are roughly the same as those of hiPSC-HB. In particular, cells cultured in hepatoblast maintenance medium containing WNT3A, EGF, and DEX (hereinafter also referred to as “YAW+EGF+DEX”) show gene expression profiles similar to those of hiPSC-HB.

[0262] In candidate culture media for proliferation, when WNT3A was used in addition to Y-27632 and A8301, and when additional growth factors (FGF2, HGF, EGF) were added, a high proliferation rate was observed. Figure 6-2 "Proliferation rate"). Furthermore, the expression levels of hepatoblast-related genes in cultured cells showed similar results to those in hepatoblasts cultured under "YAW+EGF+DEX" conditions (…). Figure 6-2 In particular, when EGF was used as a growth factor and DEX as a glucocorticoid was further used, a high proliferation rate was observed while maintaining the expression levels of hepatoblast-related genes. Figure 6-2 (“WNT+EGF+DEX”).

[0263] To confirm the feasibility of replacing WNT3A with CHIR99021 in the candidate proliferation medium "YAW+EGF+DEX", the concentration of added CHIR99021 was set at 0.5 μM, 1 μM, 2 μM, or 3 μM. The results showed that the expression levels of hepatoblast-related genes in cells cultured in media supplemented with 0.5 μM ("CHIR-0.5" in the figure) and 1 μM ("CHIR-1" in the figure) of CHIR99021 were similar to those in hiPSC-HB before proliferation culture, as in the case of supplemented WNT3A ("WNT3A-50" in the figure). Figure 6-3 ).

[0264] When WNT3A, EGF, and DEX were used, the cell proliferation rate continued to increase even after multiple passages, and the proliferation rate did not change depending on the type of iPS cell line used or the preservation method. Figure 7 ).

[0265] Furthermore, to explore ROCK inhibitors, TGFβ / Smad inhibitors, and WNT3 signaling activators that could be used in candidate culture media for proliferation, various compounds were used to confirm the proliferation capacity of hiPSC-HB in the sixth generation. Thiazolvid (…) was used as a ROCK inhibitor. Figure 6-4In the middle, "Thiazovivin" and Rho Kinase Inhibitor IV ( Figure 6-4 (in Chinese, "Rho") Figure 6-4 As an inhibitor of the TGFβ signaling pathway ( Figure 6-5 ), using SB 431542, SB 505124, RepSox, LY364947, and LY2157299. As activators of the Wnt signaling pathway, BIO, TWS119, SB216763, and Recombinant R-Spondin 1 were used. Figure 6-6 In Chinese, “R-Spondin” Figure 6-6 After 7 days of culture, cells were peeled off, counted, and the proportion of cells relative to the number on day 1 of culture was calculated as the proliferation rate. The results showed that the proliferative capacity of hiPSC-HB was maintained regardless of the compound used. Figures 6-4 to 6-6 ).

[0266] <Example 3. Gene expression profile of hepatocytes in the proliferation culture stage> To evaluate whether hepatoblasts in the proliferation culture stage were identical to those in pre-proliferation hiPSC-HB cells, changes in passage number and gene expression profiles were assessed over time. Cells were passaged using medium supplemented with Y27623, A8301, WNT3A, EGF, and DEX. After one passage (P1, ...), the cells were... Figure 8 A) Cells that have undergone 5 passages (P5, Figure 8 B) Cells that have undergone 10 passages (P10, Figure 8 C) and cells that have undergone 15 passages (P15, Figure 8 The expression levels of CD13 and EpCAM were analyzed in D. The results showed that even after 15 passages, the proportion of EpCAM-high cells in CD13-positive cells remained at a high level of over 98%, showing the same expression profile as hiPSC-HB before proliferation culture. Figure 8 D).

[0267] Furthermore, analysis of the expression patterns of characteristic genes in hepatoblasts revealed that even after 15 passages, the gene expression profile did not change significantly. Figure 9Specifically, in any of the hepatoblasts at P1, P5, P10, and P15 (represented in the figure as "HB P0", "HB P1", "HB P5", "HB P10", and "HB P15"), all genes—including membrane proteins SLC4A4, TNFSF4, and IL1RAPL1; NRP1 transcription factors SOX9 and ONECUT1; and intracellular proteins PLEKHA2, RNF207, and LYST—were highly expressed. On the other hand, the expression of these genes was not detected in other cell types (represented in the figure as "hiPSC", "DE", "HE", and "MH"). Therefore, it can be said that the expression of SLC4A4, TNFSF4, IL1RAPL1, NRP1, SOX9, ONECUT1, PLEKHA2, RNF207, and LYST is characteristic of hepatoblasts. Figure 3 The results show that the hiPSC-HB of the present invention is characterized by expressing, in addition to AFP, DLK, ALB, TBX3, C-MET and EpCAM, which are typical gene markers of hepatoblasts, SLC4A4, TNFSF4, IL1RAPL1, NRP1, SOX9, ONECUT1, PLEKHA2, RNF207, NID1 and LYST.

[0268] Furthermore, based on the results of the major component analysis of the gene expression profile, it was confirmed that the gene expression profiles of P1, P5, P10, and P15 are the same, and the expression profile of hiPSC-HB did not change significantly even after multiple passages. Figure 10 ).

[0269] The above results indicate that hiPSC-HB can be passaged more than 15 times while maintaining its original characteristics, and the cell number can be increased to 10. 16 above.

[0270] <Example 4. In vitro differentiation induction of hiPSC-HB cells into hepatocytes and bile duct cells> (Purpose) In vitro differentiation of hepatoblasts (hiPSC-HB) differentiated from iPS cells into hepatocytes and bile duct cells was induced, and their properties were evaluated.

[0271] (method) 1. Induction of differentiation into hepatocytes (HB-Hep) When hiPSC-HB reached 90% confluence, the culture medium was replaced with hepatocyte differentiation induction medium, and the cells were cultured in the hepatocyte differentiation induction medium for 10 days. As the medium for inducing hepatocyte differentiation, DMEM medium (Wako, 043-30085) supplemented with 5% FBS, 10 ng / mL oncogene M, and 100 nM DEX was used. Furthermore, the above-mentioned medium is a generally applicable medium for differentiating hepatoblasts into hepatocytes.

[0272] 2. Induction of differentiation into HB-Cholangiocytes When hiPSC-HB reached 90% confluence, the cells were recovered at a concentration of 1×10⁶. 4 cells / cm 2 Cells were seeded at a density of 1 × 10⁻⁶ in iMatrix-511 coated culture dishes and cultured in cholangiocarcinoma differentiation induction medium for 10 days. The cholangiocarcinoma differentiation induction medium was SFD medium supplemented with 10 ng / mL EGF (molecular weight: approx. 6.2 kDa; Sigma, E9644), 20 ng / mL HGF, 50 ng / mL WNT3A, 100 ng / mL R-spondin 1 (molecular weight: approx. 14 kDa; R&D Systems, 4645-RS / CF), 50 ng / mL FGF10 (molecular weight: approx. 19 kDa; R&D Systems, 345-FG), 3 μM retinoic acid (Wako, 182-01116), 10 μM Y-27632, and 10 μM trichomoniasis. 4 cells / cm 2 The culture medium was inoculated at a density of [specific value] into culture dishes coated with iMatrix-511. The culture medium was changed every 2 days.

[0273] 3. Evaluation of cell properties Cells were recovered on day 10 of culture in each differentiation-inducing medium for qPCR analysis. In addition to the primers used in Example 1, the primers shown in Table 3 were also used in the qPCR analysis. The fold change was calculated as a relative value to the expression level in hiPSC-HB.

[0274] [Table 3] Table 3: Additional primers used in the qPCR analysis of Example 4

[0275] 4. Statistical Analysis Statistical analysis was performed as described in Example 1. Tests related to the qPCR analysis in this example were performed using the Mann-Whitney t-test (two-sided test).

[0276] (result) The results are shown in Figure 11 .

[0277] Cultured in hepatocyte differentiation-inducing medium, the expression levels of liver maturation genes containing α-1 antitrypsin (A1AT), ALB, carbamoyl phosphate synthase 1 (CPS1), cytochrome P450 3A4 (CYP3A4), and glucose-6-phosphatase catalytic subunit (G6PC) were significantly increased, indicating that differentiation into hepatocytes was effectively promoted. Figure 11 A). Furthermore, culturing in bile duct cell differentiation-inducing medium significantly increased the expression of bile duct cell-specific genes such as keratin 19 (KRT19), γ-glutamyl transferase 1 (GGT1), aquaporin 1 (AQP1), and somatostatin receptor 2 (SSTR2), indicating that it effectively promoted differentiation into bile duct cells. Figure 11 B). On the other hand, in these cells, the expression level of AFP, an immature gene in the liver, is reduced.

[0278] <Example 5. Differentiation induction from hiPSC-HB to liver tissue in vivo> (Purpose) Hepatoblasts differentiated from iPS cells (hiPSC-HB) were transplanted into mice to confirm their differentiation into liver tissue and the function of the differentiated liver tissue.

[0279] (method) 1. Establishment of a mouse model of liver failure Adult male HSVtk gene-transplanted immunodeficient NOG (TK-NOG) mice (8–10 weeks old, Invivo Science) were used in the experiment. To induce liver injury, mice were administered ganciclovir (GCV: Tanabe Mitsubishi Pharmaceutical) intraperitoneally at 15 mg / kg on day 12 (D-12) and day 14 (D-14) before transplantation (Figure 12A). Mice were housed in a specific pathogen-free (SPF) animal facility with temperature control and a 12-hour light / dark cycle. Water and food were provided freely throughout the experiment. All animal experiments and care were conducted in accordance with guidelines (PA22-05) approved by the Facility Review Committee of the Animal Research Center, University of Tokyo.

[0280] 2. Transplantation of hiPSC-HB into a mouse model of liver failure In mice prior to transplantation, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using DRI-CHEM (Fujifilm, Tokyo, Japan) according to the manufacturer's protocol. All mice were anesthetized with isoflurane (Wako, 095-06573) at this time.

[0281] 1×10 6 HiPSC-HB cells were transplanted into TK-NOG mice via intrasplenic injection. Serum was collected every 4 weeks post-transplantation to measure human ALB levels. Mice were sacrificed at the time points required for tissue analysis.

[0282] 3. Measurement of ALB levels in humans Human ALB was measured using a human albumin ELISA kit (Bethyl Laboratories, E80-129).

[0283] 4. Evaluation of cell maturation properties after transplantation Mouse livers were recovered 40 weeks after hiPSC-HB transplantation for qPCR analysis. In addition to the primers used in Example 1, primers binding to FAH, CYP2C9, and CYP2C19 were prepared and used according to standard methods for the qPCR analysis. The fold change was calculated as a relative value to the expression level in hiPSC-HB.

[0284] 5. Statistical Analysis Statistical analysis was performed as described in Example 1. Tests related to the qPCR analysis in this example were performed using Dunnett's multiple comparisons test (two-sided test).

[0285] (result) Human ALB production increases over time from 8 to 40 weeks after hiPSC-HB transplantation. Figure 12-1 This result indicates that hiPSC-HB can survive long-term in the liver of the recipient and has the ability to reproliferate.

[0286] Furthermore, to investigate the maturation of hiPSC-HB in transplanted tissue at 40 weeks post-transplantation, qPCR analysis using human-specific primers was performed. The results showed that in reconstructed liver tissue composed of transplanted human cells (“hiPSC-HBengraftment”), the expression of immature human liver genes (AFP and KRT19) was significantly reduced, while the expression of human liver function genes such as ALB, ornithine carbamoyltransferase (OTC), fumarate acetoacetate hydrolase (FAH), CYP3A4, CYP2C9, and CYP2C19 was significantly increased. Their expression levels were comparable to those in primary human hepatocytes (PHH). Figure 12-2 ).

[0287] Furthermore, immunostaining and gene expression analysis were used to investigate whether the transplanted hiPSC-HB integrated into the mouse liver. The results confirmed that the hiPSC-HB grafts integrated into the mouse liver (data not shown).

[0288] The above results demonstrate that transplantation of hiPSC-HB into mice with liver failure resulted in the re-proliferation of transplanted cells within the liver, leading to maturation into adult hepatocytes. This suggests the potential therapeutic benefit of hiPSC-HB for human liver failure.

[0289] <Example 6. Increased Liver Repair Capacity of hiPSC-HB Through Human OSM Exposure> (background) In Example 5, when hiPSC-HB was transplanted into a mouse model of liver injury, the secretion of human ALB decreased from week 1 to week 8 after transplantation, and the replacement rate of human tissue remained at approximately 1% at week 40. These results indicate that further improvements in the tissue-building capacity of transplanted hiPSC-HB are needed for the treatment of human liver disease.

[0290] OSM (oncogenes M) is known to play a crucial role in promoting hepatocyte proliferation and maturation during fetal liver development, and its role in promoting hepatoblast proliferation and maturation has also been reported. To evaluate whether OSM treatment in mice and humans has different effects on hiPSC-HB, the inventors exposed hiPSC-HB to human and mouse OSM, respectively, and analyzed gene expression profiles. The results showed that human OSM treatment significantly increased the expression of liver function genes (ALB, CPS1, CYP3A4, and G6PC), and enhanced ALB production. On the other hand, no similar promoting effect was observed in the group exposed to mouse OSM. Furthermore, phosphorylation of STAT3, a downstream signal of the human OSM receptor expressed in hiPSC-HB, was specifically activated only by human OSM. Transcriptome analysis showed that hiPSC-HB treated with human OSM exhibited abundant expression of features associated with the farnesoid X receptor pathway, nuclear receptor metabolic pathway, and pregnane X receptor pathway, which play important roles in hepatocyte function (data not shown). These results indicate that differentiation of hiPSC-HB requires stimulation by human OSM.

[0291] Based on the above results, it is clear that mice need to express human OSM to accurately evaluate the treatment of liver injury via hiPSC-HB transplantation in mouse models. Therefore, the following method was designed: mice were exogenously supplemented with human OSM and subjected to an environment similar to that of hiPSC-HB transplantation into a human liver to evaluate the treatment of liver function injury in vivo. Furthermore, it was considered that stimulating hiPSC-HB with human OSM prior to transplantation might further improve its survival rate. Therefore, the following study was conducted.

[0292] (Methods and Results) 1. Based on TK-NOG hOSM Preparation of a mouse model of lethal liver failure To produce TK-NOG that expresses human OSM in tissues such as the liver. hOSM Mice, 11 days prior to transplantation (D-11), were given 1×10 9 AAV8-hOSM (Vectorbuilder) was administered via the tail vein of TK-NOG mice at a titer of GC / mL. This confirmed that the mice produced human OSM at levels close to the physiological concentrations of human OSM in serum (data not shown). To establish a liver failure model, TK-NOG mice were subjected to... hOSMMice were administered retrorsine (Sigma, R382) intraperitoneally twice at a dose of 70 mg / kg at 42 days prior to transplantation (D-42) and 28 days prior to transplantation (D-28). Retrorsine is a pyrrolizidine alkaloid known to inhibit endogenous hepatocyte regeneration. The treatment was based on TK-NOG. hOSM A lethal liver failure model in mice ( Figure 13 A). Histological analysis was performed using the same method as in Example 5. The results showed that the livers of mice given senna showed enlarged hepatocytes, and Hnf4a was observed 2 weeks after GCV administration. + Ki67 + The cell population has decreased.

[0293] 2. HiPSC-HB Human OSM Pretreatment hiPSC-HB at 1×10 4 cells / cm 2 The cells were seeded at a density in new culture dishes, and human OSM was added at a concentration of 10 ng / mL to the hepatocyte maintenance medium (YAW+EGF+DEX) containing WNT3A, EGF and DEX prepared in Example 2, and cultured in the medium for more than 7 days.

[0294] 3. The therapeutic effect of hiPSC-HB transplantation on liver disease like Figure 13 As shown in B, in the cases of transplantation of hiPSC-DE (n=3) and transplantation of hepatocytes differentiated from hiPSC-HB (hiPSC-HB-Hep) (n=3), the production level of human ALB from transplanted cells did not change or decreased compared with the level at transplantation. However, in the case of transplantation of hiPSC-HB into mice (n=7), the production level of human ALB increased after transplantation and remained at a high level. Figure 13 B). This indicates that hiPSC-HB can survive long-term in the liver of mice expressing human OSM, and can reproliferate and mature in vivo. On the other hand, it shows that even when either cells that differentiate earlier than hiPSC-HB (hiPSC-DE) or cells that have further differentiated (hiPSC-HB-Hep) are transplanted, the liver function improvement effect obtained by hiPSC-HB transplantation is not obtained.

[0295] Of the mice whose liver failure was induced, 12 out of 13 died within two months. Figure 14In A, “Sham”, in addition to significant weight loss and liver weight reduction, elevated serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-Bil), and direct bilirubin (D-Bil) levels were also observed. Figure 14 B, “Sham”, observed disruption of liver tissue structure. On the other hand, in the case of hiPSC-HB transplantation, TK-NOG was significantly improved, similar to what was observed in mice transplanted with primary hepatocytes (PHH). hOSM Survival rate of liver failure model ( Figure 14 A) Body weight and liver weight significantly recovered, and serum levels of ALT, AST, T-Bil, and D-Bil significantly decreased. Figure 14 B). The human cell clusters derived from hiPSC-HB expanded over time. The proportion of human cell clusters in the recipient's liver was 3.49% on average at week 6 post-transplantation, but exceeded 10% on average at week 8 post-transplantation, and reached 27.13% on average at week 14 post-transplantation, showing extremely high survival rate.

[0296] The appearance of liver tissue composed of human cells constructed in mice by reproliferation and maturation of hiPSC-HB was observed using transmission electron microscopy. Figure 15 As a result, bile capillaries (BC: bile canaliculi) form between the apical membranes of human hepatocytes. Figure 15 A), observed hepatic sinusoids surrounded by the basement membrane of human hepatocytes (A). Figure 15 B), the Disse space between the hepatic sinusoids and hepatocytes ( Figure 15 C, Dissespace) and tight junctions between hepatocytes (Tight junction) Figure 15 D). This indicates that the transplantation of hiPSC-HB resulted in the formation of complex liver tissue with ultrafine structures visible in normal adult liver tissue.

[0297] Mice transplanted with hiPSC-HB were monitored for more than 40 weeks, and no tumor formation was observed during this period.

[0298] The above demonstrates that transplanting hiPSC-HB cells differentiated or proliferated using the method of this invention can achieve the same level of liver function improvement as transplantation of primary hepatocytes obtained from other individuals. Since hiPSC-HB cells can be generated from autologous iPS cells, this indicates that hiPSC-HB is extremely useful as a liver transplant cell that does not pose a risk of rejection, can survive long-term, and can reconstruct highly tissue-rich liver tissue.

[0299] 4. The effects of human OSM The results are shown in Figure 16.

[0300] To further investigate whether human OSM enhances the reproliferative capacity of hiPSC-HB, human OSM was exposed to hiPSC-HB through three methods: in vitro, in vivo, or a combination of in vitro and in vivo methods. Figure 16-1 In vitro, human OSM was added to the proliferation medium of hiPSC-HB. In vivo, TK-NOG constructed in Example 6 was used. hOSM .

[0301] It is worth noting that in the groups exposed to human OSM in vitro and in vivo, human ALB production in hiPSC-HB transplanted mice reached its maximum, increasing approximately 100-fold over 20 weeks post-transplantation. Figure 16-2 Furthermore, even with transplantation of hiPSC-HB from different passages (P2 and P10), there was no difference in efficacy (data not shown). Significant improvement in liver failure status and hALB was also observed with transplantation of hiPSC-HB from two other hiPSC strains. + hGAPDH + The cell viability rates reached 29.56% and 33.98%, respectively. These results strongly demonstrate that hiPSC-HB transplantation effectively reconstructs damaged livers and rescues mice from liver failure.

[0302] Consistent with observations in human livers, rifampicin-induced hCYP3A4 expression and regeneration of human hepatocytes associated with 70% hepatectomy were detected in living hiPSC-HB-derived cells. Furthermore, to confirm the liver function of the reproliferating hepatocytes in vivo, mice with liver failure who had not received hiPSC-HB were intraperitoneally injected with a lethal dose of 350 mg / kg of acetaminophen. Surprisingly, 4 out of 5 mice with reproliferated hiPSC-HB survived, and their liver function significantly recovered. This indicates that reproliferated hiPSC-HB in the mouse liver plays a protective role against acetaminophen-induced acute liver failure. Moreover, transmission electron microscopy revealed ultrastructural features of adult liver tissue in reproliferated hiPSC-HB, including bile ducts formed between the apical membranes of human hepatocytes, hepatic sinusoids surrounded by the basement membrane of human hepatocytes, the Disse lumen between the sinusoids and hepatocytes, and tight junctions between hepatocytes.

[0303] The above results indicate that by transplanting hiPSC-HB into individuals with liver injury, it can mature into human hepatocytes with the structural characteristics of liver failure and functional efficacy. It is expected that cell preparations containing hiPSC-HB may have a therapeutic effect on liver failure.

[0304] <Example 7. Changes in the properties of hiPSC-HB caused by human OSM exposure> Example 6 demonstrates that by culturing hiPSC-HB in a proliferation medium supplemented with human OSM for more than 7 days, cells exhibiting the same level of liver function improvement as transplanted primary hepatocytes obtained from other individuals can be prepared. To investigate the distribution of hiPSC-HB cells cultured in medium supplemented with human OSM for more than 7 days, cells were recovered on day 7 of culture in each differentiation induction medium and subjected to RNA-seq analysis. The fold change was calculated as a relative value to the expression level of hiPSC-HB cultured in the proliferation medium. The results showed that, by culturing in a medium supplemented with human OSM, in addition to the expression of typical hepatoblast markers (AFP, DLK, ALB, TBX3, C-MET, CER1, EpCAM, etc.) and genes such as SLC4A4, TNFSF4, IL1RAPL1, NRP1, SOX9, ONECUT1, PLEKHA2, RNF207, NID1, and LYST found in the hiPSC-HB of this invention, the expression of newly discovered genes listed in Table 4, such as REG3A, SAA1, SAA2, HP, SPINK1, LBP, KCNG2, ANXA10, and ITIH3, was significantly increased.

[0305] [Table 4] Table 4: Genes whose expression is increased by human OSM exposure

[0306] All publications, patents and patent applications referenced in this specification are incorporated herein by reference in their entirety.

Claims

1. A method for the proliferation of hepatoblasts, characterized in that, include: The proliferation culture process involves proliferating and culturing hepatocytes in a culture medium containing ROCK inhibitors, TGFβ signaling pathway inhibitors, Wnt signaling pathway activators, and growth factors.

2. A method for manufacturing a population of hepatocytes, characterized in that, include: The proliferation culture process involves proliferating and culturing hepatocytes in a culture medium containing ROCK inhibitors, TGFβ signaling pathway inhibitors, Wnt signaling pathway activators, and growth factors.

3. The method according to claim 1 or 2, characterized in that, The growth factor is one or more proteins selected from the group consisting of FGF2, EGF, IGF, VEGF and PDGF.

4. The method according to any one of claims 1 to 3, characterized in that, ROCK inhibitors include Y-27632, Rho Kinase Inhibitor IV, or thiazolinone.

5. The method according to any one of claims 1 to 4, characterized in that, TGFβ signaling pathway inhibitors are selected from one or more inhibitors in the group consisting of A8301, SB431542, SB505124, LY364947, LY2157299 and RepSox.

6. The method according to any one of claims 1 to 5, characterized in that, The Wnt signal transduction pathway activator is one or more substances selected from the group consisting of WNT3A, CHIR99021, SB216763, BIO, TWS119, SB216763, and Recombinant R-Spondin 1.

7. The method according to any one of claims 1 to 6, characterized in that, The culture medium for hepatoblast proliferation also contains steroids.

8. The method according to claim 7, characterized in that, Steroids are glucocorticoids or their derivatives.

9. The method according to claim 8, characterized in that, Glucocorticoids or their derivatives include dexamethasone.

10. The method according to any one of claims 1 to 9, characterized in that, Hepatoblasts express IL1RAPL1, NRP1, SOX9, ONECUT1, NID1, and LYST.

11. The method according to claim 10, characterized in that, Hepatoblasts further express one or more genes selected from the group consisting of SLC4A4, TNFSF4, PLEKHA2, and RNF207.

12. A method for manufacturing a cell preparation for treating liver disease, characterized in that, Includes the method according to any one of claims 1 to 11.

13. A method for manufacturing a cell preparation for treating liver disease, characterized in that, include: The first step is to induce pluripotent stem cells to differentiate into defined endoderm cells. The second step is to induce the differentiation of the shaped endoderm cells into hepatocytes. as well as The third step involves proliferating and culturing hepatoblasts to create a hepatoblast population. The first step includes the following steps: culturing in a serum-free medium containing Y-27632 during the early stage of culture; and culturing in a medium containing B-27 supplement, activin A, and WNT signaling pathway activator after the middle stage of culture. The second step includes the following steps: culturing in a serum-free medium containing FGF2, HGF, oncogene M, dexamethasone, and nicotinamide. The third step includes a proliferation culture process, in which hepatocytes are proliferated and cultured in a hepatocyte proliferation culture medium containing ROCK inhibitors, TGFβ signaling pathway inhibitors, Wnt signaling pathway activators and growth factors.

14. The method for manufacturing the cell preparation according to claim 13, characterized in that, Following the third step, a maturation promotion process is also included, in which oncogene M is added to the proliferated and cultured hepatoblasts and cultured.

15. A treatment method for liver disease, characterized in that, The process includes the following steps: The cell preparation, which contains hepatocytes proliferated and cultured in a hepatocyte proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and growth factors, is administered to the target population.

16. The treatment method according to claim 15, characterized in that, The cell preparation comprises further addition of oncogene M to proliferating and cultured hepatoblasts to promote cell maturation.

17. A method for manufacturing hepatocytes, characterized in that, include: The proliferation culture step involves proliferating and culturing hepatocytes in a hepatocyte proliferation medium containing ROCK inhibitors, TGFβ signaling pathway inhibitors, Wnt signaling pathway activators, and growth factors; and the hepatocyte induction step involves inducing differentiation from the proliferated and cultured hepatocytes into hepatocytes.

18. A method for manufacturing bile duct cells, characterized in that, include: The proliferation culture step involves proliferating and culturing hepatocytes in a hepatocyte proliferation medium containing ROCK inhibitors, TGFβ signaling pathway inhibitors, Wnt signaling pathway activators, and growth factors; and the bile duct cell induction step involves inducing differentiation from proliferated and cultured hepatocytes into bile duct cells.

19. A cell preparation for treating liver disease, characterized in that, It contains cells expressing IL1RAPL1, NRP1, SOX9, ONECUT1, NID1, and LYST.

20. The cell preparation according to claim 19, characterized in that, Cells are cells that further express one or more genes selected from the group consisting of SLC4A4, TNFSF4, PLEKHA2, RNF207, REG3A, SAA1, SAA2, HP, SPINK1, LBP, KCNG2, ANXA10, ITIH3, ITIH4, MTUS1, CRP, CEACAM5, C4B, ATP10B, NNMT, LCN2, LOC145837, ORM1, ORM2, PC, SERPINA7, SOCS3, C4A, SERPINA3, C8A, CD38, TMEM45B, CDA, and UGT2B4.

21. A method for the proliferation of hepatocytes, characterized in that, include: The proliferation culture process involves proliferating and culturing hepatocytes in a culture medium containing ROCK inhibitors, TGFβ signaling pathway inhibitors or Wnt signaling pathway activators, and growth factors.

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