Method for proliferating hepatoblasts, and method for treating liver disease using hepatoblasts

Abstract

The present invention addresses the problem of providing: a method for preparing hepatic lineage cells in a large amount and with high purity; and a method for preparing liver system cells which can achieve high graft survival rate in a living body.　The present invention provides: a hepatoblast proliferation promoter and a culture medium, each of which contains a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor; and a proliferation / culture method using the hepatoblast proliferation promoter or the culture medium.

Description

Method for proliferation of hepatoblasts and method for treating liver disease using hepatoblasts

[0001] The present invention relates to a method for proliferating hepatoblasts, a method for producing a hepatoblast population, a pharmaceutical composition containing hepatoblasts for use in treating liver diseases, a method for treating liver diseases using hepatoblasts, and the like.

[0002] The liver is a highly regenerative organ that can recover from injury. This is due to the plasticity of hepatocytes, which account for approximately 70% of liver tissue. However, if the liver is damaged excessively to the point that its regenerative capacity is exceeded, or if the liver is damaged under conditions in which the regenerative capacity itself is reduced or impaired due to aging or other factors, liver function will be significantly impaired, leading to liver failure. This liver failure is a serious disorder with a high mortality rate.

[0003] Liver transplantation is one of the treatments for liver failure. While liver transplantation has long been recognized as an effective treatment that can cure liver failure, a shortage of transplantable donor livers has made it difficult for patients to receive treatment when needed. Cell replacement therapy, a more convenient treatment method that does not require extensive surgery, has been explored. However, because cell replacement therapy involves administering donor liver-derived hepatocytes, including hepatocytes and fetal hepatocytes / hepatoblasts, it also faces the problem of being dependent on the number of donor livers. Furthermore, depending on the donor-recipient combination, these methods can result in low engraftment rates one year after transplantation, limiting the long-term therapeutic effect, even when the recipient's immune system is suppressed to reduce rejection.

[0004] In this context, cell replacement therapy and liver transplantation using human pluripotent stem cells are seen as promising methods for enabling treatment independent of the number of donor livers. In particular, autologous iPS cells and genome-edited pluripotent stem cells are expected to have low immunogenicity and are less likely to cause rejection in recipients. Based on these advantages, various studies have been conducted to date to generate hepatocytes and liver-like organoids using human pluripotent stem cells. However, when these cells and organoids are applied in vivo, their in vivo repopulation capacity has been limited, and it has been shown that the transplanted cells account for less than 1% of the recipient's liver. Generally, for transplanted cells to achieve therapeutic effects in patients with liver failure, transplanted cells must account for more than 10% of the hepatocytes in the recipient's liver (Non-Patent Document 1).

[0005] To date, a method for inducing differentiation of hepatoblasts, the precursor cells of hepatocytes, from pluripotent stem cells has been reported (Patent Document 1). Hepatoblasts are highly proliferative liver progenitor cells present in fetal livers and have been shown to be a potential cell source for cell replacement therapy in liver disease. Mouse allotransplantation experiments have confirmed that fetal liver-derived hepatoblasts have the ability to repopulate livers with liver damage. However, compared with transplantation of hepatocytes derived from mature livers, the proportion of transplanted hepatoblasts in the recipient liver was significantly lower (Non-Patent Document 2). Furthermore, hepatoblasts are a cell type normally found only in fetuses, making it difficult to secure sufficient numbers of cells for cell transplantation. Therefore, cell replacement therapy using hepatoblasts has not yet been put to practical use.

[0006] For these reasons, cell replacement therapy and liver transplantation using hepatocytes derived from pluripotent stem cells have many advantages but have not yet reached practical use, and there has been a need to establish a method for preparing hepatic lineage cells that achieves a high engraftment rate in the body.

[0007] WO2021 / 060380

[0008] Puppi, J., et al., Cell Transplant 21, 1-10 (2012)Dhivya H. et al., The American Journal of Pathology 175(4), 1483-1492 (2009)

[0009] The present inventors sought to establish a cell transplantation method that achieves a high in vivo engraftment rate. To test the usefulness of pluripotent stem cell-derived hepatoblasts in cell transplantation, it was necessary to prepare highly pure hepatoblasts in large quantities. However, with conventional culture methods, hepatoblasts differentiate into hepatocytes or bile duct cells over the course of the culture period, making it difficult to prepare highly pure and large quantities of hepatoblasts while maintaining their undifferentiated state. Therefore, the present invention aims to provide a method for preparing highly pure and large quantities of hepatoblasts, and further, to provide a method for preparing liver lineage cells that achieves a high in vivo engraftment rate.

[0010] As a result of extensive research, the present inventors have found that hepatoblasts can be stably proliferated in an undifferentiated state for 15 or more passages by culturing them in a medium with a specific composition. Furthermore, they have found that transplanting the hepatoblasts thus prepared into the liver of a mouse model of liver failure stably engrafts in vivo, complements liver tissue, and restores liver function.

[0011] That is, the present invention is based on these novel findings and provides the following [1] to

[38] . [1] A method for expanding hepatoblasts, comprising a step of expanding and culturing hepatoblasts in a hepatoblast proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor. [2] A method for producing a hepatoblast cell population, comprising a step of expanding and culturing hepatoblasts in a hepatoblast proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor. [3] The method described in [1] or [2], wherein the growth factor is one or more proteins selected from the group consisting of FGF2, EGF, IGF, VEGF, and PDGF. [4] The method described in any of [1] to [3], wherein the ROCK inhibitor is Y-27632, Rho Kinase Inhibitor IV, or thiazovivin. [5] The method described in [4], wherein the ROCK inhibitor is Y-27632. [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. [7] The method according to [6], wherein the TGFβ signaling pathway inhibitor is A8301. [8] The method according to any one of [1] to [3], wherein the Wnt signaling pathway activator is one or more substances selected from the group consisting of WNT3A, CHIR99021, SB216763, BIO, TWS119, SB216763, and Recombinant R-Spondin 1. [9] The method according to [8], wherein the Wnt signaling pathway activator is WNT3A.

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

[11] The method according to

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

[12] The method according to

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

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

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

[14] The method according to

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

[15] A method for producing a cell preparation for use in the treatment of liver disease, the method comprising the method according to any one of [1] to

[14] .

[16] A method for producing a cell preparation for use in the treatment of liver disease, comprising: a first step of inducing the differentiation of pluripotent stem cells into definitive endoderm cells; a second step of inducing the differentiation of the definitive endoderm cells into hepatoblasts; and a third step of expanding and culturing the hepatoblasts to produce a hepatoblast cell population, wherein the first step comprises culturing the hepatoblasts in a serum-free culture medium containing Y-27632 in the early culture phase and culturing them in a medium containing B-27 supplement, activin A, and a WNT signaling pathway activator in the intermediate culture phase or later, the second step comprises culturing the hepatoblasts in a serum-free medium containing FGF2, HGF, oncostatin M, dexamethasone, and nicotinamide, and the third step comprises expanding and culturing the hepatoblasts in a hepatoblast growth medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor. [16-1] The method for producing the cell preparation described in

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

[17] A method for producing the cell preparation described in

[16] or [16-1], further comprising, after the third step, a maturation promotion step of adding oncostatin M to the hepatoblasts that have been cultured for expansion and culturing.

[18] A method for treating liver disease, comprising a step of administering to a subject a cell preparation containing hepatoblasts that have been cultured for expansion in a hepatoblast growth medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor.

[19] The method for treating liver disease described in

[18] , wherein the cell preparation contains hepatoblasts that have been cultured for expansion and whose maturation has been promoted by further adding oncostatin M.

[20] A method for producing hepatocytes, comprising: a growth and culture step of growing and culturing hepatoblasts in a hepatoblast growth medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor; and a hepatocyte induction step of inducing differentiation of the cultured hepatoblasts into hepatocytes.

[21] A method for producing cholangiocytes, comprising: a growth and culture step of growing and culturing hepatoblasts in a hepatoblast growth medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor; and a cholangiocyte induction step of inducing differentiation of the cultured hepatoblasts into cholangiocytes.

[22] A cell preparation for use in the treatment of liver disease, comprising cells expressing IL1RAPL1, NRP1, SOX9, ONECUT1, NID1, and LYST.

[23] The cell preparation of

[22] , wherein the cells 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.

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

[25] The treatment method described in

[24] , wherein the cells 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.

[26] A cell for use in a method for treating liver disease, characterized in that the cell expresses IL1RAPL1, NRP1, SOX9, ONECUT1, NID1, and LYST.

[27] The cell according to

[26] , further expressing 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.

[28] A method for proliferating hepatoblasts, comprising a step of proliferating and culturing hepatoblasts in a hepatoblast proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor or a Wnt signaling pathway activator, and a growth factor.

[29] The method according to

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

[30] The method according to

[28] or

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

[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.

[32] The method according to any one of

[28] to

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

[33] The method according to any one of

[28] to

[32] , wherein the hepatoblast proliferation medium further contains a glucocorticoid or a derivative thereof.

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

[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.

[36] The hepatoblast proliferation promoter according to

[34] or

[35] , further comprising a glucocorticoid or a derivative thereof.

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

[38] A culture medium for hepatoblast proliferation, comprising the hepatoblast proliferation promoter according to any of

[34] to

[36] .

[0012] This specification includes the disclosures of Japanese Patent Application Nos. 2024-059423 and 2025-013968, from which the present application claims priority.

[0013] According to the method for expanding hepatoblasts of the present invention, hepatoblasts can be expanded and cultured while maintaining an undifferentiated state. Furthermore, according to the method for producing a hepatoblast population of the present invention, hepatoblasts can be expanded and cultured while maintaining an undifferentiated state, thereby producing a cell population of hepatoblasts. Furthermore, the method for expanding hepatoblasts of the present invention provides a cell preparation containing hepatoblasts for use in treating liver disease, a method for treating liver disease using hepatoblasts, etc.

[0014] The present invention also provides a hepatoblast proliferation promoter, a hepatoblast proliferation promotion kit, and a hepatoblast proliferation medium, which are used for proliferating and culturing hepatoblasts while maintaining the hepatoblasts in an undifferentiated state.

[0015] FIG. 1 is a schematic diagram illustrating the process of hepatoblast differentiation and hepatoblast-mediated liver tissue regeneration according to the present invention. FIG. 1A shows the differentiation of human iPS cells into definitive endoderm cells, followed by hepatoblasts and hepatocytes. FIG. 1B shows the regeneration of liver tissue by transplantation of expanded hepatoblasts. FIG. 2 shows the results of qPCR analysis in Example 1. In the figure, "hiPSC" indicates the results for human iPS cells, "hiPSC-DE" indicates the results for definitive endoderm cells (Definitive Endoderm) induced from human iPS cells, and "hiPSC-HB" indicates the results for hepatoblasts induced from human iPS cells. In the figure, "Fold changes" indicates the fold change calculated as a relative value to the expression level obtained from hiPSCs, and the horizontal dashed line indicates the level where the fold change is 1. In the figure, the circles on each bar graph represent the respective data points. FIG. 3 shows the results of transcriptome analysis in Example 1. Each box indicates the expression level of each gene (row) in each cell (column), with darker colors generally indicating higher expression levels. Each row label indicates the name of the signature gene for the hepatoblast of interest, and each column label indicates the cell type of interest. In the figure, "hiPSC-HE" indicates the results for hepatic endoderm induced from human iPS cells, "hiPSC-Hep" indicates the results for hepatocytes induced from human iPS cells, and "PHH" indicates the results for primary hepatocytes. Figure 4 shows the flow cytometry results from Example 1. Figure 4A shows an exemplary graph plotting the expression levels of CD13 and EpCAM in hepatoblasts induced with WNT3A or CHIR99021. In Figure 4A, each plot shows data for a single cell. Data surrounded by a solid box indicates data for cells with high EpCAM expression (EpCAM-high), and data surrounded by a dashed box indicates data for cells with low EpCAM expression (EpCAM-low). In Figure 4A, the data shown to the lower left of the solid and dashed squares are those obtained when an isotype control antibody was used. Figure 4B shows the proportion of EpCAM-low cells among CD13-positive cells, and Figure 4C shows the proportion of EpCAM-high cells among CD13-positive cells.In Figures 4B and 4C, each circle represents data from one experiment, and error bars indicate standard deviation. Figure 5 shows the results of flow cytometry in Example 1. Figure 5A shows the same diagram as the left side of Figure 4A. Figure 5B shows the results of comparing the distribution of CD90 expression levels in EpCAM-low cells with that in the case of using an isotype control antibody, and Figure 5C shows the results in EpCAM-high cells. Figure 6-1 shows the results of qPCR analysis at the end of the first passage in Example 2. In the figures, "YAC" indicates the candidate expansion medium containing Y-27632, A8301, and CHIR99021, and "YAW" indicates the candidate expansion medium containing Y-27632, A8301, and WNT3A. "Fold changes" indicates the fold change calculated as a relative value to the expression level obtained in hepatoblasts induced from hiPSCs (hiPSC-HB). In the figures, the name of each series indicates the candidate maintenance medium used. In the figure, circles on each bar graph represent data points, and error bars indicate standard deviations. Figure 6-2 shows the results of cell proliferation rate at the end of the first passage and the results of qPCR analysis in Example 2. In the figure, "Fold changes" indicates the fold change calculated as a relative value to the expression level in hepatoblasts cultured using a growth medium containing Y27632, A8301, WNT3A, EGF, and DEX, and the horizontal dashed line indicates the level where the fold change is 1. In the figure, the name of each series indicates the candidate maintenance medium used. In the figure, the error bars indicate standard deviations. Figure 6-3 shows the results of qPCR analysis at the end of the first passage in Example 2. In the figure, "Related Expression" indicates the fold change calculated as a relative value to the expression level in hepatoblasts cultured using a growth medium containing Y27632, A8301, WNT3A, EGF, and DEX. In the figure, the name of each series indicates the candidate maintenance medium used. In the figure, "WNT3A-50" indicates the results when WNT3A was used at a concentration of 50 ng / mL, and "CHIR-0.5," "CHIR-1," "CHIR-2," and "CHIR-3" indicate the results when CHIR99021 was used at concentrations of 0.5 μM, 1 μM, 2 μM, and 3 μM, respectively. Error bars indicate standard deviation.Figure 6-4 shows the results of examining the proliferation potential of hiPSC-HBs at passage 6 using various ROCK inhibitors. In the figure, "Proliferation rate" indicates the proliferation rate when the number of cells on Day 1 is set to 1, and the horizontal axis indicates the name and concentration of the added substance. "Rho" indicates the results when Rho Kinase Inhibitor IV was used. Error bars indicate standard deviation. Figure 6-5 shows the results of examining the proliferation potential of hiPSC-HBs at passage 6 using various TGFβ / Smad inhibitors. In the figure, "Proliferation rate" indicates the proliferation rate when the number of cells on Day 1 is set to 1, and the horizontal axis indicates the name and concentration of the added substance. Error bars indicate standard deviation. Figure 6-6 shows the results of examining the proliferation potential of hiPSC-HBs at passage 6 using various WNT3 signaling activators. In the figure, "Proliferation rate" indicates the proliferation rate when the number of cells on Day 1 is set to 1, and the horizontal axis indicates the name and concentration of the added substance. In the figure, "R-Spondin" indicates the results when recombinant R-Spondin 1 was used. Error bars indicate standard deviation. Figure 7 shows the relationship between passage number and cumulative cell proliferation rate in Example 2. The label for each data series indicates the name of each iPS cell line used to induce hepatoblast differentiation. In the figure, numbers accompanied by "#" indicate different lots, and "Cryopreservation" indicates that cells awakened from cryopreservation were used. Figure 8 shows the relationship between passage number and EpCAM expression level in Example 3. Results are shown after one passage (A), five passages (B), ten passages (C), and fifteen passages (D), respectively. In the figure, the percentage shown below the solid-line square indicates the proportion of EpCAM-high cells among CD13-positive cells. Figure 9 shows the results of transcriptome analysis in Example 3. Each square indicates the expression level of each gene (row) in each cell (column), with darker colors generally indicating higher expression levels. Each row label indicates the name of the signature gene of the hepatoblast of interest, and each column label indicates the passage number of the cell of interest.In the figure, "hiPSC" indicates the results for human iPS cells, "DE" indicates the results for definitive endoderm cells induced from human iPS cells, "HE" indicates the results for hepatic endoderm cells induced from human iPS cells, "HB P0" indicates the results for hepatoblasts before passage, "HB P1" indicates the results after one passage, "HB P5" indicates the results after five passages, "HB P10" indicates the results after ten passages, and "HB P15" indicates the results after 15 passages, and "MH" indicates the results for mature hepatocytes. Figure 10 shows the results of principal component analysis in Example 3. In the figure, the ratio between the vertical and horizontal axes indicates the contribution rate of each component. Figure 11 shows the results of qPCR analysis in Example 4. In the figure, "Fold changes" indicates the fold change calculated as a relative value to the expression level obtained in hepatoblasts induced from hiPSCs, and the horizontal dashed line indicates a fold change level of 1. In the figure, "HB" indicates the results for hepatoblasts induced from hiPSCs, "HB-Hep" indicates the results for hepatocytes differentiated from HBs, and "HB-Cholangiocyte" indicates the results for cholangiocytes differentiated from HBs. In the figure, the circles on each bar graph represent data points, and the error bars represent standard deviations. Figure 12-1 shows the results of measuring human ALB serum levels after transplantation of hepatoblasts (hiPSC-HBs) into TK-NOG mice in Example 5. In the figure, each circle on the graph represents a data point, and the error bars represent standard deviations. Figure 12-2 shows the results of qPCR analysis in Example 5. In the figure, "Fold changes" indicates the fold change calculated as a relative value to the expression level obtained in hepatoblasts induced from hiPSCs. In the figure, "hiPSC-HB" indicates the results for hepatoblasts induced from hiPSCs, "hiPSC-HB engraftment" indicates the results for liver tissue composed of human cells reconstituted after hiPSC-HB transplantation, and "PHH" indicates the results for primary human hepatocytes. In the figure, the circle on each bar graph represents each data point, and the error bar represents the standard deviation. Figure 13 shows an outline of transplantation of hepatoblasts (hiPSC-HB) into mice in Example 6 (A) and the measurement results of serum levels of human ALB (B). In Figure 13A, "TK-NOG. hOSM" indicates TK-NOG mice transduced with AAV8-hOSM, and "GCV" indicates the timing of ganciclovir administration. In Figure 13B, "hiPSC-HB-Hep" shows the results for hepatocytes differentiated from hepatoblasts induced from hiPSCs. Figure 14 shows the survival curve (A) for mice transplanted with hepatoblasts in Example 6, and the measurement results of serum levels of ALT, AST, total bilirubin (T-Bil), and direct bilirubin (D-Bil) (B). In the figure, "Sham" indicates mice with liver failure not transplanted with hepatoblasts, "hiPSC-HB" indicates mice with liver failure transplanted with hepatoblasts, "PHH" indicates mice with liver failure transplanted with primary hepatocytes, and "Normal" indicates mice without liver failure induction. In the figure, circles on each bar graph indicate data points, and error bars indicate standard deviation. Figure 15 shows the results of transmission electron microscopy observation in Example 6. In the figure, "BC" indicates bile canaliculi, "ER" indicates endoplasmic reticulum, and "M" indicates mitochondria. The scale bars in each figure indicate 10 μm (A), 2 μm (B), and 1 μm (C and D), respectively. Figure 16-1 shows an outline of the in vitro and / or in vivo human OSM (hOSM) stimulation treatments for hepatoblast transplantation performed in Example 6. In the figure, "TK-NOG hOSM " indicates TK-NOG mice transfected with AAV8-hOSM, "hOSM" indicates in vitro administration of OSM, and "AAV8 hOSM" indicates in vivo production of hOSM using AAV8. Figure 16-2 shows the results of measuring human ALB serum levels in Example 6. Each series shows the results for each hOSM-stimulated treatment group in Figure 16-1. In the figure, error bars indicate standard deviation.

[0016] <Definitions> Terms used in this specification are defined below.

[0017] "Pluripotent stem cells" refer to cells that have the multipotency (pluripotency) to differentiate into all types of cells that make up a living organism. Typically, pluripotent stem cells can continue to proliferate indefinitely while maintaining pluripotency when cultured in vitro under appropriate conditions. Here, "pluripotency" refers to the ability to differentiate into cells of all types of germ layers that make up an individual (in vertebrates, the three germ layers are ectoderm, mesoderm, and endoderm). Pluripotent stem cells as used herein include both naive and primed types.

[0018] "iPS cells" are cells created by a culture method that includes introducing pluripotency-inducing factors into somatic cells such as human skin or blood cells and treating them with low-molecular-weight compounds. These cells have the ability to grow almost indefinitely and differentiate into cells of various tissues and organs. They are called "induced pluripotent stem cells."

[0019] Hepatic endoderm cells (HE) are precursor cells formed during early liver development and are a cell type derived from the endoderm during embryonic development. In response to specific signals (e.g., FGF and BMP), HE differentiates into hepatoblasts, which then develop into mature hepatocytes and bile duct cells.

[0020] Hepatoblasts (hereinafter sometimes referred to as "HBs") are tissue stem cells derived from the foregut endoderm during the fetal period and can differentiate into both bile duct epithelial cells and mature hepatocytes. Hepatoblasts are not typically observed in adults, but are a cell type observed only during fetal liver development. They typically express markers such as alpha-fetoprotein (AFP), delta-like Notch ligand (DLK), and sometimes albumin (ALB), T-box transcription factor 3 (TBX3), hepatocyte growth factor receptor (c-MET), and epithelial cell adhesion molecule (EpCAM), but do not express markers such as keratin 19 (KRT19), and sometimes OCT4 and CD90.

[0021] "Hepatocytes" are cells that make up approximately 60% of the liver and constitute the liver parenchyma. As used herein, hepatocytes encompass not only mature hepatocytes but also hepatic progenitor cells, as well as all differentiation stages of cells that have been committed to hepatocyte differentiation. Mature hepatocytes are terminally differentiated cells that perform various liver-specific functions, such as protein and cholesterol synthesis, metabolism of biomolecules such as proteins, carbohydrates, and lipids, and drug detoxification. They typically express markers such as ALB and, in some cases, alpha-1 antitrypsin (A1AT), carbamoyl phosphate synthase 1 (CPS1), cytochrome P450 3A4 (CYP3A4), and glucose-6-phosphatase catalytic subunit (G6PC), but do not express markers such as AFP, DLK, and, in some cases, KRT19, EpCAM, and CD90.

[0022] "Cholangiocytes" are synonymous with biliary epithelial cells and are epithelial cells that make up the bile duct. Cholangiocytes regulate the flow and properties (including ion concentration and pH) of bile secreted by hepatocytes. They typically express markers such as KRT19 and, in some cases, gamma-glutamyltransferase 1 (GGT1), aquaporin 1 (AQP1), somatostatin receptor 2 (SSTR2), and EpCAM, but do not express markers such as AFP, DLK, and, in some cases, A1AT.

[0023] "Proliferation" refers to increasing the number of cells, and "expansion culture" refers to increasing the number of cells while maintaining their original properties without differentiation. The "expansion culture step" refers to a step of expanding and culturing hepatoblasts in a hepatoblast proliferation medium, and may also include a passaging step.

[0024] The term "cell population" refers to a homogeneous group of cells obtained by expansion culture.

[0025] "Differentiation" refers to the specialization and increased specificity of cell morphology and / or function. In this specification, differentiation particularly refers to the restriction of the differentiation fate of a cell and the reduction in the number of cell types that can be generated from that cell.

[0026] The term "ROCK inhibitor" refers to an inhibitor of a signal transduction pathway mediated by ROCK (Rho-associated coiled-coil forming kinase). As used herein, ROCK inhibitors include both drugs that directly inhibit the function of ROCK and drugs that do not directly inhibit the function of ROCK but inhibit its signal transduction pathway, and any ROCK inhibitor known in the art can be used.

[0027] The term "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. The TGFβ signaling pathway inhibitor herein encompasses both inhibitors of the classical TGFβ signaling pathway mediated by Smads and inhibitors of the non-classical TGFβ signaling pathway, and any TGFβ signaling pathway inhibitor known in the art can be used.

[0028] The term "Wnt signaling pathway" refers to both the canonical Wnt signaling pathway, which inhibits the degradation and stabilization of β-catenin through binding of Wnt to Wnt receptors such as Frizzled (e.g., Fizzled, Celsr, Vangl, etc.), and other non-canonical Wnt signaling pathways. As used herein, the term "Wnt signaling pathway activator" refers to a drug that can activate either or both of the canonical Wnt signaling pathway and the non-canonical Wnt signaling pathway.

[0029] The term "growth factor" refers to a molecule that promotes cell proliferation and differentiation within an organism, and is a concept that encompasses both low-molecular-weight molecules such as vitamins and high-molecular-weight molecules such as proteins. In this specification, "growth factor" particularly refers to a protein that promotes cell proliferation and differentiation within an organism.

[0030] "Steroid drug" refers to a drug that has a steroid skeleton in its chemical structure.

[0031] The "hepatoblast induction step" refers to a step of inducing differentiation from cells from which hepatoblasts are derived (for example, pluripotent stem cells) into hepatoblasts.

[0032] The "hepatoblast induction step" refers to a step of inducing differentiation from cells from which hepatoblasts are derived (for example, pluripotent stem cells) into hepatoblasts.

[0033] 1. Expansion and Culture of Hepatoblasts and Production of a Hepatoblast Population The present invention relates to a method for expanding hepatoblasts or a method for producing a hepatoblast population. The medium used in the method for expanding hepatoblasts or the method for producing a hepatoblast population of the present invention is a basal medium supplemented with a ROCK inhibitor, a TGFβ signaling pathway inhibitor and / or a Wnt signaling pathway activator, and growth factors. Culturing in this medium can promote hepatoblast proliferation while maintaining hepatoblast properties. During the proliferation process, when cells reach a certain level of confluency, appropriate passage is performed, and expansion culture is continued to further expand the cell number. Therefore, the expansion culture of the present invention also includes a passaging step. In one embodiment, the method for expanding hepatoblasts of the present invention includes an expansion culture step in which hepatoblasts are expanded and cultured in a hepatoblast proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor. In one embodiment, the method for expanding hepatoblasts of the present invention includes an expansion culture step in which hepatoblasts are expanded and cultured in a hepatoblast proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, and a growth factor. In one embodiment, the method for expanding hepatoblasts of the present invention comprises a step of expanding and culturing hepatoblasts in a hepatoblast proliferation medium containing a ROCK inhibitor, a Wnt signaling pathway activator, and a growth factor. In one embodiment, the method for producing a hepatoblast population of the present invention comprises a step of expanding and culturing hepatoblasts in a hepatoblast proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor. In one embodiment, the method for producing a hepatoblast population of the present invention comprises a step of expanding and culturing hepatoblasts in a hepatoblast proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, and a growth factor. In one embodiment, the method for producing a hepatoblast population of the present invention comprises a step of expanding and culturing hepatoblasts in a hepatoblast proliferation medium containing a ROCK inhibitor, a Wnt signaling pathway activator, and a growth factor.

[0034] Hepatoblasts are tissue stem cells derived from the foregut endoderm present during the fetal stage and can differentiate into both bile duct epithelial cells and mature hepatocytes. They typically express markers such as alpha-fetoprotein (AFP), delta-like Notch ligand (DLK), and sometimes albumin (ALB), T-box transcription factor 3 (TBX3), hepatocyte growth factor receptor (c-MET), and epithelial cell adhesion molecule (EpCAM). They are also known not to express markers such as keratin 19 (KRT19), and sometimes OCT4 and CD90.

[0035] The origin of the hepatoblasts used in the present invention is not particularly limited, but may be, for example, hepatoblasts isolated from animal tissues, organs, or individuals, hepatoblasts induced to differentiate from pluripotent stem cells, or a mixture thereof.

[0036] When hepatoblasts are derived from pluripotent stem cells, the origin of the pluripotent stem cells is not particularly limited. For example, pluripotent stem cells may be derived from any vertebrate, including humans (e.g., mammals, primates, etc.), and the animal from which they are derived may be healthy or suffering from a disease. For example, cells derived from mammals are preferably used, and cells derived from primates such as humans are particularly preferably used. In one embodiment, the hepatoblasts used in the present invention are derived from pluripotent stem cells derived from humans or vertebrates. In one embodiment, the hepatoblasts used in the present invention are derived from pluripotent stem cells derived from humans.

[0037] When hepatoblasts are induced to differentiate from pluripotent stem cells, any pluripotent stem cells known in the art can be appropriately selected, obtained, or prepared according to the purpose, and used; the type of pluripotent stem cell is not particularly limited. Examples include induced pluripotent stem cells (iPS cells) derived from somatic cells, embryonic stem cells (ES cells) derived from early embryos, embryonic germ cells (EG cells) derived from primordial germ cells, germline stem cells (GS cells) derived from testes, Muse cells derived from mesenchymal tissue, somatic stem cells (mesenchymal stem cells and neural stem cells derived from bone marrow, adipose tissue, dental pulp, placenta, fetal membrane, umbilical cord blood, amnion, chorion, etc.), or mixtures thereof. For example, iPS cells and ES cells (e.g., iPS cells and ES cells from mammals (particularly primates such as humans)) can be preferably used. The method for preparing the pluripotent stem cells used can be appropriately selected depending on the type of stem cells used and is not particularly limited. In one embodiment, the hepatoblasts used in the method for expanding hepatoblasts and the method for producing a hepatoblast cell population of the present invention are derived from iPS cells. In one embodiment, the hepatoblasts used in the method for expanding hepatoblasts and the method for producing a hepatoblast population of the present invention are derived from human iPS cells or vertebrate iPS cells. In one embodiment, the hepatoblasts used in the method for expanding hepatoblasts and the method for producing a hepatoblast population of the present invention are derived from human iPS cells.

[0038] For example, when hepatoblasts expanded using the method for expanding hepatoblasts and the method for producing a hepatoblast population of the present invention are used to treat a specific individual, hepatoblasts with a reduced risk of transplant rejection can be used. In this case, for example, hepatoblasts induced to differentiate from iPS cells obtained from the somatic cells of the target individual, hepatoblasts derived from an individual whose major histocompatibility complex (MHC) genotype (or, in the case of a human, human leukocyte antigen (HLA) genotype) is identical or substantially identical to that of the target individual, or hepatoblasts induced to differentiate from pluripotent stem cells derived from such an individual can be used.

[0039] The method for producing a hepatoblast population of the present invention or the hepatoblasts used in the method for producing a hepatoblast population may express, in addition to the expression of the typical markers described above (AFP, DLK, ALB, TBX3, c-MET, CER1, EpCAM, etc.), 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 method for producing a hepatoblast population of the present invention or the hepatoblasts used in the method for producing a hepatoblast population are characterized by expressing SLC4A4, TNFSF4, IL1RAPL1, NRP1, SOX9, ONECUT1, PLEKHA2, RNF207, NID1, and LYST.

[0040] <Culture Medium> The culture medium used in the method for expanding hepatoblasts of the present invention (also referred to herein as "hepatoblast expansion medium") comprises a basic culture medium (hereinafter referred to as "basal culture medium"), and further comprises essential components such as a ROCK inhibitor, a TGFβ signaling pathway inhibitor and / or a Wnt signaling pathway activator, and a growth factor, and optionally a steroid. The culture medium used in the method for expanding hepatoblasts of the present invention preferably contains both a TGFβ signaling pathway inhibitor and a Wnt signaling pathway activator, but can also be used in the method for expanding hepatoblasts of the present invention even if it contains only one of a TGFβ signaling pathway inhibitor and a Wnt signaling pathway activator. Each of the medium components is described in detail below.

[0041] The basal medium may be any medium for vertebrate cell culture, such as any medium for mammalian cell culture known in the art, either alone or in combination, with the addition of medium additives as needed, such as Minimum Essential Medium Eagle (MEM) and any modified versions thereof. For example, specific mammalian cell culture media include Dulbecco's Modified Eagle's Medium (DMEM), Advanced DMEM, Iscove's Modified Dulbecco's Medium (IMDM), Ham's F-12 medium or modifications thereof (e.g., Ham's F-12 K medium, etc.), Eagle's Minimum Essential Medium, Alpha Modification (α-MEM), Improved Minimum Essential Medium, Glasgow Minimum Essential Medium (GMEM), William's E medium, Connaught Medical Research Laboratories Medium 1066, McCoy's 5A Medium, RPMI 1640 medium, or combinations thereof (e.g., DMEM / F-12 medium). The basal medium may be a combination of multiple media. The volume ratio when combining multiple media is not particularly limited. For example, two or more media may be mixed in equal volumes, or one medium may be mixed in a larger amount than the other media. When the volumes of the mixed media are different, the content and mixing ratio of the main medium (the medium with the largest volume) and the other media are not particularly limited. For example, the main medium can be at least 1 / 4, 1 / 3, 1 / 2, 2 / 3, or 3 / 4 of the total. Furthermore, for example, the main medium can be mixed in a ratio of 1x or more, 1.5x or more, 2x or more, 2.5x or more, or 3x or more of the volume of the other media. The type of main medium is not particularly limited.For example, Iscove's modified Dulbecco's medium can be suitably used as the main medium.

[0042] The basal medium may further contain additives such as proteins or compounds. When the additive is a protein, it may be derived from a specific organism, a recombinant protein, or an artificially synthesized protein. When the protein is derived from a specific organism, the species is not particularly limited. For example, proteins derived from the same or closely related organisms as the organism from which the hepatoblasts are derived can be used. For example, proteins derived from mammals are suitable, and proteins derived from primates such as humans are particularly suitable. The medium preferably contains an organic buffer, L-glutamine or a derivative thereof, and sodium pyruvate. When multiple media are used in combination, it is sufficient that these components are present after combination. For example, a medium combining IMDM containing the organic buffer HEPES but without L-glutamine and Ham's F-12 or its modified medium (e.g., Ham's F-12 K medium) containing L-glutamine but without HEPES can be used. Furthermore, if the above components are not present or are insufficient in the medium after mixing, they can be added externally. Any organic compound with pH buffering properties can be used as the organic buffer. Suitable organic buffers include, for example, Good's buffers, and specific examples include, but are not limited to, HEPES, MOPS, TES, Bis-Tris, PIPES, MES, etc. If necessary, any inorganic buffer such as sodium bicarbonate may be included.

[0043] 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 medium of this embodiment may be a serum-free medium as a whole, or serum may be added to form a serum-containing medium. In one embodiment, the basal medium used in the present invention is a serum-free medium.

[0044] The types of ROCK inhibitor, TGFβ signaling pathway inhibitor, Wnt signaling pathway activator, growth factor, and steroid agent basal medium used in the hepatoblast proliferation medium of this specification are not particularly limited, and may be any of those commonly used by those skilled in the art.

[0045] <ROCK Inhibitor> The specific type of ROCK inhibitor used in the present invention is not particularly limited, and for example, either a ROCK1 inhibitor or a ROCK2 inhibitor can be used. Note that the ROCK1 inhibitor here does not necessarily have to be capable of selectively inhibiting ROCK1, and broadly encompasses drugs that exhibit inhibitory activity against ROCK1. Specific examples thereof include 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, and also 4-[(1R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1-carboxamide or a salt thereof (e.g., dihydrochloride) such as Y-27632. Examples of compounds that can inhibit ROCK1 include compounds capable of inhibiting ROCK1, such as hydroxyfasudil hydrochloride, RKI-1447, GSK-429286A, AT13148, GSK-269962A hydrochloride, BAY-549, and Chroman 1; and compounds that can specifically inhibit ROCK2, such as Fasudil (1-(5-isoquinolinesulfonyl)homopiperazine) or a salt thereof (e.g., the dihydrochloride salt) and H-1152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine) or a salt thereof (e.g., the dihydrochloride salt). Expression inhibitors that suppress the expression of ROCK or its downstream factors can also be used as ROCK inhibitors herein. Examples of expression inhibitors herein include antisense nucleic acids, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and their expression vectors. For example, a ROCK1 inhibitor, more specifically, Y-27632 or a salt thereof can be suitably used as a ROCK inhibitor.

[0046] Multiple types of ROCK inhibitors can be used in combination. The type of combination is not particularly limited, but for example, multiple types of ROCK function inhibitors may be used in combination, or a ROCK function inhibitor and a ROCK expression suppressor may be used in combination.

[0047] The content ratio of the ROCK inhibitor is not particularly limited, as long as the concentration at which the drug used exhibits activity is sufficient. The lower limit of the final concentration of the ROCK inhibitor (e.g., a ROCK1 inhibitor such as Y-27632) includes, for example, 0.001 μM, 0.01 μM, 0.1 μM, 0.5 μM, 1 μM, or 5 μM, and the upper limit includes, for example, 200 μM, 100 μM, 50 μM, or 20 μM. 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.

[0048] <TGFβ Signaling Pathway Inhibitors> The type of TGFβ signaling pathway inhibitor used in the present invention is not particularly limited, and both classical and non-classical TGFβ signaling pathway inhibitors can be used. Examples of classical TGFβ signaling pathway inhibitors include TGFβ receptor inhibitors, Smad phosphorylation inhibitors, Smad nuclear translocation inhibitors, and activity or expression inhibitors 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 by them is not particularly limited. For example, an inhibitor that exhibits inhibitory activity against one or more of ALK1 to ALK7 (e.g., one or more of ALK4, ALK5, and ALK7) can be used. Specific examples include A8301, SB431542, SB505124, SB525334, D4476, LY2109761, LY2157299, LY364947, GW788388, RepSox, SD-208, TEW-7197, LDN-212854, or a combination thereof. In one embodiment, the TGFβ signaling pathway inhibitor used in the present invention is one inhibitor or a combination of two 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 the present invention is an inhibitor selected from the group consisting of A8301, SB431542, SB505124, LY364947, and RepSox.

[0049] The content ratio of the TGFβ signaling pathway inhibitor is not particularly limited, as long as the concentration at which the drug used exhibits activity is sufficient. The lower limit of the final concentration of the TGFβ signaling pathway inhibitor (e.g., a TGFβ receptor inhibitor such as A8301) can be, for example, 0.001 μM, 0.005 μM, 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, or 1 μM, and the upper limit can be, for example, 100 μM, 50 μM, 10 μM, 5 μM, or 1 μM. 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.

[0050] <Wnt signaling pathway activator> The Wnt signaling pathway activator used in the present invention is not particularly limited, and examples thereof include planar cell polarity (PCP) pathway activators, Wnt / Ca pathway activators, and the like. 2+ Pathway activators, activators of Wnt receptor binding, Wnt (WNT3A, etc.) and its substitutes, Notum pectinacetylesterase inhibitors (LP-922056, Notum pectinacetylesterase-1), Notum inhibitors (ARUK3001185, 8BTC, etc.), JNK activators (R-spondin 1), Wnt expression promoters (Wnt expression vectors, γ-Glutamylvaline, etc.), GSK3β inhibitors, β-catenin stabilizers (Axin-LRP6 complex formation promoters (lycorine and its derivatives, etc.), Axin-β-catenin complex formation promoters (SKL2001, etc.)), phosphodiesterase 5 inhibitors (tadalafil, vardenafil, mirodenafil and its derivatives, etc.), other drugs (BML-284, SM-04554, Dalosirvat, methyl vanillate, etc.), promoters of expression of molecules in the Wnt signaling pathway, etc. may be used.

[0051] Examples of GSK3β inhibitors include, but are not limited to, small molecule inhibitors such as CHIR99021 (Laduviglusib) and its hydrochloride, CHIR98014, Tideglusib, 6-bromoindirubin-3-oxime (BIO), 6-bromoindirubin-3-acetoxime (BIO acetoxime), SB415286, SB216763, TWS119, Tideglusib, A1070722, LY2090314, AZD1080, 1-Azakenpaullone, AR-A014418, IM-12, Indirubin, and TDZD-8, GSK3β expression inhibitors, and combinations thereof.

[0052] Wnt or its analogs can be suitably used as Wnt signaling pathway activators, and in such cases, they may be derived from a specific organism (e.g., human) or recombinant. When a Wnt receptor agonist such as WNT3A is used as the Wnt signaling pathway activator, the agonist is contained at a final concentration 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, 5 nM or more, or 10 μM or less, 1 μM or less, 500 nM or less, 100 nM or less, 50 nM or less, or 10 nM or less. For example, when a GSK3β inhibitor is used as the Wnt signaling pathway activator, the inhibitor is contained at a final concentration 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, 5 nM or more, and a final concentration of 10 μM or less, 1 μM or less, 500 nM or less, 100 nM or less, 50 nM or less, or 10 nM or less. In one embodiment, the Wnt signaling pathway activator used in the present 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 the present invention is Wnt3A. In one embodiment, the concentration of Wnt3A used in the present invention is 0.5 ng / mL to 5 μg / mL. In one embodiment, the concentration of Wnt3A used in the present invention is 5 ng / mL to 500 ng / mL.

[0053] <Growth Factors> Any growth factor known in the art can be used as the growth factor for use in the present invention. Specific examples include, but are not limited to, fibroblast growth factors (FGFs: FGF1 to FGF23, including basic FGF (bFGF, FGF2)), epidermal growth factor (EGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), substitutes thereof, and combinations thereof. Growth factors may be derived from specific organisms (e.g., humans) or may be recombinant. For example, the growth factor in the hepatoblast proliferation promoter of this embodiment can preferably include EGF or a substitute thereof.

[0054] The content ratio of the growth factor is not particularly limited, as long as the concentration at which the drug used exhibits activity is a suitable concentration. For example, the growth factor (e.g., EGF) can be contained at a molar concentration of 0.1 times or more, 0.2 times or more, 0.4 times or more, 0.5 times or more, 1 times or more, 1.1 times or more, 1.2 times or more, 1.3 times or more, or 1.35 times or more relative to the Wnt signaling pathway activator. In addition, the growth factor (e.g., EGF) can be contained at a molar concentration of 10 times or less, 5 times or less, 3 times or less, 2 times or less, 1.5 times or less, or 1.4 times or less relative to the Wnt signaling pathway activator. The lower limit of the final concentration of a growth factor (e.g., EGF) includes, for example, 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, and the upper limit includes, for example, 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 the present 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 the present invention is FGF2. In one embodiment, the growth factor used in the present invention is FGF2. In one embodiment, the concentration of FGF2 used in the present invention is 0.01 μM to 10 μM. In one embodiment, the concentration of FGF2 used in the present invention is 10 ng / mL.

[0055] <Steroid Agent> In the method for proliferating hepatoblasts or the method for producing a hepatoblast cell population of the present invention, the medium for hepatoblast proliferation may further contain a steroid agent. Examples of steroid agents that can be used in the present invention include glucocorticoids and their analogs and derivatives. For example, glucocorticoids, or their analogs, derivatives, or substitutes can be suitably used as the steroid agent. The specific type of glucocorticoid or its analog or derivative to be used is not particularly limited, but examples include long-acting steroids with a biological half-life of 36 hours or more (dexamethasone (DEX), betamethasone, paramethasone, etc.), intermediate-acting steroids with a biological half-life of 12 hours or more (prednisolone, methylprednisolone, triamcinolone, etc.), short-acting steroids with a biological half-life of less than 12 hours (cortisone acetate, fludrocortisone acetate, hydrocortisone, cortisone, etc.), and natural glucocorticoids (cortisone acetate, fludrocortisone acetate, hydrocortisone, cortisone, etc.) and salts thereof. For example, the steroid in the hepatoblast proliferation promoter of this embodiment can preferably include a long-acting steroid such as dexamethasone (DEX). Furthermore, multiple steroids can be used in combination as the steroid in the present invention. In this case, a combination of multiple glucocorticoids may be used, or a glucocorticoid may be used in combination with other steroid drugs.

[0056] The content of the steroid agent is not particularly limited, as long as the concentration at which the agent used exhibits activity is sufficient. The lower limit of the final concentration of the steroid agent (e.g., a glucocorticoid such as DEX) can be, for example, 0.001 μM, 0.005 μM, 0.01 μM, 0.05 μM, or 0.1 μM, and the upper limit can be, for example, 10 μM, 5 μM, 1 μM, 0.5 μM, 0.3 μM, 0.2 μM, or 0.1 μM. In one embodiment, the steroid agent used in the present invention is a glucocorticoid or a derivative thereof. In one embodiment, the glucocorticoid or a derivative thereof used in the present invention is dexamethasone. In one embodiment, the concentration of dexamethasone used in the present invention is 0.1 μM.

[0057] <Other Components> The hepatoblast proliferation medium used in the method for proliferating hepatoblasts or the method for producing a hepatoblast population of the present invention may further contain any other components, such as substances (additives) commonly used by those skilled in the art, such as antioxidants, serum or serum substitutes, vitamins, albumin, amino acids, antibiotics, supplements, and sugars.

[0058] When antioxidants are included in the hepatoblast growth medium, any molecule known in the art with antioxidant activity can be included. Specific types of antioxidants include, but are not limited to, glutathione and its precursors, L-ascorbic acid and its derivatives, 2-mercaptoethanol or its substitute (monothioglycerol), etc. Specific glutathione precursors include, but are not limited to, L-cysteine ​​or its derivatives (e.g., N-acetyl-L-cysteine), γ-glutamylcysteine ​​or its derivatives, etc. For example, other components of the pharmaceutical composition of this embodiment can preferably include L-cysteine ​​or its derivatives (e.g., N-acetyl-L-cysteine).

[0059] When serum or serum substitute is included in the medium for hepatoblast growth, any serum or serum substitute known in the art can be included. Examples of serum include fetal bovine serum (FBS), human serum, sheep serum, or a mixture thereof. Examples of serum substitutes include Knockout serum. TM serum replacement (KSR), XF212 XerumFree, CDM-HD serum replacement, StemSure serum replacement, Nu-Serum TM These include, but are not particularly limited to, the following:

[0060] When an antibiotic is contained in the hepatoblast proliferation medium, examples of antibiotics that can be used include, but are not limited to, penicillin, streptomycin, penicillin-streptomycin, sulfa preparations, phenethicillin, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, methacycline, and minocycline. The amount of antibiotic added is not particularly limited, and can be, for example, the concentration recommended by the manufacturer.

[0061] When supplements are included in the hepatoblast growth medium, e.g., B27 TM supplement, N2 supplement, etc. can be used.

[0062] When sugars are included in the medium for hepatoblast proliferation, for example, monosaccharides such as glucose, galactose, mannose, fructose, etc., disaccharides such as sucrose, maltose, lactose, etc., water-soluble or water-insoluble polysaccharides such as hyaluronic acid, gellan gum, chitin, and chitosan, etc. can be used.

[0063] Other substances commonly used by those skilled in the art include, for example, vitamins, albumin, amino acids, inorganic salts, stabilizers, and buffers. These substances may be substances commonly selected by those skilled in the art and can be used in the manner and at the concentrations commonly used by those skilled in the art.

[0064] <Expansion culture step> The expansion culture step is a step of expanding and culturing hepatoblasts in a hepatoblast proliferation medium, which may include a passaging step. Typically, the expansion culture step refers to culturing hepatoblasts seeded on a culture plate at a given cell density in a hepatoblast proliferation medium under appropriate temperature, humidity, and CO2 concentration.

[0065] The culture method may be any culture method known in the art, and is not particularly limited. For example, static culture using a culture plate, static culture using a cell factory or cell stack, static culture using a bag, or culture using a bioreactor of any type (hollow fiber type, wave type, vertical water wheel agitation type, up and down reciprocating type, stirred bed type, etc.) may be used. Furthermore, surface coating may be applied to the culture, and microcarriers may be used.

[0066] The cell density in the growth culture step is not particularly limited, but may be, for example, 10 2 cells / cm 2 ~10 6 cells / cm 2 , 10 3 cells / cm 2 ~10 5 cells / cm 2 Examples of the density include:

[0067] The surface of the culture plate may be coated as needed. The extracellular matrix used for coating is not particularly limited. Examples include laminin-1 to -12, collagen, fibronectin, vitronectin, Matrigel, Geltrex, and iMatrix-511.

[0068] The appropriate temperature is usually within the range of about 30-40°C, for example, 37°C. The humidity is usually within the range of about 70-100%, for example, about 95-100%. The CO2 concentration is usually within the range of about 1-10%, for example, about 5%. The O2 concentration is not particularly limited, and the cells can be cultured at normal oxygen concentrations (18-22%) or low oxygen concentrations (0-10%).

[0069] The culture period is not particularly limited, and can be until the cells have grown to a desired number or confluency, for example, 2 to 15 days, 3 to 15 days, 7 to 15 days, 7 to 14 days, or 7 to 12 days.

[0070] The medium can be replaced as needed. The frequency of medium replacement is not particularly limited. For example, it can be replaced regularly or irregularly. When replaced regularly, it can be replaced, for example, once every 1 to 5 days, once every 1 to 4 days, once every 1 to 3 days, once every 1 to 2 days, or once every 2 days. When replaced irregularly, the timing of medium replacement can be determined based on, for example, changes in the pH or turbidity of the medium. The composition of the medium may be the same as or different from the medium used before replacement, but preferably, the medium is replaced with a fresh medium without changing its composition before and after replacement.

[0071] The time for passaging can be determined appropriately depending on the culture conditions and is not particularly limited. For example, this step can be performed when the cell confluency reaches a certain level. The specific confluency at which passaging is performed is not particularly limited. Specifically, passaging can be performed when the confluency is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0072] The specific method of subculture is not particularly limited. Typically, cells are detached from a culture dish, dispersed in a culture medium, and then reseeded onto a new dish. Cell detachment and dispersion may be performed by mechanical stimulation such as pipetting, or by chemical stimulation such as the action of an enzyme such as trypsin. Specific examples of detachment agents include EDTA (e.g., 1 to 10 mM EDTA), TryPLE, and the like. TM Select, TrypLE TM Express Enzyme, Accutase TM , Collagenase, Dispase, Trypsin, Trypsin / EDTA, Trypsin / Collagenase, ReLeSR TMor a combination thereof, but are not particularly limited thereto. The cell density at the time of seeding, the coating of the dish, etc. can be performed in accordance with the description regarding the expansion culture step. Furthermore, the composition of the medium may be the same as or different from the medium used before the passaging, but preferably the composition of the medium before and after the passaging is the same. The culture conditions after the passaging treatment can be performed in accordance with the description regarding the expansion culture step. The conditions at the time of seeding and / or the culture conditions after the passaging treatment may be the same as or different from those used in the expansion culture step. The passaging step can be performed multiple times. The conditions used in each passaging step may be the same or different.

[0073] <Preparation of pluripotent stem cells> The pluripotent stem cells used in the present invention may be prepared from cells isolated from an individual, tissue, etc., and the preparation method can be selected appropriately depending on the type of stem cell used and is not particularly limited.

[0074] Pluripotent stem cells other than iPS cells are usually prepared by culturing cells isolated from individuals or tissues under culture conditions that allow the maintenance of pluripotency.

[0075] Numerous methods for preparing iPS cells have been investigated by those skilled in the art, and any of these methods can be used. iPS cells are typically induced by introducing specific reprogramming factors (DNA or proteins) into somatic cells (including skin cells, bone marrow cells, gastrointestinal cells, liver cells, somatic stem cells, etc.). Examples of reprogramming factors include Oct family genes (e.g., Oct3 / 4), Klf family genes (e.g., Klf4, Klf2), Sox family genes (e.g., Sox1, Sox2, Sox3, Sox15, Sox17), Myc family genes (e.g., c-Myc (including T58A mutant), N-Myc, L-Myc), Nanog family genes (e.g., Nanog), Lin family genes (e.g., Lin28, Lin28b), and other known genes (e.g., Fbx15, ERas, ECAT15-2, Tcl1, β-catenin, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, p53shRNA, Glis1). Specific combinations of reprogramming factors include, for example, a combination of Oct3 / 4, Sox2, Klf4, and c-Myc; a combination of Oct3 / 4, Sox2, and Klf4; a combination of Oct4, Sox2, Nanog, and Lin28; or a combination of Oct3 / 4, Sox2, Klf4, c-Myc, Nanog, and Lin28.

[0076] Pluripotent stem cells can be cultured using known methods without any particular limitations. For example, they can be cultured in a culture vessel coated with an extracellular matrix together with feeder cells capable of producing factors necessary for maintaining pluripotency and self-renewal. The necessity of coating can be determined appropriately depending on the culture vessel and culture method used, and culture can also be performed without coating. For example, molecules described below in relation to the proliferation culture step of this embodiment can be used as the extracellular matrix for coating. Culture methods can include, for example, adhesion culture or suspension culture, and can be selected appropriately depending on the purpose. For example, suspension culture can be used to form spheroids, etc. Culture can be performed by static culture or a culture method that applies any mechanical stimulation (rotation culture, rotation culture, etc.).

[0077] The medium to be used is not particularly limited, and any medium commonly used by those skilled in the art can be used. In the present invention, a medium provided as a medium particularly suitable for pluripotent stem cells may be used. For example, media for ES cells and iPS cells include mTeSR1 medium, TeSR1 medium (Stem Cell Technologies), and Essential 8 medium. TM Medium, Essential 6 TM Culture medium (Gibco), StemPro (R) -34 SFM (Life Technologies), StemFlex TM Medium (Gibco), StemFit AK02N (Ajinomoto), etc. are commercially available.

[0078] The need for feeder cells can be appropriately determined depending on the medium and additives used. For example, when a feeder-free medium is used, feeder cells are not required. 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.

[0079] Other culture conditions are not particularly limited, and any additional treatments can be performed during culture. In particular, when feeder cells are used, treatments to suppress cell proliferation (mitomycin C treatment, irradiation, etc.) may be performed.

[0080] <Hepatoblast induction step> The hepatoblast induction step is a step of inducing and culturing pluripotent stem cells to induce their differentiation into hepatoblasts, and includes a first step of inducing the differentiation of the pluripotent stem cells into definitive endoderm cells and a second step of inducing the differentiation of the definitive endoderm cells into hepatoblasts. After the second step of the hepatoblast induction step, a proliferation and culture step can be carried out.

[0081] The first step may include culturing the cells in a serum-free culture medium containing 10 μM Y-27632 during the early culture period, and culturing them in a serum-free medium containing B-27 supplement, activin A, and a WNT signaling pathway activator during the intermediate culture period or later. The second step may include culturing them in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide. As used herein, the term "early culture period" refers to days 0, 1, 2, 3, and 4 after the start of culture. As used herein, the term "intermediate culture period" refers to days 1, 2, 3, 4, and 5 after the start of culture. In one embodiment, the first step may include culturing the cells in a serum-free culture medium containing 10 μM Y-27632 during the early culture period, and culturing them in a serum-free medium containing B-27 supplement, activin A, and a WNT signaling pathway activator during the intermediate culture period or later. The second step may include culturing in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide. In one embodiment, the first step may include culturing in a serum-free culture medium containing 10 μM Y-27632 on day 0 of culture, and culturing in a serum-free medium containing B-27 supplement, activin A, and a WNT signaling pathway activator from day 2 onwards. The second step may include culturing in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide. In one embodiment, the first step may include culturing in a serum-free culture medium containing 10 μM Y-27632 on day 0 of culture, and culturing in a serum-free medium containing B-27 supplement, activin A, and a WNT signaling pathway activator from day 3 onwards. The second step may include culturing in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide. The method for inducing differentiation of pluripotent stem cells into hepatoblasts is not particularly limited, and any method known in the art can be used. Specifically, for example, pluripotent stem cells can be first induced to differentiate into definitive endoderm cells, and the induced definitive endoderm cells can then be further differentiated into hepatoblasts, but this method is not particularly limited.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.

[0082] The pluripotent stem cells used in this process may be newly prepared from cells derived from a healthy individual or a patient, or may be obtained from a biobank or the like, and the type of pluripotent stem cells is not particularly limited. The method for newly preparing pluripotent stem cells can be a method commonly used by those skilled in the art. When obtaining from a biobank or the like, for example, iPS cells or ES cells can be obtained from the RIKEN BioResource Center Cell Materials Division (RIKEN BRC CELL BANK), ATCC (American Type Culture Collection), iPS Academia Japan, Inc., etc. Commonly available cell lines include, but are not limited to, 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. Pluripotent stem cells may be used in combination with one type of stem cell. Pluripotent stem cells may also be used after long-term storage. For example, pluripotent stem cells can be thawed after cryopreservation. In this case, the storage temperature and storage period are not particularly limited. For example, specific storage temperatures include temperatures below 0°C, -20°C or lower, -30°C or lower, -40°C or lower, -50°C or lower, -79°C or lower, -80°C or lower, -100°C or lower, -130°C or lower, -150°C or lower, -180°C or lower, and -196°C or lower. The temperature may be constant during the storage period, or may change naturally or artificially. The storage period is not particularly limited, and may be, for example, 1 day or more, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 30 days or more, 1 month or more, 2 months or more, 3 months or more, etc.

[0083] The serum-free medium or other components used, or the culture conditions, are not particularly limited, and reference may be made to media and culture conditions commonly used by those skilled in the art, or to the basal medium and the like described in the section <Expansion and Culture of Hepatoblasts and Production of Hepatoblast Population>. Furthermore, any additional treatment may be performed during the culture.

[0084] The step of inducing the differentiation of pluripotent stem cells into definitive endoderm cells (first step) and the step of inducing the differentiation of definitive endoderm cells into hepatoblasts (second step) will be described below as examples.

[0085] <Induction of Differentiation from Pluripotent Stem Cells to Definitive Endoderm Cells> In this step, the differentiation of the pluripotent stem cells into definitive endoderm cells is induced.

[0086] The specific method for inducing differentiation in this step is not particularly limited, but for example, differentiation of pluripotent stem cells into definitive endoderm cells can be induced by culturing pluripotent stem cells in a medium containing a Wnt signaling pathway activator and / or a TGFβ family protein.

[0087] The types and concentrations of the Wnt signaling pathway activator and TGFβ family protein used in this step are as described above. For example, CHIR99021 or WNT3A can be used as the Wnt signaling pathway activator, and for example, activin and / or BMP4 can be used as the TGFβ family protein.

[0088] Growth factors may be added to the differentiation-inducing medium. The growth factors and their concentrations are as described above under "Growth Factors." Suitable growth factors include, for example, FGF2 and VEGF.

[0089] The basal medium and other ingredients should conform to the description of the growth culture process. For example, B-27 as a supplement TMSupplements can be used, and RPMI 1640 medium and / or serum-free medium can be preferably used as the basal medium. Additives such as sodium butyrate may also be used. The culture period is not particularly limited and can be, for example, 1 day or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more, such as 1 to 150 days, 1 to 90 days, 1 to 50 days, 1 to 30 days, 1 to 20 days, or 3 to 10 days. A ROCK inhibitor (e.g., Y-27632) may be added only on day 0 of culture. The medium composition may be changed one or more times during this step. For example, as the culture days progress, the medium may be replaced with one containing a higher concentration of TGF-β family proteins and growth factors. The oxygen concentration in this step is not particularly limited. For example, it may be hypoxic, normoxic, or a combination thereof.

[0090] Induction of differentiation into definitive endoderm cells can be determined based on whether any properties of definitive endoderm cells are retained and / or whether properties of pluripotent stem cells are lost. For example, this can be determined by the expression of definitive endoderm cell markers and / or pluripotent stem cell markers. Definitive endoderm cell markers and pluripotent stem cell markers can be any marker known in the art and are not particularly limited. Specific definitive endoderm cell markers include FOXA2, SOX17, CXCR4 (CD184), GATA4, GATA6, CD117, CER1, etc. Specific pluripotent stem cell markers include OCT4, NANOG, SOX2, CD9, DNMT3B, GABRB3, GAL, GDF3, IFITM1, PODXL, TDGF1, ZFP42, etc.

[0091] In one embodiment, the hepatoblast induction process includes a first step of inducing differentiation of pluripotent stem cells into definitive endoderm cells, which first step includes culturing in a serum-free culture medium containing 10 μM Y-27632 on day 0 of culture, and culturing in a serum-free medium containing B-27 supplement, activin A, and a WNT signaling pathway activator on day 1 of culture or thereafter.

[0092] In one embodiment, the hepatoblast induction process includes a first step of inducing differentiation of iPS cells into definitive endoderm cells, which first step includes culturing the iPS cells in a serum-free culture medium containing 10 μM Y-27632 on day 0 of culture, and culturing the iPS cells in a serum-free medium containing B-27 supplement, activin A, and a WNT signaling pathway activator on day 1 of culture or later.

[0093] <Induction of Differentiation from Definitive Endoderm Cells to Hepatoblasts> In this step, definitive endoderm cells are induced to differentiate into hepatoblasts. This step is usually performed after the definitive endoderm cell induction step, but if definitive endoderm cells are obtained, this step can be performed without the above step. In addition, in this step, for example, hepatoblasts may be induced to differentiate through differentiation of definitive endoderm cells into other cell types (e.g., hepatic endoderm cells, foregut progenitor cells).

[0094] The basal medium and culture conditions used are not particularly limited, and may refer to those commonly used by those skilled in the art or the medium and culture conditions described in the section <Expansion culture of hepatoblasts and production of hepatoblast cell population>. In addition, any additional treatment may be performed during culture.

[0095] The specific method for inducing differentiation in this step is not particularly limited. For example, differentiation into hepatoblasts can be induced by culturing definitive endoderm cells in a serum-free medium containing one or more selected from the group consisting of a steroid, a TGFβ family protein, dimethyl sulfoxide, an inflammatory cytokine, and a growth factor. For example, the medium may contain dimethyl sulfoxide, a growth factor, a TGFβ family protein and a growth factor, dimethyl sulfoxide and a growth factor, or a steroid, an inflammatory cytokine, and a growth factor.

[0096] The steroids and growth factors are as described in the section entitled "Proliferation and Culture of Hepatoblasts and Production of Hepatoblast Cell Populations." The serum-free medium and the concentrations of steroids and growth factors are as described in the section entitled "Proliferation and Culture of Hepatoblasts and Production of Hepatoblast Cell Populations." For example, glucocorticoids such as DEX can be suitably used as steroids, and FGFs (FGF1, FGF2, FGF4, FGF10, etc.), VEGF, and / or HGF can be suitably used as growth factors. For example, activin and / or BMP4 can be suitably used as TGFβ family proteins.

[0097] The inflammatory cytokines used are not particularly limited. 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. IL-6 family proteins include oncostatin M (OSM), IL-6, IL-11, IL-27, IL-35, IL-39, LIF, CT-1, CNTF, CLCF1, and combinations thereof. For example, oncostatin M can be preferably used.

[0098] The concentration of the inflammatory cytokine (e.g., an IL-6 family protein such as OSM) is not particularly limited as long as it is an effective amount. The lower limit of the concentration includes, 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, and the upper limit includes, for example, 10 nM, 5 nM, 1 nM, 0.6 nM, 0.5 nM, 0.45 nM, 0.4 nM, and 0.35 nM.

[0099] The culture conditions are the same as those described for the growth culture step of this embodiment. For example, vitamin B, such as nicotinamide, or a derivative thereof, or vitamin C, such as L-ascorbic acid, or a derivative thereof, can be suitably used as a medium additive. The culture period is not particularly limited, and can be, for example, 1 day or more, 3 days or more, 5 days or more, 6 days or more, or 7 days or more, such as 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, or 3 days to 10 days.

[0100] During this step, the composition of the medium may be changed one or more times. The oxygen concentration during this step is not particularly limited. For example, the medium may be hypoxic, normoxic, or a combination thereof.

[0101] Induction of differentiation into hepatoblasts can be determined based on whether any properties of hepatoblasts are acquired and / or whether properties of definitive endoderm cells are lost. For example, this can be determined by the expression of hepatoblast markers and / or definitive endoderm cell markers. Hepatoblast markers can be any marker known in the art, including, but not limited to, AFP, ALB, CD324, CYP3A7, DLK1, PROX1, TBX3, CDH6, CTNND2, DLK1, EpCAM, FOXA1, GATA4, GATA6, GPRC5B, HHEX, HNF1B, HNF4A, ID3, MCAM, MET, ONECUT1, PROM1, SLC12A2, SOX9, SPP1, STAT1, KRT19, E-cadherin, LIV2, CD13, CD133, and the like. Definitive endoderm cell markers can be, for example, any of the definitive endoderm cell markers described above in this embodiment.

[0102] In one embodiment, the hepatoblast induction process includes a second step of inducing the differentiation of definitive endoderm cells into hepatoblasts, which second step includes culturing the cells in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide.

[0103] <Maturation promotion step> This step is an optional step in which a treatment for promoting the maturation of hepatoblasts is carried out. This step can be carried out simultaneously with or after the expansion and culture step. Furthermore, this step can be carried out as a pretreatment, particularly when hepatoblasts expanded and cultured by the method of the present invention are to be transplanted into an individual organism.

[0104] In this step, hepatoblasts are exposed to an expression promoter of genes expressed in the late stage of liver development in the species from which the hepatoblasts are derived. The type of gene expressed in the late stage of liver development is not particularly limited, as long as it is a gene related to liver maturation. Suitable examples include IL-6 family proteins, tumor necrosis factor (TNF) superfamily proteins such as TNF-α, and JAK-STAT3 pathway activators. Examples of JAK-STAT3 pathway activators include FGF19, the inflammatory cytokines described in the hepatoblast induction step of this embodiment, and overexpression vectors for JAK-STAT3 pathway-related genes. Alternatively, one or more substances selected from the group consisting of growth factors (FGFs such as FGF2 and FGF10, HGF, etc.), steroids (glucocorticoids such as dexamethasone), and Wnt signaling pathway activators (Wnt signaling pathway activators such as GSK3β inhibitors) may be used. The components and their concentrations are as described above. In one embodiment, the substance used in the hepatoblast maturation promotion step is oncostatin M.

[0105] During this step, the composition of the medium may be changed one or more times. The oxygen concentration during this step is not particularly limited. For example, the medium may be hypoxic, normoxic, or a combination thereof.

[0106] In the case of human-derived hepatoblasts (including hepatoblasts differentiated from human-derived pluripotent stem cells), the hepatoblasts are exposed to a human-derived protein. The human-derived protein may be produced in another organism or chemically synthesized, as long as it has the same amino acid sequence as the human protein. The exposure method is not particularly limited, but examples include adding the target protein to the culture medium, culturing the target protein with cells expressing the target protein, or a combination thereof. The exposure period is also not particularly limited, but may be, for example, until STAT3 activity increases (e.g., until a significant increase occurs) or until the expression levels of mature hepatocyte markers such as ALB, CPS1, CYP3A4, and G6PC increase. Specifically, the exposure period may be, for example, 2 or more days, 3 or more days, 4 or more days, 5 or more days, 6 or more days, or 7 or more days. For example, after exposure, cells may further express 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), C4B (Complementary Protein). C4B), ATP10B (ATPase Phospholipid Transporting 10B), NNMT (NicotinamideN-Methyltransferase), LCN2 (Lipocalin 2), LOC145837, ORM1 (Orosomucoid 1), ORM2 (Orosomucoid 2), PC (Pyruvate 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).

[0107] <Effects> The hepatoblast proliferation method of the present invention can achieve cell proliferation while maintaining the undifferentiated state of hepatoblasts without causing overexpression of oncogenes. Furthermore, the hepatoblast production method of the present invention can produce a homogeneous cell population containing highly pure hepatoblasts based on stable cell proliferation. Typically, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more of the cells in the produced cell population are hepatoblasts. In one embodiment, the purity of the hepatoblast population produced by the hepatoblast production method of the present invention is 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more. In one embodiment, the purity of the hepatoblast population produced by the hepatoblast production method of the present invention is 85% or more. In one embodiment, the purity of the hepatoblast population produced by the hepatoblast production method of the present invention is 90% or more. In one embodiment, the purity of the hepatoblast population produced by the hepatoblast production method of the present invention is 95% or more. In one embodiment, the purity of the hepatoblast population produced by the hepatoblast production method of the present invention is 97% or more. In one embodiment, the purity of the hepatoblast population produced by the hepatoblast production method of the present invention is 99% or more. When administered to an individual, the produced hepatoblast population can be engrafted in vivo for a long period of time and exhibit the ability to repair liver tissue in vivo.

[0108] 2. Method for Producing Hepatocytes or Bile Duct Cells The present invention further provides a method for producing hepatocytes or cholangiocytes. The method for producing hepatocytes or cholangiocytes of the present invention makes it possible to produce a large number of hepatocytes or cholangiocytes from hepatoblasts that have been subjected to expansion culture.

[0109] The method for producing hepatocytes or bile duct cells of the present invention may include a pluripotent stem cell preparation step, a hepatoblast induction step, a proliferation and culture step, and a differentiation induction step. The hepatoblast induction step is a step of inducing and culturing pluripotent stem cells and inducing their differentiation into hepatoblasts, and includes a first step of inducing the differentiation of the pluripotent stem cells into definitive endoderm cells and a second step of inducing the differentiation of the definitive endoderm cells into hepatoblasts. The pluripotent stem cell preparation step and the hepatoblast induction step are optional steps in the method of this embodiment, while the proliferation and culture step and the differentiation induction step are essential steps.

[0110] <Method for producing hepatocytes> Differentiation into hepatocytes can be induced by culturing hepatoblasts in a hepatocyte induction medium. Although not particularly limited, 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.

[0111] The hepatocyte induction medium used can be any medium known in the art for use in inducing differentiation into hepatocytes. For example, a medium containing one or more selected from the group consisting of inflammatory cytokines, steroids, and growth factors can be used. As the inflammatory cytokine, for example, any of the inflammatory cytokines described in the section <Induction of Differentiation from Definitive Endoderm Cells to Hepatoblasts> can be used. As the steroid and growth factor, any of the steroids and growth factors described in the section <Expansion Culture of Hepatoblasts and Production of Hepatoblast Cell Populations> can be used. For example, IL-6 family proteins such as OSM can be used as the inflammatory cytokine, glucocorticoids such as DEX can be used as the steroid, and HGF can be used as the growth factor. The concentrations of the inflammatory cytokines, steroids, growth factors, etc. are not particularly limited and can be any concentrations commonly used by those skilled in the art.

[0112] The culture conditions are not particularly limited. For example, the culture can be carried out in accordance with the description of the expansion culture step of the first embodiment. The culture period is not particularly limited, and the culture can be carried out until differentiation into hepatocytes is induced. For example, the culture period can be 1 day or more, 3 days or more, 5 days or more, 7 days or more, 9 days or more, or 10 days or more. Specifically, the culture period can be, for example, 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.

[0113] Induction of differentiation into hepatocytes can be determined based on whether or not the cells possess any properties possessed by hepatocytes, whether or not the properties possessed by hepatoblasts have been lost, and / or whether or not the cells lack any properties possessed by cholangiocytes. For example, this can be determined by the expression of hepatocyte markers, hepatoblast markers, and / or cholangiocyte markers. Hepatocyte markers can be any marker known in the art and are not particularly limited. Specific examples include A1AT, ALB, CPS1, CYP3A4, SERPINA1, AHSG, APOA4, ASGR1, KRT8, FABP1, FGG, G6PC, PCK2, RBP4, TAT, TF, TDO2, HHEX, and HNF1A. Hepatoblast markers can be, for example, any of the hepatoblast markers described above in the first aspect, and cholangiocyte markers can be, for example, any of the hepatocyte markers described below in this aspect.

[0114] <Method for producing cholangiocytes> Differentiation into cholangiocytes can be induced by culturing hepatoblasts in a cholangiocyte induction medium. Methods described in, for example, 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, without particular limitation.

[0115] The cholangiocyte induction medium used can be any medium known in the art for use in inducing differentiation into cholangiocytes. For example, a medium containing a Wnt signaling pathway activator, a ROCK inhibitor, a TGFβ family protein (e.g., activin, TGFβ), an adenylate cyclase activator, and / or a growth factor can be used. The Wnt signaling pathway activator, ROCK inhibitor, and growth factor can be the ROCK inhibitor and growth factor described in "Expansion Culture of Hepatoblasts and Production of Hepatoblast Cell Populations," respectively. For example, the Wnt signaling pathway activator can be one or more of Wnt (e.g., WNT3A), its analogs, and JNK activators (e.g., R-spondin 1); the ROCK inhibitor can be a ROCK1 inhibitor (e.g., Y-27632); and the growth factor can be one or more of EGF, HGF, and FGF (e.g., FGF10). The Wnt concentration is not particularly limited, but can be, for example, 0.1 nM to 5 nM, 0.5 nM to 3 nM, 1 nM to 2 nM, or 1 nM to 1.5 nM. The concentration of the JNK activator is not particularly limited, and can be, for example, 0.1 nM to 50 nM, 1 nM to 20 nM, 1 nM to 10 nM, 5 nM to 10 nM, 6 nM to 8 nM, etc. The concentrations of the ROCK inhibitor and growth factor are not particularly limited, and can be, for example, the final concentrations described in the first aspect.

[0116] The adenylate cyclase activator is not particularly limited, and any known drug that improves the activity of adenylate cyclase can be used. Specific examples of the adenylate cyclase activator include forskolin, forskolin analogs (colforsin daropate, etc.), cholera toxin, carbacyclin, isoproterenol, analogs thereof, or salts thereof, biogenic amines such as octopamine, steroid hormones (glucagon, prostaglandins, vasopressin, etc.), polypeptides such as pituitary adenylate cyclase-activating polypeptide (PACAP) and parathyroid hormone, nucleotide derivatives such as 8-bromo-cAMP, and combinations thereof. The concentration of the adenylate cyclase activator such as forskolin is not particularly limited as long as it is an effective amount, and can be, for example, 0.1 μM to 100 μM, 1 μM to 50 μM, 1 μM to 20 μM, 5 μM to 15 μM, etc.

[0117] As the basal medium, any medium described in the first embodiment can be used, but for example, DMEM is preferably used. Furthermore, other components, additives, and medium additives described in the first embodiment can be appropriately added. For example, a vitamin A derivative such as retinoic acid can be preferably added.

[0118] The culture conditions are not particularly limited. For example, the culture can be carried out in accordance with the description of the expansion culture step of the first embodiment. The culture period is not particularly limited, and the culture can be carried out until differentiation into bile duct cells is induced. For example, the culture period can be 1 day or more, 3 days or more, 5 days or more, 7 days or more, 9 days or more, or 10 days or more. Specifically, the culture period can be, for example, 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.

[0119] Induction of differentiation into cholangiocytes can be determined based on whether or not the cells possess any of the properties possessed by cholangiocytes, whether or not the properties possessed by hepatoblasts have been lost, and / or whether or not the cells lack any of the properties possessed by hepatocytes. For example, this determination can be based on the expression of cholangiocyte markers, hepatoblast markers, and / or hepatocyte markers, and is not particularly limited. Any marker known in the art can be used as the cholangiocyte marker. Specific examples include KRT19, GGT1, KRT7, AQP1, HES1, and SSTR2. For example, any of the hepatoblast markers described above in the first aspect can be used as the hepatocyte marker, and for example, any of the hepatocyte markers described above in this aspect can be used as the hepatoblast marker.

[0120] 3. Agent for Promoting Hepatoblast Proliferation and Kit for Promoting Hepatoblast Proliferation The present invention also provides an agent for promoting hepatoblast proliferation and a kit for promoting hepatoblast proliferation.

[0121] The hepatoblast proliferation promoter of the present invention comprises, as essential components, a ROCK inhibitor, a TGFβ signaling pathway inhibitor, and / or a Wnt signaling pathway activator, and a growth factor, and optionally, a steroid. The ROCK inhibitor, TGFβ signaling pathway inhibitor, Wnt signaling pathway activator, growth factor, or steroid may contain the substances and concentrations described in the section "Expansion and Culture of Hepatoblasts and Production of Hepatoblast Cell Population." In one embodiment, the hepatoblast proliferation promoter of the present invention comprises, as essential components, a ROCK inhibitor, a TGFβ signaling pathway inhibitor, and a growth factor. In one embodiment, the hepatoblast proliferation promoter of the present invention comprises, as essential components, a ROCK inhibitor, a Wnt signaling pathway activator, and a growth factor.

[0122] The kit for promoting hepatoblast proliferation of the present invention includes, as essential components, a ROCK inhibitor, a TGFβ signaling pathway inhibitor, and / or a Wnt signaling pathway activator, as well as a hepatoblast proliferation promoter containing a growth factor and, as optional components, a steroid and other ingredients, and may additionally include a culture medium, a culture medium additive, etc. In one embodiment, the kit for promoting hepatoblast proliferation of the present invention includes, as essential components, a ROCK inhibitor, a TGFβ signaling pathway inhibitor, and a growth factor. In one embodiment, the kit for promoting hepatoblast proliferation of the present invention includes, as essential components, a ROCK inhibitor, a Wnt signaling pathway activator, and a growth factor.

[0123] The kit of the present invention may further include a container. The material of the container is not limited as long as it is made of a material that does not contaminate the contents or is not contaminated by the contents. Examples of materials that can be used for the container include plastics such as polypropylene and polystyrene, glass, and paper with a specially coated surface.

[0124] In the kit of the present invention, each active ingredient can be contained in a container. In the kit of the present invention, each active ingredient can be contained together in a single container or divided into multiple containers. When divided into multiple containers, the containers may be divided by ingredient, or the containers may be divided by other criteria (such as the amount used per time), or a combination thereof.

[0125] The kit of the present invention may include, for example, a means for adding to a medium (such as a dropper, micropipette, syringe, injection needle, or sprayer). The kit of the present invention may also include instructions for use, if necessary.

[0126] The kit of the present invention has the advantage that the proliferation and culture of hepatoblasts can be easily carried out.

[0127] 4. Cell preparations containing hepatoblasts for use in treating liver disease or methods for treating liver disease The present invention also provides cell preparations containing hepatoblasts for use in treating liver disease or methods for treating liver disease. The cell preparations containing hepatoblasts for use in treating liver disease of the present invention contain hepatoblasts as an essential component, and optionally contain a solvent or carrier. The cell preparations containing hepatoblasts for use in treating liver disease of the present invention can be used to treat subjects with liver disease.

[0128] <Cell preparation containing hepatoblasts for use in the treatment of liver disease> The cell preparation containing hepatoblasts for use in the treatment of liver disease of the present invention (hereinafter referred to as the "cell preparation of the present invention") contains a hepatoblast population produced by the hepatoblast production method of the present invention.

[0129] The origin of the hepatoblasts contained in the cell preparation of the present invention is not particularly limited. For example, the hepatoblasts may be derived from the target species or individual to be administered or treated. In one embodiment, the hepatoblasts contained in the pharmaceutical composition of the present invention are derived from humans.

[0130] The pharmaceutical compositions of the present invention are characterized in that, in addition to typical genes expressed in hepatoblasts (e.g., AFP, DLK, ALB, TBX3, c-MET, EpCAM, KRT19), one or more genes selected from the group consisting of SLC4A4, TNFSF4, IL1RAPL1, NRP1, SOX9, ONECUT1, PLEKHA2, RNF207, NID1, and LYST are expressed. In one embodiment, the pharmaceutical compositions of the present invention express one or more genes selected from the group consisting of SLC4A4, TNFSF4, IL1RAPL1, NRP1, SOX9, ONECUT1, PLEKHA2, RNF207, NID1, and LYST. In another embodiment, the pharmaceutical compositions of the present invention express 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 SLC4A4, TNFSF4, PLEKHA2, and RNF207. In one embodiment, the cell preparation of the present invention further expresses one or more genes selected from the group consisting of SLC4A4, TNFSF4, IL1RAPL1, NRP1, SOX9, ONECUT1, PLEKHA2, RNF207, and LYST, in addition to typical genes expressed in hepatoblasts (e.g., AFP, DLK, ALB, TBX3, C-MET, EpCAM, KRT19). In one embodiment, the cell preparation of the present invention further expresses IL1RAPL1, NRP1, SOX9, ONECUT1, NID1, and LYST, in addition to typical genes expressed in hepatoblasts (e.g., AFP, DLK, ALB, TBX3, C-MET, EpCAM, KRT19). In one embodiment, the cell preparation of the present invention expresses typical genes expressed in hepatoblasts (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.

[0131] The cell preparation of the present invention can be produced by a production method comprising a hepatoblast induction step, which includes a first step of inducing the differentiation of pluripotent stem cells (e.g., iPS cells) into definitive endoderm cells and a second step of inducing the differentiation of the definitive endoderm cells into hepatoblasts, and a third step of producing a hepatoblast cell population by expansion and culture of the hepatoblasts. In the treatment of human liver diseases, a cell preparation containing the cells obtained by the first to third steps can be used, but it is expected that improved therapeutic outcomes can be achieved by further using a hepatoblast cell population cultured in a serum-free medium containing oncostatin M in the maturation promotion step.

[0132] The method for producing a cell preparation of the present invention may further comprise a maturation promoting step of culturing a hepatoblast population with the addition of oncostatin M. In one embodiment, the method for producing a cell preparation for use in treating a liver disease of the present invention comprises a first step of inducing the differentiation of pluripotent stem cells into definitive endoderm cells, a second step of inducing the differentiation of the definitive endoderm cells into hepatoblasts, a third step of producing a hepatoblast population by expansion culture of the hepatoblasts, and a maturation promoting step of culturing the hepatoblast population in a serum-free medium containing oncostatin M.

[0133] The first step may include culturing the hepatoblasts in a serum-free culture medium containing Y-27632 (e.g., at a concentration of 10 μM) at the initial stage of culture, and culturing them in a serum-free medium containing B-27 supplement, activin A, and a WNT signaling pathway activator at the intermediate stage or later of culture. The second step may include culturing them in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide. The third step may include an expansion culture step of expanding the hepatoblasts in a hepatoblast growth medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor.

[0134] In one embodiment, a method for producing a cell preparation for use in treating liver diseases comprises: a first step of inducing differentiation from pluripotent stem cells into definitive endoderm cells; a second step of inducing differentiation from definitive endoderm cells into hepatoblasts; and a third step of expanding and culturing the hepatoblasts to produce a hepatoblast cell population. The first step comprises culturing the hepatoblasts in a serum-free culture medium containing 10 μM Y-27632 in the early culture period, and culturing the hepatoblasts in a serum-free medium containing B-27 supplement, activin A, and a WNT signaling pathway activator in the intermediate culture period or later. The second step comprises culturing the hepatoblasts in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide. The third step comprises an expansion and culture step of expanding and culturing the hepatoblasts in a hepatoblast growth medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor.

[0135] In one embodiment, the method of the present invention for producing a cell preparation for use in treating liver diseases comprises: a first step of inducing the differentiation of iPS cells into definitive endoderm cells; a second step of inducing the differentiation of the definitive endoderm cells into hepatoblasts; a third step of expanding and culturing the hepatoblasts to produce a hepatoblast population; and a maturation promotion step of culturing the hepatoblast population in a serum-free medium containing oncostatin M, wherein the first step comprises culturing the hepatoblast population in a serum-free culture medium containing 10 μM Y-27632 on day 0 of culture and culturing the hepatoblast population in a serum-free medium containing B-27 supplement, activin A, and a WNT signaling pathway activator on day 1 or later of culture; the second step comprises culturing the hepatoblasts in a serum-free medium containing FGF2, HGF, OSM, dexamethasone, and nicotinamide; and the third step comprises an expansion and culture step of expanding and culturing the hepatoblasts in a hepatoblast growth medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor.

[0136] The cells used in the cell preparation of this embodiment may be subjected to any treatment before storage. Specific treatments are not particularly limited, and may include, for example, selection, transformation, genetic manipulation, or a combination thereof. In one embodiment, the cells used in the cell preparation of this embodiment may be further genetically manipulated after expansion culture. In one embodiment, the cells used in the cell preparation of this embodiment may be further transformed after expansion culture.

[0137] The state of cells in the cell preparation of this embodiment may be a single cell state or a cell aggregate state such as a spheroid. A single cell state is preferred. As used herein, "single cell state" refers to a state in which cells exist singly and are not aggregated. The proportion of single cells 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 cells in the cell preparation can be measured using any method. For example, cells can be dispersed in a buffer solution (e.g., PBS) and a randomly selected number of cells are observed under a microscope to determine whether or not they aggregate.

[0138] The cells in the cell preparation may be floating or in contact with the inner wall of the container, etc. Preferably, the cells are floating. As used herein, "floating" means that the cells are not fixed to the inner wall of the container containing the cell preparation by adhesion or the like.

[0139] The number of cells contained in the cell preparation of the present invention per unit dose is not particularly limited. Generally, the number of cells varies depending on the type of cell, the route of administration, the purpose of administration, and the type of carrier, which is another component described below. Therefore, it may be determined appropriately taking into consideration each condition. For example, it is sufficient that a sufficient number of cells is contained in a single dose of the cell preparation. The specific number of hepatoblasts per unit dose is not particularly limited, but may be, for example, 10 3 pcs / mL or more, 10 4 pcs / mL or more, 10 5 pcs / mL or more, 10 6 pcs / mL or more, 10 7 pcs / mL or more, 108 pcs / mL or more, 10 9 It is sufficient that the preparation contains at least 100 hepatoblasts / mL. When administered in multiple doses, the total amount should contain a sufficient number of cells. When the cell preparation of the present invention is diluted and administered, it is sufficient that the diluted preparation contains a sufficient number of cells to achieve the desired effect.

[0140] Any cells other than the hepatoblast cell population can be additionally contained. The type of additional cells is not particularly limited. Preferably, the cells are derived from the same species or the same individual as the hepatoblast cells.

[0141] <Solvent> The cell preparation of this embodiment may contain a pharmaceutically acceptable solvent as needed. "Pharmaceutically acceptable solvent" refers to a solvent commonly used in the pharmaceutical formulation field. Examples include water and aqueous solutions. Aqueous solutions include, for example, physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer. Examples of adjuvants include inorganic salts such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and organic acid salts such as citrate, gluconate, and succinate, as well as low-concentration nonionic surfactants and polyoxyethylene sorbitan fatty acid esters.

[0142] The pH of the solution is not particularly limited as long as the cells to be preserved remain viable. Specific pH values ​​are, for example, 3.5 to 8.5, 4 to 8, 4.5 to 7.5, or 5 to 7.5.

[0143] The osmotic pressure of the solution is not particularly limited as long as it allows cells to survive. For example, the solution may be hypotonic (less than 250 mOsm / L), isotonic (250 mOsm / L to 380 mOsm / L), or hypertonic (more than 380 mOsm / L). The osmotic pressure may be expressed as the osmotic pressure ratio with respect to physiological saline (e.g., 306 mOsm / L).

[0144] The solution of the present invention may be prepared by the patient himself or may be commercially available. For example, preferred solutions include Ringer's solution (lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution, etc.), Ringer's basal solution, and any other solution used as an infusion solution.

[0145] The viscosity of the solution is not particularly limited as long as the cells to be preserved remain viable. For example, the viscosity is preferably 8 mPas or more and 18 mPas or less at the temperature during cell suspension and / or storage. The viscosity of the non-freezing preservation solution can be measured, for example, using a TV-20 viscometer (Toki Sangyo Co., Ltd.) at a rotation speed of 10 rpm.

[0146] <Carrier> The cell preparation of the present invention may contain a pharmaceutically acceptable carrier as needed. The term "pharmaceutically acceptable carrier" refers to additives commonly used in the pharmaceutical technology field. Examples include excipients, binders, disintegrants, emulsifiers, flow additives, lubricants, etc.

[0147] Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, pluronics, etc. (R) , kaolin, silicic acid, or a combination thereof.

[0148] Examples of binders include starch paste using vegetable starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone, and combinations thereof.

[0149] Disintegrants include, for example, the above-mentioned starches, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, or salts thereof.

[0150] Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0151] Examples of flow regulators and lubricants include silicates, talc, stearates or polyethylene glycol.

[0152] In addition to the above, if necessary, the composition may appropriately contain solubilizers, suspending agents, diluents, dispersants, surfactants, soothing agents, stabilizers, absorption promoters, bulking agents, moisturizing agents, humectants, humectants, adsorbents, flavoring agents, disintegration inhibitors, coating agents, colorants, preservatives, antiseptics, antioxidants, buffers, pH adjusters, isotonic agents, and the like that are commonly used in pharmaceutical compositions, cell preparations, and the like.

[0153] The carrier is used to avoid or suppress the decomposition of the active ingredient by enzymes and the like in the subject's body, as well as to facilitate formulation and administration methods and maintain the dosage form and medicinal efficacy, and may be used appropriately as needed.

[0154] The cell preparation of the present invention is preferably in a sterile state. The method for achieving a sterile state can be any method known in the art and is not particularly limited.

[0155] <Dosage Form> The dosage form of the cell preparation of the present invention is a liquid. Liquid preparations include any preparations that have fluidity. Specific examples include injections, suspensions, gels, etc. The specific volume, etc., of each dosage form is not particularly limited as long as it is within the range known in the art. The cell preparation of the present invention can be produced by a method commonly used in the art.

[0156] <Method for treating liver diseases with a cell preparation containing hepatoblasts> The cell preparation of the present invention can be used to treat various liver diseases.

[0157] <Administration Method> The cell preparation of the present invention is administered parenterally. Parenteral administration can be further divided into systemic administration and local administration. Local administration includes, for example, intraportal administration, tissue administration, intralesional administration, organ administration, intrasplenic administration, subcutaneous administration, intradermal administration, intravenous administration, and intramuscular administration. Systemic parenteral administration includes intracirculatory administration (e.g., intravenous administration (intravenous injection), intraarterial administration, and intralymphatic administration), intraperitoneal administration, etc. For example, the cell preparation of the present invention can be administered locally, in which case it can be administered directly to the target site by, for example, injection. It can also be used, for example, by incorporating it into a gel, suspension, or any other appropriate substance at the time of application. Systemic administration can be performed, for example, by intravenous injection into the circulatory system. The dosage may be any amount effective for the cells to be effective. The effective amount is appropriately selected depending on the subject information, as described above.

[0158] The application method and dosage of the cell preparation of the present invention may vary depending on information about the subject. As used herein, "subject information" refers to various information about the characteristics and condition of the subject. For example, when the subject is a human individual, information may include age, weight, sex, general health condition, presence or absence of disease, progression and severity of disease, drug sensitivity, presence or absence of concomitant drugs, and resistance to treatment.

[0159] The cell preparation of the present invention can be stored before application. The specific storage method, storage period, and storage temperature are not particularly limited.

[0160] Furthermore, the cell preparation of the present invention may be applied as is, or may be applied after any additional treatment. Specific additional treatments include, but are not limited to, application by transplantation of a reservoir containing the cell preparation of the present invention, or application by transplantation of tissues, organs, or organoids formed by further culturing the cell preparation. Further culturing can be carried out for the purpose of increasing cell number, cell differentiation, transformation, gene transfer into cells, etc.

[0161] The cell preparation of the present invention can also be used in combination with one or more known cell preparations.

[0162] <Applicable Subject> As used herein, the term "applicable subject" refers to a subject to which the cell preparation of the present invention is administered or treated. The applicable subject of the cell preparation of the present invention is not particularly limited, and may be a healthy individual or a person suffering from or in a state of some disease. The disease or state in this case is not particularly limited, but examples include diseases and states associated with liver damage. Specifically, the cell preparation of the present invention can be used as a cell preparation for improving, treating, or preventing one or more liver disorders selected from the group consisting of end-stage liver disease, cirrhosis, diabetes, obesity, acute hepatitis, chronic hepatitis, fatty liver, liver fibrosis, portal hypertension, regenerative failure, hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis, autoimmune hepatitis, liver dysfunction, hepatic blood flow disorder, acute liver failure, liver fibrosis, liver cancer, hepatic metabolic disease, traumatic liver injury, drug-induced liver injury, and liver damage due to liver failure.

[0163] The purpose of application is not particularly limited, and can be used for purposes such as amelioration, treatment, and prevention of diseases and conditions.

[0164] <Effects> By administering the cell preparation of this embodiment, the introduced hepatoblasts can be stably engrafted in the body for a long period of time, and at least a portion of them can function similarly to endogenous cells. Therefore, the cell preparation of this embodiment can be used in cell replacement therapy.

[0165] For example, the introduced hepatoblasts stably engraft in vivo for a period of 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, or 10 months or more.

[0166] The percentage of introduced cells in the recipient liver is typically 10% or more at 2 months or more after transplantation. For example, the percentage of introduced cells in the recipient liver is 10% or more, 15% or more, 20% or more, 25% or more, or 27% or more at 14 weeks or more after transplantation.

[0167] Furthermore, according to the cell preparation of this embodiment, no tumors are formed as a result of transplantation for a period of, for example, two or more months, three or more months, four or more months, five or more months, six or more months, seven or more months, eight or more months, nine or more months, or ten or more months, and therefore, a therapeutic method with a low risk of tumor formation can be provided.

[0168] The use of hepatoblasts prepared by the method described herein offers several advantages over cell replacement therapy using primary cultured mature hepatocytes. First, hepatoblasts derived from pluripotent stem cells can be stably produced regardless of the donor, allowing for a stable supply when treatment is needed. Second, the introduced hepatoblasts generate new mature hepatocytes in the recipient's body, which can be expected to have a longer-term therapeutic effect than mature hepatocytes, which have a limited number of replication cycles. Third, hepatoblasts are thought to be more resistant to liver injury than mature hepatocytes, resulting in more robust therapeutic effects after introduction. Fourth, the small size of hepatoblasts reduces the risk of immediate blood-mediated inflammatory response (BIMIR) in the portal vein region, which is often observed immediately after transplantation of mature hepatocytes.

[0169] Due to these properties, the present invention provides a promising new option for the treatment of liver failure.

[0170] In order to gain a better understanding of the present invention, specific examples are provided below for reference purposes and are not intended to be limiting of the invention.

[0171] Example 1. Induction of differentiation into hepatoblasts and evaluation of the properties of hepatoblasts (Objective) To induce differentiation of iPS cells into hepatoblasts and to examine the properties of the induced hepatoblasts.

[0172] (Methods) 1. iPS Cells. Human iPS cell lines used were M48 (CiRA Foundation), QHJI (CiRA Foundation), and 1383D6 (Riken). Human iPS cell lines were cultured in StemFit AK02N medium (Ajinomoto, RCAK02N) at 37°C and 5% CO2 on plates coated with iMatrix-511 (Nippi, 892011) mixed with PBS at a ratio of 1:150. Every 7 days, cells were dispersed using Accutase (Innovative Cell Technologies, AT104-500) and subcultured as single cells. M48 cells were cryopreserved at -196°C for 30 days in cryopreservation medium (Cellbanker1) and then reactivated.

[0173] Cultured human iPS cells were seeded in StemFit AK02N medium supplemented with 10 μM Y-27632 (Wako, 030-24026) (day 0). The medium was replaced with fresh StemFit AK02N medium on day 1 of culture, and then replaced every two days.

[0174] 2. Induction of differentiation into hepatoblasts 2-1. Induction of differentiation into definitive endoderm cells As in 1., human iPS cells (hiPSCs) were cultured at 1.5 x 10 on an iMatrix-511-coated dish. 5 cells / cm 2Cells were seeded at a density of 1000 μg / ml (day 0). They were cultured for 7 days in RPMI 1640 medium (Wako, 189-02025) supplemented with 1% B-27 supplement (50x) (Gibco, 17504-001), 100 ng / mL activin A (molecular weight: approximately 26 kDa; Ajinomoto, 18585-81), and 2 μM CHIR99021 (Cayman, 13122) or 50 ng / mL WNT3A (molecular weight: approximately 42 kDa; R&D Systems, 5036-WN-500). Only on day 0 of culture were 10 μM Y-27632 added to the medium. From days 1 to 3 of culture, 500 μM sodium butyrate (NaB; Wako, 193-01522) was added to the medium. Medium was changed daily. The differentiated cells obtained on day 7 of culture were definitive endoderm cells (hiPSC-DE).

[0175] 2-2. Hepatoblast Differentiation Induction After day 7 of culture, the medium was changed to hepatoblast differentiation medium. The hepatoblast differentiation medium was serum-free (SFD) medium supplemented with 100 nM dexamethasone (DEX: Sigma, D2915), 10 mM nicotinamide (Sigma, N636), 0.5 mM L-ascorbic acid 2-phosphate (Sigma, A8960), 10 ng / mL oncostatin M (OSM; molecular weight: approximately 30 kDa; R&D Systems, 295-OM), 10 ng / mL fibroblast growth factor 2 (FGF2; molecular weight: approximately 17 kDa; Wako, 060-04543), and 20 ng / mL hepatic growth factor (HGF; molecular weight: approximately 79 kDa; REPROCELL, 03-0019). Medium was changed daily.

[0176] The serum-free differentiation medium (SFD medium) used was a mixture of 375 mL of Iscove's modified Dulbecco's medium (Life Technologies), 125 mL of Ham's F-12K medium (Life Technologies), 5 mL of B27 supplement (Life Technologies), and 2.5 mL of N2 supplement (Life Technologies), to which 0.05% bovine serum albumin (Sigma-Aldrich), 2 mM L-glutamine (Life Technologies), 1% penicillin-streptomycin (Life Technologies), 0.45 mM monothioglycerol solution (Wako Pure Chemical Industries), and 0.5 mM L-ascorbic acid (Sigma-Aldrich) was added.

[0177] On day 13 of culture, differentiated hepatoblasts (iPSC-HBs) were collected and subjected to evaluation of the properties of hepatoblasts as described in 4.

[0178] 3. Preparation of other cells Human iPS cell-derived hepatic endoderm cells and hepatocytes were prepared by the following method. Primary hepatocytes (PHH) used were human adherent hepatocytes (Bioreclamation IVT, IVT-F00995-P or IVT-M00995-P).

[0179] 3-1. Preparation of Hepatic Endoderm Cells Definitive endoderm cells induced as described above were harvested after 6 days of culture and cultured in hepatic endoderm differentiation medium for 5 days to prepare differentiated hepatic endoderm cells (hiPSC-HE). The hepatic endoderm differentiation medium used was DMEM 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.

[0180] 3-2. Preparation of Hepatocytes hiPSC-HE were cultured for 10 days in hepatocyte differentiation medium to prepare differentiated hepatocytes (hiPSC-Hep). The hepatocyte differentiation medium used was DMED medium containing 10 ng / mL oncostatin M (OSM: R&D Systems, 295-OM), 100 nM dexamethasone (DEX: Sigma, D2915), and 5% FBS.

[0181] 4. Evaluation of Hepatoblast Properties 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 with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368814) according to the manufacturer's protocol. qPCR was performed using the CFX96 Real-Time System machine (BioRad) with TB Green Premix Ex Taq II (Takara, RR820D) according to the manufacturer's protocol.

[0182] The primers used to detect the expression of each gene are shown in Table 1. The expression level of each gene was calculated using the ΔΔCT method using ACTB as a housekeeping gene, and the fold change was calculated as a relative value to the expression level obtained from hiPSCs.

[0183] The number of samples was n = 8 for each of hiPSC, hiPSC-DE, and hiPSC-HB.

[0184]

[0185] 4-2. Flow Cytometry. Isolated cells were dispersed with 0.05% trypsin / EDTA (Gibco) and washed with DMEM containing 10% fetal bovine serum (FBS), followed by PBS. Cells were then stained with labeled antibodies for 30 minutes on ice. The samples were then washed and resuspended in FACS buffer. Flow cytometry was performed using a BD FACS Celesta Cell Analyzer or Aria III (BD Biosciences), and collected data were analyzed using FlowJo 10.7.1 (BD Biosciences) software. Standard isotype control antibodies from Biogen or BD Science were used.

[0186] The antibodies used were: FITC mouse anti-human EpCAM (BioLegend, 324203) APC / CY7 anti-human CD13 (BioLegend, 301709) BV421 mouse anti-human CD90 (BD Bioscience, 562556).

[0187] 4-3. Transcriptome Analysis. RNA integrity was assessed for the total RNA obtained as described above using an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA samples with an RNA Integrity Number (RIN) greater than 9.0 were subjected to RNA sequencing (RNA-Seq) analysis. RNA-Seq libraries were prepared from 100 ng of total RNA using the Ion AmpliSeq Transcriptome Human Gene Expression kit (Thermo Fisher Scientific, A26326) according to the manufacturer's protocol. Library sequencing was performed on an Ion Proton system using the Ion PI Hi-Q Sequencing 200 kit and Ion PI Chip v3 (Thermo Fisher Scientific, A26772). Sequencing reads were mapped to hg19_AmpliSeq_Transcriptome_ERCC_v1 using the Torrent Mapping Alignment Program. Subsequently, QC metrics and normalized read counts per gene were obtained using the AmpliSeqRNA plugin v5.2.0.3 and Torrent Suite Software v5.2.2 (Thermo Fisher Scientific). Data analysis was performed using GeneSpring (Agilent Technologies).

[0188] 5. Statistical Analysis GraphPad Prism 8.0 (GraphPad Software) was used for statistical analysis. No data were excluded from the analysis. The significance level was set at p<0.05. Flow cytometry results were tested using the Mann-Whitney t-test (two-tailed), and qPCR analysis results in this example were tested using Dunnett's multiple comparison test.

[0189] (Results) The results are shown in Figures 2 to 5.

[0190] As shown in Figure 2, gene expression analysis by qPCR revealed that the expression of typical hepatoblast-associated 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 higher in hiPSC-HBs than in pre-differentiation iPSCs (hiPSCs) and definitive endoderm cells after first differentiation (hiPSC-DE). This suggests that the hiPSC-HBs used in this study exhibit a hepatoblast-like gene expression profile.

[0191] Furthermore, comprehensive transcriptome analysis confirmed that hiPSC-HBs strongly expressed all hepatoblast signature genes, demonstrating that hiPSC-HBs are hepatoblast-like cells, as shown in Figure 3. Furthermore, when compared with the expression profiles of hepatoblast-specific markers during human liver organogenesis, the gene expression profile of hiPSC-HBs was similar to that of human embryonic liver lineage cells at 5 weeks post-conception (data not shown).

[0192] Furthermore, during the process of inducing differentiation from hiPSCs to hiPSC-HBs, we compared the induction efficiency of hiPSC-HBs when using WNT3A and when using the GSK3β inhibitor CHIR99021 to induce differentiation from hiPSCs to hiPSC-DE (Figure 4).

[0193] CD13-positive cells, a marker for fetal liver stem cells, were extracted by flow cytometry and analyzed for the expression of the hepatocyte marker epithelial cell adhesion molecule (EpCAM). As shown in Figure 4, the percentage of cells expressing low levels of EpCAM (EpCAM-low) was significantly lower with CHIR99021 (0.3±0.2%) than with WNT3A (6.5±1.6%) (Figures 4A and 4B). Therefore, the use of CHIR99021 resulted in a highly homogeneous cell population with a high EpCAM expression (EpCAM-high) of 99.7±0.2% (Figures 4A and 4C).

[0194] To characterize the cells contained in the EpCAM-low and EpCAM-high cell populations, we examined the expression of CD90, a mesenchymal cell marker, in these cell populations. As shown in Figure 5, the expression of CD90 was generally higher in the EpCAM-low cell population compared to the isotype control antibody alone, suggesting that the EpCAM-low cell population is a CD90-expressing mesenchymal cell population (Figure 5B). On the other hand, the expression of CD90 in the EpCAM-high cell population was low and comparable to the isotype control antibody, suggesting that the EpCAM-high cell population is a CD90-nonexpressing cell population (Figure 5C). These results suggest that the EpCAM-low cell population is enriched in mesenchymal stem cells rather than hepatoblasts, while the EpCAM-high cell population is a hepatoblast-like cell population. These results demonstrate that the use of CHIR99021 in the differentiation of hiPSCs into hiPSC-DE can produce a highly purified hepatoblast-like EpCAM-high cell population.

[0195] Example 2: Investigation and evaluation of a method for expanding and culturing hepatoblasts (hiPSC-HBs) (Objective) To use hepatoblasts differentiated from iPS cells (hiPSC-HBs) for the treatment of human liver disorders, it is necessary to prepare large quantities of hiPSC-HBs. Therefore, we investigated culture conditions for expanding hiPSC-HBs while maintaining their undifferentiated state.

[0196] (Method) 1. Examination of proliferation culture hiPSC-HB differentiated from hiPSC by the method using CHIR99021 described in Example 1 was dispersed in 0.05% trypsin / EDTA (Gibco) and cultured at 1 × 10 4 cells / cm 2The cells were seeded onto new dishes at a density of 10 μM and cultured in candidate growth medium. The candidate growth medium was SFD medium supplemented with 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), with the additional ingredients listed in Table 2. Medium was changed every two days.

[0197]

[0198] 2. Passaging in expansion culture hiPSC-HB cells were passaged when they reached approximately 90% confluence. TrypLE Express Enzyme (Gibco, 12604013) was used to detach and disperse the cells. 1 × 10 4 cells / cm 2 Cells were seeded onto new dishes at a density of 100 μg / cm2, and a maintenance candidate medium with the same composition as the culture medium before passage was used. At each passage, the number of seeded cells and the total number of cells immediately before the next passage were counted, 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 multiplying the number of hepatoblasts before passage (1) by the proliferation rate at each passage.

[0199] 3. Evaluation of Cell Properties qPCR analysis, flow cytometry, transcriptome analysis, and statistical analysis were performed as in Example 1. In qPCR analysis, fold changes were calculated as relative values ​​to the expression levels in hiPSC-HB before maintenance culture.

[0200] 4. Statistical Analysis Statistical analysis was performed by the method described in Example 1. Testing of the results of qPCR analysis was performed by Dunnett's multiple comparison test.

[0201] (Results) The results are shown in FIGS. 6-1 to 6-3 and 7.

[0202] When the candidate proliferation medium contained the WNT3 signaling activator CHIR99021 in addition to Y-27632 and the TGFβ / Smad inhibitor A8301, as well as EGF and FGF2, 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 the expansion culture (Figure 6-1, "YAW+EGF+FGF2"). On the other hand, when WNT3A was used in addition to Y-27632 and A8301, and the growth factors EGF and / or FGF2 and DEX were added (Figure 6-1, "YAW+EGF+FGF2," "YAW+EGF," and "YAW+EGF+DEX"), the gene expression profile was generally similar to that of hiPSC-HB cells. In particular, cells cultured in a hepatoblast maintenance medium containing WNT3A, EGF, and DEX (hereinafter also referred to as "YAW+EGF+DEX") exhibited a gene expression profile similar to that of hiPSC-HB.

[0203] When WNT3A was used in addition to Y-27632 and A8301 in the candidate proliferation medium, a high proliferation rate was observed when additional growth factors (FGF2, HGF, EGF) were added (Figure 6-2, "Proliferation rate"). Furthermore, the expression levels of hepatoblast-related genes in the cultured cells were similar to those of hepatoblasts cultured under the "YAW + EGF + DEX" condition (Figure 6-2). In particular, when EGF was used as a growth factor and the glucocorticoid DEX was also added, a high proliferation rate was observed while maintaining the expression levels of hepatoblast-related genes (Figure 6-2, "WNT + EGF + DEX").

[0204] To confirm the feasibility of replacing WNT3A with CHIR99021 in the candidate proliferation medium "YAW+EGF+DEX," we added CHIR99021 at concentrations of 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 medium supplemented with 0.5 μM ("CHIR-0.5") and 1 μM ("CHIR-1") CHIR99021 were similar to those in hiPSC-HBs before proliferation, as were the cases when WNT3A was added ("WNT3A-50") (Figure 6-3).

[0205] 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 storage method (Figure 7).

[0206] Furthermore, to identify potential ROCK inhibitors, TGFβ / Smad inhibitors, and WNT3 signaling activators for potential growth media, we examined the proliferation ability of hiPSC-HBs at passage 6 using various compounds. Thiazovivin (Figure 6-4, "Thiazovivin") and Rho Kinase Inhibitor IV (Figure 6-4, "Rho") were used as ROCK inhibitors (Figure 6-4). SB 431542, SB 505124, RepSox, LY364947, and LY2157299 were used as TGFβ signaling pathway inhibitors (Figure 6-5). BIO, TWS119, SB216763, and Recombinant R-Spondin 1 (Figure 6-6, "R-Spondin") were used as Wnt signaling pathway activators (Figure 6-6). After 7 days of culture, cells were detached and counted. The proliferation rate was calculated as the percentage of the number of cells counted relative to the number on day 1 of culture. As a result, the proliferation and culture ability of hiPSC-HB was maintained when any of the compounds was used (Figures 6-4 to 6-6).

[0207] Example 3. Gene Expression Profile of Hepatoblasts During Expansion Culture To assess whether hepatoblasts during expansion culture were similar to hiPSC-HB cells before expansion culture, we assessed the change in gene expression profile over time as a function of passage number. Cells were passaged using medium supplemented with Y27623, A8301, WNT3A, EGF, and DEX. Expression levels of CD13 and EpCAM were analyzed in cells after one passage (P1, Figure 8A), five passages (P5, Figure 8B), ten passages (P10, Figure 8C), and fifteen passages (P15, Figure 8D). Even after 15 passages, the proportion of EpCAM-high cells among CD13-positive cells remained high at over 98%, demonstrating a profile similar to that of hiPSC-HB cells before expansion culture (Figure 8D).

[0208] Furthermore, analysis of the expression patterns of hepatoblast signature genes revealed no significant changes in the gene expression profile even after 15 passages (Figure 9). Specifically, in hepatoblasts at P1, P5, P10, and P15 (represented by "HB P0," "HB P1," "HB P5," "HB P10," and "HB P15," respectively), the membrane proteins SLC4A4, TNFSF4, IL1RAPL1, and NRP1, the transcription factors SOX9 and ONECUT1, and the intracellular proteins PLEKHA2, RNF207, and LYST were all highly expressed. In contrast, expression of these genes was not observed in other cell types (represented by "hiPSC," "DE," "HE," and "MH"). These findings suggest that the expression of SLC4A4, TNFSF4, IL1RAPL1, NRP1, SOX9, ONECUT1, PLEKHA2, RNF207, and LYST is characteristic of hepatoblasts. Combined with the results in Figure 3, it was shown that the hiPSC-HBs of the present invention are characterized by the expression of SLC4A4, TNFSF4, IL1RAPL1, NRP1, SOX9, ONECUT1, PLEKHA2, RNF207, NID1, and LYST in addition to the typical hepatoblast gene markers AFP, DLK, ALB, TBX3, c-MET, and EpCAM.

[0209] Furthermore, the results of principal component analysis of the gene expression profiles confirmed that the gene expression profiles of P1, P5, P10, and P15 were similar, and the expression profiles of hiPSC-HB did not change significantly even after multiple passages (Figure 10).

[0210] The above results indicate that hiPSC-HB can be passaged for more than 15 times while retaining its original phenotype, and the number of cells can be increased by 10 16 This shows that it can be expanded further.

[0211] Example 4: Induction of differentiation of hepatocytes and bile duct cells from hiPSC-HB in vitro (Objective) To induce differentiation of hepatoblasts (hiPSC-HB) differentiated from iPS cells into hepatocytes and bile duct cells in vitro and evaluate their properties.

[0212] (Method) 1. Induction of differentiation into hepatocytes (HB-Hep) When hiPSC-HB reached 90% confluence, the medium was replaced with hepatocyte differentiation medium and cultured for 10 days. The hepatocyte differentiation medium used was DMEM medium (Wako, 043-30085) supplemented with 5% FBS, 10 ng / mL oncostatin M, and 100 nM DEX. This medium is commonly used for differentiating hepatoblasts into hepatocytes.

[0213] 2. Induction of differentiation into cholangiocytes (HB-cholangiocytes) When hiPSC-HB reached 90% confluence, the cells were harvested and plated onto iMatrix-511-coated dishes at a density of 1 × 10 4 cells / cm 2The cells were seeded at a density of 1 × 10 cells per well and cultured for 10 days in cholangiocyte differentiation medium. The cholangiocyte differentiation medium was SFD medium supplemented with 10 ng / mL EGF (molecular weight: approximately 6.2 kDa; Sigma, E9644), 20 ng / mL HGF, 50 ng / mL WNT3A, 100 ng / mL R-spondin 1 (molecular weight: approximately 14 kDa; R&D Systems, 4645-RS / CF), 50 ng / mL FGF10 (molecular weight: approximately 19 kDa; R&D Systems, 345-FG), 3 μM retinoic acid (Wako, 182-01116), 10 μM Y-27632, and 10 μM forskolin. The cells were cultured at a density of 1 × 10 cells per well in cholangiocyte differentiation medium. 4 cells / cm 2 Cells were seeded onto iMatrix-511-coated dishes at a density of 1000 μg / ml. Medium was changed every 2 days.

[0214] 3. Evaluation of cell properties Cells were harvested on day 10 of culture in each differentiation-inducing medium and subjected to qPCR analysis. The primers used for qPCR analysis were those used in Example 1 and those listed in Table 3. The fold change was calculated as a relative value to the expression level in hiPSC-HB.

[0215]

[0216] 4. Statistical Analysis Statistical analysis was performed as described in Example 1. Tests for the qPCR analysis in this example were performed using the Mann-Whitney t-test (two-tailed test).

[0217] (Results) The results are shown in FIG.

[0218] Culture in hepatocyte differentiation medium significantly increased the expression of hepatic maturation genes, including α-1-antitrypsin (A1AT), ALB, carbamoylphosphate synthase 1 (CPS1), cytochrome P450 3A4 (CYP3A4), and glucose-6-phosphatase catalytic subunit (G6PC), demonstrating the promotion of hepatocyte differentiation (Figure 11A). Culture in cholangiocyte differentiation medium also significantly increased the expression of cholangiocyte-specific genes, including keratin 19 (KRT19), γ-glutamyltransferase 1 (GGT1), aquaporin 1 (AQP1), and somatostatin receptor 2 (SSTR2), demonstrating the promotion of cholangiocyte differentiation (Figure 11B). Meanwhile, expression of the liver immaturity gene AFP was decreased in these cells.

[0219] Example 5. Induction of differentiation of liver tissue from hiPSC-HB in vivo (Objective) Hepatoblasts differentiated from iPS cells (hiPSC-HB) were transplanted into mice, and differentiation into liver tissue and the function of the differentiated liver tissue were confirmed.

[0220] (Methods) 1. Generation of Liver Failure Model Mice Adult male HSVtk transgenic immunodeficient NOG (TK-NOG) mice (8-10 weeks old, In vivo Science) were used for this experiment. To induce liver damage, mice were intraperitoneally administered 15 mg / kg of ganciclovir (GCV; Mitsubishi Tanabe Pharma) 12 days (D-12) and 14 days (D-14) before transplantation (Figure 12A). Mice were housed in a specific pathogen-free (SPF) animal facility with a temperature-controlled, 12-hour light / dark cycle. Water and food were available ad libitum throughout the study period. All animal experiments and care were conducted in accordance with guidelines (PA22-05) approved by the Institutional Review Board of the Animal Research Center, The University of Tokyo.

[0221] 2. Transplantation of hiPSC-HBs into a Liver Failure Model Mouse Before transplantation, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using DRI-CHEM (Fujifilm, Tokyo, Japan) according to the manufacturer's protocol. All mice were anesthetized with isoflurane (Wako, 095-06573).

[0222] 1×10 6 The hiPSC-HB cells were transplanted into TK-NOG mice via intrasplenic injection. Serum was collected every 4 weeks after transplantation to measure human ALB levels. Mice were sacrificed at the required time points for tissue analysis.

[0223] 3. Measurement of human ALB levels Human ALB was measured using a human albumin ELISA quantification set (Bethyl Laboratories, E80-129).

[0224] 4. Evaluation of the maturation properties of transplanted cells Livers were collected from mice 40 weeks after hiPSC-HB transplantation and subjected to qPCR analysis. Primers used for qPCR analysis were those used in Example 1, as well as primers binding to FAH, CYP2C9, and CYP2C19, prepared according to standard methods. Fold changes were calculated relative to the expression levels in hiPSC-HB.

[0225] 5. Statistical Analysis Statistical analysis was performed as described in Example 1. Tests for the qPCR analysis in this example were performed using Dunnett's multiple comparisons test (two-tailed test).

[0226] (Results) Human ALB production increased over time from 8 to 40 weeks after hiPSC-HB transplantation (Figure 12-1). This result suggests that hiPSC-HB cells have the ability to engraft and repopulate in the recipient liver for a long period of time.

[0227] To examine the maturation of hiPSC-HBs in the transplanted tissue 40 weeks after transplantation, we performed qPCR analysis using human-specific primers. The results showed that in the reconstructed liver tissue composed of transplanted human cells (hiPSC-HB engraftment), the expression of human immature liver genes (AFP and KRT19) was significantly reduced, while the expression of human liver function genes, such as ALB, ornithine transcarbamylase (OTC), fumarylacetoacetate hydrolase (FAH), CYP3A4, CYP2C9, and CYP2C19, was significantly increased. These expression levels were comparable to those of primary human hepatocytes (PHHs) (Figure 12-2).

[0228] Furthermore, we investigated whether the transplanted hiPSC-HBs were integrated into the mouse liver by immunostaining and gene expression analysis, and the results confirmed that the hiPSC-HB grafts were integrated into the mouse liver (data not shown).

[0229] These results demonstrate that transplantation of hiPSC-HBs into mice with liver failure resulted in the transplanted cells repopulating the liver and maturing into adult hepatocytes, suggesting that hiPSC-HBs may be useful in treating liver failure in humans.

[0230] Example 6. Exposure to human OSM enhances the liver repair capacity of hiPSC-HBs. (Background) In Example 5, when hiPSC-HBs were transplanted into a mouse model of liver damage, human ALB secretion decreased between weeks 1 and 8 after transplantation, and by week 40 after transplantation, the human tissue replacement rate was only approximately 1%. These results suggest that further improvement of the tissue-building capacity of transplanted hiPSC-HBs is necessary to treat human liver disease.

[0231] OSM (oncostatin M) is known to play a key role in promoting hepatocyte proliferation and maturation during fetal liver development. It has also been reported to promote hepatoblast proliferation and maturation. To assess whether treatment with mouse and human OSM differentially affects hiPSC-HBs, we analyzed gene expression profiles of hiPSC-HBs exposed to human and mouse OSM separately. Human OSM treatment significantly increased the expression of liver function genes (ALB, CPS1, CYP3A4, and G6PC) and enhanced ALB production. However, similar stimulatory effects were not observed in the mouse OSM-exposed group. Furthermore, phosphorylation of STAT3, a downstream signaling pathway of the human OSM receptor expressed in hiPSC-HBs, was specifically activated only by human OSM. Transcriptome analysis showed that human OSM-treated hiPSC-HBs exhibited enriched expression of signatures related to the farnesoid X receptor pathway, nuclear receptor meta-pathway, and pregnane X receptor pathway, which play important roles in hepatocyte function (data not shown). These results suggest that stimulation with human OSM is required for hiPSC-HB differentiation.

[0232] Based on the above results, we concluded that in order to accurately evaluate the therapeutic effect of hiPSC-HB transplantation on mouse liver damage in a mouse model, the mice must express human OSM. Therefore, we devised a method to evaluate the therapeutic effect of mouse liver dysfunction in vivo by exogenously supplementing mice with human OSM, thereby simulating the environment during hiPSC-HB transplantation into the human liver. Furthermore, we hypothesized that stimulating hiPSC-HB with human OSM before transplantation might further enhance engraftment ability. Therefore, we performed the following study.

[0233] (Methods and Results) 1. TK-NOG hOSM Generation of a mouse model of lethal liver failure TK-NOG expressing human OSM in liver and other tissues hOSM To generate mice, 1 × 10 9AAV8-hOSM (Vectorbuilder) was administered to TK-NOG mice via the tail vein at a titer of GC / mL. These mice were confirmed to produce human OSM at levels close to physiological concentrations in serum (data not shown). To establish the liver failure model, TK-NOG mice were injected with AAV8-hOSM (Vectorbuilder). hOSM Retrorsine (Sigma, R382), a pyrrolizidine alkaloid known to inhibit endogenous hepatocyte regeneration, was intraperitoneally administered to mice at a dose of 70 mg / kg twice, 42 days (D-42) and 28 days (D-28) before transplantation. hOSM A mouse model of fatal liver failure was established (Fig. 13A). Histological analysis was performed using the same method as in Example 5. As a result, the livers of mice administered with leucocytamine exhibited enlarged hepatocytes, and two weeks after GCV administration, Hnf4a + Ki67 + The cell population decreased.

[0234] 2. Pretreatment of hiPSC-HB with human OSM hiPSC-HB cells were cultured at 1×10 4 cells / cm 2 The cells were seeded onto new dishes at a density of 100 μg / mL and cultured for 7 days or more in the hepatoblast maintenance medium (YAW+EGF+DEX) containing WNT3A, EGF, and DEX prepared in Example 2, supplemented with human OSM at a concentration of 10 ng / mL.

[0235] 3. Therapeutic Effects of hiPSC-HB Transplantation on Liver Disease As shown in Figure 13B, when hiPSC-DE (n = 3) or hepatocytes differentiated from hiPSC-HB (hiPSC-HB-Hep) were transplanted (n = 3), the level of human ALB production from the transplanted cells remained unchanged or decreased compared to the level at the time of transplantation. However, when hiPSC-HB (n = 7) were transplanted into mice, the level of human ALB production increased and remained at a high level after transplantation (Figure 13B). This indicates that in an environment where human OSM is expressed in the mouse liver, hiPSC-HB can engraft for a long period of time and regrow and mature in vivo. However, the improvement in liver function observed with hiPSC-HB transplantation was not observed when cells differentiated earlier than hiPSC-HB (hiPSC-DE) or more differentiated cells (hiPSC-HB-Hep) were transplanted.

[0236] In mice with induced liver failure, 12 out of 13 died within 2 months (Fig. 14A, "Sham"), and significant weight and liver weight loss were observed, as well as increases in serum alanine transaminase (ALT), aspartate aminotransferase (AST), total bilirubin (T-Bil), and direct bilirubin (D-Bil) (Fig. 14B, "Sham"), indicating destruction of liver tissue structure. On the other hand, when hiPSC-HBs were transplanted, TK-NOG was not observed, as was observed in mice transplanted with primary hepatocytes (PHHs). hOSM The survival rate of the liver failure model was significantly improved (Fig. 14A), and body and liver weights were significantly restored, along with significant decreases in serum levels of ALT, AST, T-Bil, and D-Bil (Fig. 14B). The human cell clusters derived from hiPSC-HBs expanded over time, and the proportion of human cell clusters in the recipient liver increased from an average of 3.49% at 6 weeks post-transplantation to an average of 10.80% at 8 weeks post-transplantation, exceeding 10%. At 14 weeks post-transplantation, the engraftment rate averaged 27.13%.

[0237] The liver tissue composed of human cells reconstructed in vivo by repopulating and maturing hiPSC-HBs was observed using a transmission electron microscope (Figure 15). Bile canaliculi (BC) were formed between the apical membrane of human hepatocytes (Figure 15A), sinusoids surrounded by the basal membrane of human hepatocytes (Figure 15B), Disse spaces between sinusoids and hepatocytes (Figure 15C), and tight junctions between hepatocytes (Figure 15D). These findings demonstrate that transplantation of hiPSC-HBs results in the formation of complex liver tissue with the ultrastructure seen in normal adult liver tissue.

[0238] Mice transplanted with hiPSC-HB were monitored for over 40 weeks, and no tumor formation was observed during this period.

[0239] These results demonstrate that transplantation of hiPSC-HBs differentiated or expanded by the method of the present invention improves liver function to a similar extent as transplantation of primary hepatocytes obtained from an individual. Because hiPSC-HBs can be generated from autologous iPS cells, they are highly useful as cells for liver transplantation, capable of long-term engraftment and capable of reconstructing advanced tissues without the risk of rejection.

[0240] 4. Effect of human OSM The results are shown in Figure 16. To further investigate whether human OSM enhances the repopulation ability of hiPSC-HBs, human OSM was contacted with hiPSC-HBs in three ways: in vitro, in vivo, or in vitro and in vivo (Figure 16-1). In vitro, human OSM was added to the growth medium of hiPSC-HBs. In vivo, the TK-NOG constructed in Example 6 was used. hOSM ) was used.

[0241] Notably, human ALB production was highest in hiPSC-HB transplanted mice exposed to human OSM both in vitro and in vivo, with human ALB production increasing approximately 100-fold at 20 weeks post-transplantation (Figure 16-2). Furthermore, there was no difference in the effect even when hiPSC-HBs of different passages (P2 and P10) were transplanted (data not shown). Significant improvement in liver failure was also observed when hiPSC-HBs derived from the other two hiPSC lines were transplanted, demonstrating the presence of hALB. + hGAPDH + The cell engraftment rates reached 29.56% and 33.98%, respectively. These results strongly suggest that transplantation of hiPSC-HBs can effectively reconstruct the damaged liver and rescue mice from liver failure.

[0242] Consistent with findings in human livers, rifampicin-induced hCYP3A4 expression and human hepatocyte regeneration following 70% hepatectomy were also detected in hiPSC-HB-derived engraftments. To further confirm the liver function of repopulated hepatocytes in vivo, we intraperitoneally injected repopulated mice with 350 mg / kg acetaminophen, a lethal dose for mice with liver failure not transplanted with hiPSC-HBs. Surprisingly, four out of five mice repopulated with hiPSC-HBs survived and showed significant recovery of liver function. This indicates that hiPSC-HBs repopulated in mouse livers exerted a protective effect against acetaminophen-induced acute liver failure. Furthermore, transmission electron microscopy revealed that repopulated hiPSC-HBs exhibited ultrastructural features of adult liver tissue, such as bile ducts formed between the apical membranes of human hepatocytes, sinus chambers surrounded by the basolateral membranes of human hepatocytes, Disse spaces between the sinus chambers and hepatocytes, and tight junctions between hepatocytes.

[0243] The above results suggest that transplantation of hiPSC-HBs into humans with liver damage can lead to the maturation of functionally competent human hepatocytes with histoarchitectural characteristics of liver failure, and cell preparations containing hiPSC-HBs are expected to be effective in treating liver failure.

[0244] Example 7. Changes in hiPSC-HB Properties Following Exposure to Human OSM Example 6 demonstrated that culturing hiPSC-HBs in growth medium supplemented with human OSM for 7 days or longer can produce cells that improve liver function to a similar extent as transplantation of primary hepatocytes obtained from other individuals. To investigate the profile of these hiPSC-HBs cultured in medium supplemented with human OSM for 7 days or longer, cells were harvested on day 7 of culture in each differentiation-inducing medium and subjected to RNA sequencing analysis. Fold changes were calculated relative to the expression levels in hiPSC-HBs cultured in growth medium. As a result, it was found that culturing in a medium supplemented with human OSM significantly increased 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-HBs of the present invention, as well as the expression of new genes listed in Table 4, such as REG3A, SAA1, SAA2, HP, SPINK1, LBP, KCNG2, ANXA10, and ITIH3.

[0245]

[0246] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A method for proliferating hepatoblasts, comprising a growth culture step of proliferating hepatoblasts in a hepatoblast growth medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor.

2. A method for producing a hepatoblast cell population, comprising an expansion culture step of expanding and culturing hepatoblasts in a hepatoblast cell expansion medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator and a growth factor.

3. The method according to claim 1 or 2, wherein 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, wherein the ROCK inhibitor is Y-27632, Rho Kinase Inhibitor IV or thiazovivin.

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

6. The method according to any one of claims 1 to 5, wherein the Wnt signaling 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, wherein the hepatoblast growth medium further comprises a steroid agent.

8. The method of claim 7, wherein the steroid drug is a glucocorticoid or a derivative thereof.

9. The method of claim 8, wherein the glucocorticoid or a derivative thereof is dexamethasone.

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

11. The method of claim 10, wherein the hepatoblasts further express one or more genes selected from the group consisting of SLC4A4, TNFSF4, PLEKHA2, and RNF207.

12. A method for producing a cell preparation for use in treating liver disease, comprising the method according to any one of claims 1 to 11.

13. A method for producing a cell preparation for use in the treatment of liver disease, comprising: a first step of inducing the differentiation of pluripotent stem cells into definitive endoderm cells; a second step of inducing the differentiation of the definitive endoderm cells into hepatoblasts; and a third step of expanding and culturing the hepatoblasts to produce a hepatoblast cell population, wherein the first step comprises culturing the hepatoblasts in a serum-free culture medium containing Y-27632 in the early culture period and culturing them in a medium containing B-27 supplement, activin A, and a WNT signaling pathway activator in the intermediate culture period or later; the second step comprises culturing the hepatoblasts in a serum-free medium containing FGF2, HGF, oncostatin M, dexamethasone, and nicotinamide; and the third step comprises an expansion and culture step of expanding and culturing the hepatoblasts in a hepatoblast growth medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor.

14. The method for producing a cell preparation according to claim 13, further comprising, after the third step, a maturation promotion step in which oncostatin M is added to the hepatoblasts cultured for expansion and culturing.

15. A method for treating a liver disease, comprising the step of administering to a subject a cell preparation containing hepatoblasts cultured and expanded in a hepatoblast proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor.

16. The method of treatment according to claim 15, wherein the cell preparation comprises hepatoblasts that have been cultured for expansion and whose maturation has been promoted by the addition of oncostatin M.

17. A method for producing hepatocytes, comprising: a proliferation and culture step of proliferating hepatoblasts in a hepatoblast proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor; and a hepatocyte induction step of inducing differentiation of the proliferated hepatoblasts into hepatocytes.

18. A method for producing cholangiocytes, comprising: a proliferation and culture step of proliferating hepatoblasts in a hepatoblast proliferation medium containing a ROCK inhibitor, a TGFβ signaling pathway inhibitor, a Wnt signaling pathway activator, and a growth factor; and a cholangiocyte induction step of inducing differentiation of the proliferated hepatoblasts into cholangiocytes.

19. A cell preparation for use in treating liver disease, comprising cells expressing IL1RAPL1, NRP1, SOX9, ONECUT1, NID1 and LYST.

20. The cell preparation of claim 19, wherein the cells 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 proliferating hepatoblasts, comprising a growth culture step of proliferating hepatoblasts in a hepatoblast growth medium containing a ROCK inhibitor, and a TGFβ signaling pathway inhibitor or a Wnt signaling pathway activator, and a growth factor.

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