Method for producing hepatic stem cells

Abstract

The present invention provides a method for producing hepatic stem cells, the method including a step for culturing hepatocytes in a medium containing a Wnt signaling promoting substance and an FGF signaling promoting substance. The present invention also provides a method for producing hepatic stem cells, the method including a step for culturing hepatic stem cells in a medium containing a Wnt signaling promoting substance, an FGF signaling promoting substance, and an ALK inhibitor.

Description

Method for producing hepatic stem cells

[0001] The present invention relates to a method for producing hepatic stem cells, hepatic stem cells produced by said method, and a cell transplantation therapeutic agent containing said cells.

[0002] While severe cases of congenital metabolic disorders have a poor short-term prognosis, even non-severe cases can cause central nervous system disorders. For example, urea cycle disorders, a typical example of congenital metabolic disorders, are a group of liver diseases that result in hyperammonemia due to a congenital enzyme deficiency that prevents the liver from metabolizing ammonia produced in the body during the process of taking it up and producing urea. Urea cycle disorders occur in 1 in 8,000 to 44,000 people in Japan and are designated as intractable diseases.

[0003] Liver transplantation is the only curative treatment for congenital liver diseases such as those described above. However, there is currently a severe shortage of transplantable liver grafts worldwide. In particular, in East Asian countries, including Japan, brain-dead donors are limited, and transplants rely heavily on living donors. Furthermore, living donor liver transplantation is burdensome for newborns due to the small intraperitoneal volume and the complex vascular anastomosis procedures required. Hepatocyte transplantation, in addition to liver transplantation, is considered an effective treatment for liver disease because it is minimally invasive and can be applied to newborns, for whom transplantation of even a reduced-weight donor liver is difficult. However, for transplanted hepatocytes to survive long-term in the patient's liver, they must pass through the sinusoidal endothelial cells and Kupffer cells that make up the sinusoidal endothelium, integrate into the hepatic parenchyma, compete with the patient's own hepatocytes, and proliferate at a rate that exceeds their own. However, in practice, maintaining and maintaining high-quality hepatocytes isolated from donor livers in vitro presents a challenge. Furthermore, because isolated hepatocytes have low proliferation potential, even if transplantation is successful, they do not survive long-term in the liver, resulting in limited therapeutic efficacy. Hepatocytes for transplantation can be obtained not only by isolation from donor livers, but also by differentiation from pluripotent stem cells. Previously, successful differentiation into hepatocyte-like cells (HLCs) by processing human induced pluripotent stem cells (iPSCs) has been reported (Non-Patent Document 1). However, even iPSC-derived HLCs exhibit a decline in proliferation upon maturation, similar to isolated hepatocytes (Non-Patent Document 2), and therefore the above-mentioned problem cannot be resolved.

[0004] Therefore, developing a method to confer proliferation ability to hepatocytes in vitro would be an important breakthrough for making hepatocyte transplantation an effective treatment. However, the methods reported so far require many factors, and a simpler method that makes it easier to control cells has been sought.

[0005] Si-Tayeb K, Noto FK, Nagaoka M et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 2010; 51: 297-305. Zhang R, Takebe T, Sekine K et al. Identification of proliferating human hepatic cells from human induced pluripotent stem cells. Transplant Proc 2014; 46: 1201-1204.

[0006] Therefore, an objective of the present invention is to provide a method for producing hepatic stem cells with proliferation capacity applicable to hepatic cell transplantation, as well as to provide hepatic stem cells produced by the method and cell transplantation therapeutic agents containing the cells.

[0007] As mentioned above, isolated hepatocytes and iPSC-derived hepatocytes have problems with proliferation. Based on several reports indicating that both Wnt / β-catenin signaling and FGF signaling play important roles in liver regeneration, the present inventors hypothesized that treating iPSC-derived hepatocytes with Wnt signaling promoters and FGF signaling promoters might confer proliferation potential to hepatocytes. Treatment of iPSC-derived hepatocytes, generated according to a slightly modified previously reported method, with the Wnt signaling promoter CHIR99021 and the FGF signaling promoter bFGF demonstrated the production of proliferative, undifferentiated hepatocytes (hepatic stem cells). However, after a single passage of these hepatic stem cells, we confirmed that TGF-β signaling was activated, leading to senescence and cessation of proliferation.

[0008] The present inventors found that hepatic stem cells cultured in a medium containing CHIR99021, bFGF, and the ALK inhibitor SB431542 maintained their proliferation potential. Therefore, culturing hepatic stem cells in a medium containing a Wnt signaling promoter, an FGF signaling promoter, and an ALK inhibitor produced hepatic stem cells that could be expanded by subculture. Furthermore, the hepatic stem cells were able to redifferentiate into hepatocytes. These results suggest that the Wnt signaling promoter, the FGF signaling promoter, and the ALK inhibitor primarily revert hepatocytes to more primitive endodermal cells, confer proliferative capacity, and suppress aging, respectively.

[0009] The present inventors further transplanted hepatic stem cells into the normal livers of unconditionally immunodeficient mice and confirmed that the hepatic stem cells could be engrafted in the liver. Therefore, the hepatic stem cells of the present invention were demonstrated to be suitable as transplant cells. Based on these findings, the present inventors conducted further research and completed the present invention.

[0010] That is, the present invention provides the following: [1] A method for producing hepatic stem cells, comprising the step of culturing hepatic cells in a medium containing a Wnt signaling promoter and an FGF signaling promoter. [2] The method according to [1], wherein at least one of the Wnt signaling promoters is CHIR99021. [3] The method according to [1] or [2], wherein at least one of the FGF signaling promoters is bFGF. [4] A method for producing hepatic stem cells, comprising the step of culturing hepatic stem cells in a medium containing a Wnt signaling promoter, an FGF signaling promoter, and an ALK inhibitor. [5] The method according to [4], wherein at least one of the ALK inhibitors is SB431542. [6] The method according to [4] or [5], wherein the starting hepatic stem cells are cells obtained by the method according to any one of [1] to [3]. [7] The method according to any one of [1] to [6], wherein the hepatic cells or hepatic stem cells are derived from pluripotent stem cells. [8] The method according to [7], wherein the pluripotent stem cells are derived from humans. [9] Hepatic stem cells obtained by the method according to any one of [1] to [8]. [10-1] Hepatic stem cells having all of the following properties (A) to (C): (A) derived from pluripotent stem cells; (B) expressing SOX17 and Axin2; and (C) having the ability to differentiate into hepatocytes. [10-2] Hepatic stem cells having all of the following properties (A') to (C'): (A') derived from pluripotent stem cells; (B') expressing SOX17 and / or HNF4α; and (C') having the ability to differentiate into hepatocytes.

[11] The cells according to [10-1] or [10-2], further having the property (D) being positive for at least one selected from the group consisting of LGR5, HNF4α, phosphorylated retinoblastoma protein, and unphosphorylated β-catenin.

[12] A method for producing hepatocytes, comprising the step of inducing the differentiation of the hepatic stem cells according to any one of [9] to

[11] into hepatocytes.

[13] The method according to

[12] , wherein the step of inducing differentiation of hepatic stem cells into hepatocytes comprises a step of culturing the hepatic stem cells in a medium containing an ALK inhibitor but not containing a Wnt signaling promoter.

[14] A cell transplantation therapeutic agent comprising the cells according to any one of [9] to

[11] or the cells obtained by the method according to

[12] or

[13] .

[15] The agent according to

[14] for treating liver disease.

[16] A method for treating liver disease, comprising transplanting an effective amount of the cells according to any one of [9] to

[11] or the cells obtained by the method according to

[12] or

[13] into a subject.

[17] The cells according to any one of [9] to

[11] or the cells obtained by the method according to

[12] or

[13] for use in treating liver disease.

[18] Use of the cells according to any one of [9] to

[11] or the cells obtained by the method according to

[12] or

[13] in the manufacture of a therapeutic agent for liver disease.

[0011] The present invention makes it possible to provide hepatic stem cells that have the ability to proliferate in vitro, and is expected to be applicable to hepatic cell transplantation, which has previously posed a problem in terms of long-term engraftment.

[0012] Figure 1 shows the 12-day differentiation scheme for iPSCs into hepatocyte-like cells (HLCs). Figure 2 shows flow cytometry analysis of the iPSC-to-HLC differentiation process. (A) Day 0, (B) Day 4, (C) Day 8, and (D) Day 12. Figure 3 shows Western blot analysis of the iPSC-to-HLC differentiation process. Figure 4 shows the design of Superfolder GFP knock-in into the albumin locus using the CRISPR-Cas9 system. Figure 5 shows flow cytometry and immunocytochemistry analysis of iPSCs bearing sfGFP-conjugated ALB before differentiation and HLCs after 20 days of differentiation. (A) Flow cytometry analysis before differentiation, (B) Flow cytometry analysis at day 20 after differentiation, and (C) Immunocytochemistry analysis at day 20 after differentiation. Figure 6 shows quantitative RT-PCR analysis of HLCs differentiated for 12 days and then cultured for 1 day in the presence of CHIR99021 or bFGF. (A) TCF7, (B) LEF1. Figure 7 shows Western blot analysis of HLCs differentiated for 12 days and then cultured for 2 days in the presence of CHIR99021 or bFGF. Figure 8 shows quantitative RT-PCR analysis of HLCs differentiated for 12 days and then cultured for 1 day in the presence of CHIR99021 or bFGF. (A) Sox17, (B) Foxa2, (C) HNF4α, (D) Albumin. Figure 9 shows quantitative RT-PCR analysis of HLCs differentiated for 12 days and then cultured for 1 day in the presence of CHIR99021 or bFGF. (A) cyclin D1, (B) c-Myc. Figure 10 shows Western blot analysis of HLCs after 12 days of differentiation and then cultured for two days in the presence of CHIR99021 or bFGF. Figure 11 shows immunocytochemistry of HLCs induced by 12 days of differentiation of iPSCs bearing sfGFP-conjugated ALBs and then cultured for eight days in the presence of HGF or CHIR99021. Figure 12 shows CCK-8 assay of HLCs after 12 days of differentiation and then cultured for four days in the presence of CHIR99021 and / or bFGF.Figure 13 shows quantitative RT-PCR analysis of HLCs after 12 days of differentiation followed by 1 day of culture in the presence of CHIR99021 alone or both CHIR99021 and bFGF. (A) cyclin D1, (B) c-Myc. Figure 14 shows quantitative RT-PCR analysis of HLCs after 12 days of differentiation followed by 1 day of culture in the presence of CHIR99021 alone or both CHIR99021 and bFGF. (A) Sox17, (B) HNF4α, (C) AFP, (D) Albumin. Figure 15 shows Western blot analysis of HLCs after 12 days of differentiation followed by 2 days of culture in the presence of CHIR99021 alone or both CHIR99021 and bFGF. Figure 16 shows immunocytochemical analysis of Sox17 and cyclin D in HLC cells cultured for 2 days in the presence of (A) CHIR99021 alone or (B) both CHIR99021 and bFGF after 12 days of differentiation. Figure 17 shows immunocytochemical analysis of Sox17 and phosphorylation of the retinoblastoma protein in HLC cells cultured for 2 days in the presence of (A) CHIR99021 alone or (B) both CHIR99021 and bFGF after 12 days of differentiation. Figure 18 shows flow cytometry analysis of HLC cells cultured for 8 days in the presence of CHIR99021 and bFGF after 12 days of differentiation. Figure 19 shows light microscopic images of HLC cells cultured for 8 days in the presence of CHIR99021 and bFGF after 12 days of differentiation and passaged. (A) Immediately before passaging; (B) Day 4 after passaging. Figure 20 shows quantitative RT-PCR analysis of HLCs cultured for 8 days in the presence of CHIR99021 and bFGF (total of 20 days of differentiation induction) after 12 days of differentiation induction, followed by further culture for 4 days, or cells cultured for 8 days (total of 20 days of differentiation induction) and then passaged and cultured for another 4 days. (A) TGF-β, (B) p15INK4b, (C) p21, (D) Ki67.Figure 21 shows Western blot analysis of HLCs cultured in the presence of CHIR99021 and bFGF for 8 days (total 20 days of differentiation induction) after 12 days of differentiation induction, followed by further culture for 8 days, or cells passaged after 8 days of culture (total 20 days of differentiation induction) and then cultured for another 8 days. Figure 22 shows light microscopy images and CCK-8 assays of HLCs cultured in the presence of CHIR99021 and bFGF for 8 days (total 20 days of differentiation induction) and then passaged, followed by further culture for 3 days with CHIR99021 and bFGF alone or with CHIR99021 and bFGF plus SB431542. (A) Light microscopy images of cells cultured for 3 days after passage in the presence of CHIR99021 + bFGF, (B) light microscopy images of cells cultured for 3 days after passage in the presence of CHIR99021 + bFGF + SB431542, and (C) CCK-8 assay of cells cultured for an additional 3 days after passage with CHIR99021 + bFGF alone or CHIR99021 + bFGF plus SB431542. Figure 23 shows Western blot analysis of HLCs cultured for 12 days after differentiation induction, further cultured for 8 days in the presence of CHIR99021 and bFGF (total 20 days of differentiation induction), and then cultured for an additional 2 or 4 days with CHIR99021 and bFGF alone or CHIR99021 and bFGF plus SB431542. Figure 24 shows quantitative RT-PCR analysis of HLCs after 12 days of differentiation, which were further cultured in the presence of CHIR99021 and bFGF for 8 days (total 20 days of differentiation induction) and then passaged, followed by an additional 4 days of culture with CHIR99021 and bFGF alone or with CHIR99021, bFGF, and SB431542. (A) p15INK4b, (B) p21. Figure 25 shows immunocytochemistry analysis of HLCs after 12 days of differentiation, which were further cultured in the presence of CHIR99021 and bFGF for 8 days (total 20 days of differentiation induction) and then passaged, followed by an additional 2 hours, 4 days, and 8 days of culture with CHIR99021, bFGF, and SB431542.Figure 26 shows Western blot analysis of HLCs after 12 days of differentiation induction, which were further cultured in the presence of CHIR99021 and bFGF for 8 days (total 20 days of differentiation induction) and then passaged, followed by an additional 2, 4, and 6 days of culture with CHIR99021, bFGF, and SB431542. Figure 27 shows a graph depicting the relationship between the number of passages and cell number at passage, which were performed on HLCs after 12 days of differentiation induction, which were further cultured in the presence of CHIR99021 and bFGF for 8 days (total 20 days of differentiation induction), followed by passage with CHIR99021, bFGF, and SB431542. Figure 28 shows immunocytochemistry of cells after the 21st passage in the presence of CHIR99021, bFGF, and SB431542. Figure 29 shows (A) quantitative RT-PCR analysis of p15INK4b and (B) Western blot analysis of long-term passaged HLCs cultured for 3 days with SB431542 alone, followed by 7 days of culture without CHIR99021, bFGF, or SB431542. Figure 30 shows quantitative RT-PCR analysis of long-term passaged HLCs cultured for 3 days with SB431542 alone, followed by 7 days of culture without CHIR99021, bFGF, or SB431542. (A) Sox17, (B) HNF4α, (C) AFP, (D) Albumin. Figure 31 shows fluorescence microscopy images of long-term passaged HLCs cultured for 7, 10, 14, and 18 days in the presence of SB431542 alone after differentiation induction from iPSCs with Superfolder GFP knocked into the albumin locus. Figure 32 shows Western blot analysis of long-term passaged HLC cells cultured for 25 days in the presence of SB431542 alone. Figure 33 shows (A) quantitative RT-PCR analysis and (B) Western blot analysis of long-term passaged HLC cells cultured for 25 days in the presence of SB431542 alone (rifampicin was added on day 24). Figure 34 shows fluorescence microscopy images of the livers 30 days after transplantation from immunodeficient mice transplanted with long-term passaged HLC cells differentiated from iPSCs in which Superfolder GFP had been knocked in at the albumin locus.Figure 35 shows immunocytochemistry of livers harvested from immunodeficient mice transplanted with HLCs differentiated and passaged for a long period from iPSCs expressing Superfolder GFP at the albumin locus. Figure 36 shows quantitative RT-PCR analysis of gene expression in HLCs cultured for 4 days with CHIR99021 at 2 μM, bFGF at 50 ng / ml, and SB431542 at 1 μM, 1 μM, and 0 μM. Figure 37 shows Western blot analysis of protein expression in HLCs cultured for 3 days with CHIR99021 at 2 μM, bFGF at 50 ng / ml, and SB431542 at 2 μM, 0 μM, and 0 μM (left). Figure 38 shows flow cytometry analysis of HLCs cultured for 4 days with CHIR99021 at 2 μM, 1 μM, and 0 μM, compared with long-term culture with CHIR99021 at 2 μM, bFGF at 50 ng / ml, and SB431542 at 10 μM. Figure 39 shows quantitative RT-PCR analysis of gene expression in HLCs with beta-catenin knockdown using the CRISPR-Cas9 system and controls. Figure 40 shows Western blot analysis of protein expression in HLCs with beta-catenin knockdown using the CRISPR-Cas9 system and controls (left). Figure 41 also shows quantitative RT-PCR analysis of gene expression in HLCs in which Sox17 was knocked down using the CRISPR-Cas9 system, compared with the control. Figure 42 shows Western blot analysis of protein expression in HLCs in which beta-catenin was knocked down using the CRISPR-Cas9 system, compared with the control.Also shown is a CCK-8 assay to compare the number of engrafted cells between HLCs in which beta-catenin was knocked down and controls (right).

[0013] 1. Method for Producing Hepatic Stem Cells The present invention provides a method for producing hepatic stem cells from hepatocytes (hereinafter, sometimes referred to as "production method (1) of the present invention"). Specifically, the present invention provides a method for producing hepatic stem cells, comprising the step of culturing hepatocytes in a medium containing a Wnt signaling promoter and an FGF signaling promoter.

[0014] Hepatocytes (also called hepatocytes) comprise the majority (approximately 70%) of the liver in vivo. Their functions are diverse, including (1) metabolic enzyme production, (2) transporter function, (3) bile production and secretion, (4) drug or ammonia detoxification, (5) gluconeogenesis, and (6) lipid metabolism. Congenital genetic mutations inhibit the above functions of hepatocytes, resulting in metabolic disorders. As used herein, "hepatocytes" refers to cells that express FOXA2, HNF4α, AFP, ASGPR1, and albumin and perform one or more of the above functions, including hepatocytes that mature from these cells. Hepatocytes typically do not express OCT4, SOX17, or GATA4.

[0015] The hepatocytes used in production method (1) of the present invention may be, for example, those selected from a portion of the liver isolated from a living body using the above-mentioned hepatocyte markers as indicators by methods such as flow cytometry or mass cytometry, magnetic cell separation, or affinity columns on which a desired antigen is immobilized, or may be prepared by inducing differentiation from pluripotent stem cells. In production method (1) of the present invention, hepatocytes obtained from pluripotent stem cells are preferably used.

[0016] In this specification, unless otherwise specified, the term "cell" includes a "cell population." A cell population may be composed of one type of cell, or may be composed of two or more types of cells.

[0017] As used herein, the term "Wnt signaling promoter" refers to a substance that activates a signaling pathway that uses a Wnt family protein as a ligand and primarily Frizzled as a receptor. Examples of such signaling pathways include the canonical Wnt pathway and the non-canonical Wnt pathway. The canonical Wnt pathway is transduced by β-catenin. Non-canonical Wnt pathways include the planar cell polarity (PCP) pathway, Wnt / JNK pathway, Wnt / Calcium pathway, Wnt-RAP1 pathway, Wnt-Ror2 pathway, Wnt-PKA pathway, Wnt-GSK3MT pathway, Wnt-aPKC pathway, Wnt-RYK pathway, and Wnt-mTOR pathway. In the non-canonical Wnt pathway, there are common signaling factors that are also activated in signaling pathways other than Wnt, and activators of these factors are also included in the Wnt signaling promoter. Examples of Wnt signaling promoters include GSK3 inhibitors and Wnt proteins. These substances may be used alone or in combination.

[0018] Examples of GSK3 inhibitors include CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), CHIR98014 (N-6-[2-[[4-(2,4-Dichlorophenyl)-5-(1H-imidazol-1-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine), 1-Azakenpaullone (9-bromo-7,12-dihydro-pyrido[3',2':2,3]azepino[4,5-b]indol-6(5H)-one), and AZD 2858 (3-amino-6-[4-[(4-methyl-1- piperazinyl)sulfonyl]phenyl]-N-3-pyridinyl-2-pyrazinecarboxamide), BIO(6-bromo-3-[(3E)-1,3-dihydro-3-(hydroxyimino)-2H-indol-2-ylidene]-1,3- dihydro-(3Z)-2H-indol-2-one), CP21R7 (3-(3-aminophenyl)-4-(1-methyl-1H-indol3-yl)-1H-pyrrole-2,5-dione), Sotrastaurin (3-(1H-indol-3-yl)-4-[2-(4-methyl1-piperazinyl)-4-quinazolinyl]-1Hpyrrole-2,5-dione), TWS119 (3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]-phenol), Valproic Acid (2-propyl-pentanoic acid), and the like are included, and CHIR99021 is preferred. Examples of Wnt proteins include Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11, and Wnt16.These substances may be used alone or in combination.

[0019] The concentration of the Wnt signaling promoter in the medium can be appropriately set depending on the substance used. For example, when CHIR99021, a type of GSK3 inhibitor, is used as the Wnt signaling promoter, its concentration is usually 0.1 μM to 50 μM, preferably 0.5 μM to 10 μM, and more preferably 1 μM to 3 μM (in one embodiment, 1 μM). When a Wnt signaling promoter other than CHIR99021 is used, the concentration of the Wnt signaling promoter in the medium is appropriately selected.

[0020] The FGF signaling promoter is not particularly limited as long as it is a substance that can enhance the signaling pathway mediated by FGF (fibroblast growth factor). Examples of FGF signaling promoters include FGF proteins such as FGF1, bFGF (also known as FGF2), FGF3, FGF8, and FGF10, anti-FGF receptor antibodies, and FGF partial peptides. These substances may be used alone or in combination.

[0021] The FGF signaling promoter is preferably at least one selected from the group consisting of bFGF, FGF3, FGF8, FGF10, and modified forms thereof, and more preferably bFGF.

[0022] The concentration of the FGF signaling promoter in the medium can be appropriately set depending on the substance used. For example, when bFGF is used as the FGF signaling promoter, the concentration is usually 1 ng / ml to 1 μg / ml, preferably 5 ng / ml to 200 ng / ml, and more preferably 10 ng / ml to 100 ng / ml (50 ng / ml in one embodiment). When an FGF other than bFGF is used, the concentration of FGF in the medium is appropriately selected.

[0023] The culture period for Production Method (1) of the present invention is not particularly limited as long as it is sufficient to obtain hepatic stem cells, but is typically 1 to 16 days, preferably 3 to 13 days, and more preferably 5 to 10 days (particularly 8 days). During the culture period for Production Method (1) of the present invention, the cells may or may not be passaged, but preferably, the cells are not passaged. Furthermore, as shown in the Examples, no increase in proliferation-inhibitory markers can be detected in the cells even after long-term culture (at least 16 days) using Production Method (1) of the present invention. Therefore, the culture period may be 16 days or longer (e.g., 20 days or longer, 40 days or longer, or 60 days or longer).

[0024] The cell density at the time of seeding hepatocytes is not particularly limited as long as hepatic stem cells can be obtained. Typically, the cell density is 1.0 × 10 4 ~5.0×10 5 cells / cm 2 is.

[0025] The hepatocytes used in production method (1) of the present invention can also be hepatocyte-like cells (HLCs) produced by inducing differentiation from pluripotent stem cells. Methods for producing HLCs include the method reported by Si-Tayeb K et al. (Hepatology 2010; 51: 297-305) or modifications of said method. Specifically, methods including the following steps (A) to (D) can be included. Thus, production method (1) of the present invention may include at least one of steps (A) to (D): (A) culturing pluripotent stem cells in a medium containing a Wnt signaling promoter and Activin A to obtain mesendoderm cells; (B) culturing mesendoderm cells in a medium containing Activin A to obtain definitive endoderm cells; and (C) culturing definitive endoderm cells in a medium containing a BMP signaling promoter and an FGF signaling promoter to obtain hepatic endoderm cells. (D) A step of culturing hepatic endoderm cells in a medium containing HGF to obtain hepatocyte-like cells.

[0026] The pluripotent stem cells used in the present invention may be any undifferentiated cells that possess both the "self-renewal ability" (ability to proliferate while maintaining an undifferentiated state) and the "pluripotency" (ability to differentiate into all three primary germ layers). Examples of such pluripotent stem cells include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (nuclear transfer embryonic stem cells (ntES cells)), multipotent germline stem cells (mGS cells), and embryonic germ stem cells (EG cells). Preferably, these are iPS cells (more preferably, human iPS cells). When the pluripotent stem cells are ES cells or any cells derived from human embryos, they may be produced by or without the destruction of embryos. However, from an ethical standpoint, they are preferably produced without the destruction of embryos.

[0027] iPS cells are artificial stem cells derived from somatic cells that can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. They have properties similar to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal (Takahashi K. and Yamanaka S. (2006) Cell, 126:663-676; Takahashi K. et al. (2007), Cell, 131:861-872; Yu J. et al. (2007), Science, 318:1917-1920; Nakagawa M. et al., Nat. Biotechnol.26:101-106 (2008); WO 2007 / 069666). When iPS cells are used, the iPS cells may be produced from somatic cells by a method known per se, or iPS cells that have already been established and stored may be used. The reprogramming factors may be composed of genes that are specifically expressed in ES cells, their gene products, or non-coding RNAs, or genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products, or non-coding RNAs, or low-molecular-weight compounds. Examples of genes contained in the reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include WO 2007 / 069666, WO 2008 / 118820, WO 2009 / 007852, WO 2009 / 032194, WO 2009 / 058413, WO 2009 / 057831, WO 2009 / 075119, WO 2009 / 079007, WO 2009 / 091659, WO 2009 / 101084, WO 2009 / 101407, WO 2009 / 102983, WO 2009 / 114949, WO 2009 / 117439, WO 2009 / 126250, WO 2009 / 126251, WO 2009 / 126655, WO 2009 / 157593, WO 2010 / 009015, WO 2010 / 033906, WO 2010 / 033920, WO 2010 / 042800, WO 2010 / 050626, WO 2010 / 056831, WO 2010 / 068955, WO 2010 / 098419, WO 2010 / 102267, WO 2010 / 111409, WO 2010 / 111422, WO 2010 / 115050, WO 2010 / 124290, WO 2010 / 147395, WO 2010 / 147612, Huangfu D, et al. (2008), Nat.The combinations described in Biotechnol., 26:795-797, Shi Y, et al. (2008), Cell Stem Cell, 2:525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, R.L. Judson et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9 are exemplified.

[0028] ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in humans, monkeys, and other primates (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Methods for establishing and maintaining human and monkey ES cells are described, for example, in US Pat. No. 5,843,780; Thomson JA, et al. (1995), Proc. Natl. Acad. Sci. USA 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1145-1147; Suemori H. et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; Ueno M. et al. (2006), Proc. Natl. Acad. Sci. USA 103:9554-9559; Suemori H. et al. (2001), Dev. Dyn., 222:273-279; Kawasaki H. et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I. et al. (2006), Nature. 444:481-485, etc.Alternatively, ES cells can be established using only a single blastomere from an embryo at the cleavage stage prior to the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or from a developmentally arrested embryo (Zhang X. et al. (2006), Stem Cells 24: 2669-2676).

[0029] ntES cells are ES cells derived from cloned embryos by nuclear transfer technology and have almost the same properties as ES cells derived from fertilized eggs (Wakayama T. et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; Byrne J. et al. (2007), Nature, 450:497-502). Specifically, ntES (nuclear transfer ES) cells are established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with that of a somatic cell. To generate ntES cells, nuclear transfer technology (Cibelli JB et al. (1998), Nature Biotechnol., 16:642-646) is combined with ES cell generation technology (see above) (Wakayama Sayaka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, and the egg is then cultured for several hours to reprogram the embryo.

[0030] mGS cells are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various cell lineages, e.g., when transplanted into mouse blastocysts, chimeric mice can be generated (Kanatsu-Shinohara M. et al. (2003) Biol. Reprod., 69:612-616; Shinohara K. et al. (2004) Cell, 119:1001-1012). They are capable of self-renewal in culture medium containing glial cell line-derived neurotrophic factor (GDNF). Furthermore, germline stem cells can be obtained by repeated passage under culture conditions similar to those for ES cells (Takebayashi M. et al. (2008) Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 41-46, Yodosha, Tokyo, Japan).

[0031] EG cells are derived from embryonic primordial germ cells (PGCs) and have pluripotency similar to that of ES cells. They can be established by culturing PGCs in the presence of LIF, bFGF, stem cell factor, and other substances (Matsui Y. et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550-551).

[0032] The species from which the pluripotent stem cells are derived is not particularly limited, and may be cells from, for example, rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, and sheep, carnivores such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. The preferred species is human.

[0033] Prior to step (A), pluripotent stem cells (particularly human pluripotent stem cells) may be precultured in a medium containing a Rho-associated coiled-coil kinase (ROCK) inhibitor to suppress cell death (see JP 2008-99662 A). Examples of ROCK inhibitors include Y-27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride), fasudil / HA1077, SR3677, GSK269962, GSK429286A, H1152, Wf-536, thiazovivin, and salts or derivatives thereof. The concentration of the ROCK inhibitor is sufficient to suppress cell death of pluripotent stem cells. For example, for Y-27632, such a concentration is typically 0.1 to 200 μM, preferably 2 to 50 μM (5 μM in one embodiment). Treatment with a ROCK inhibitor is typically carried out for one day, and thereafter, it is preferable to continue culturing in a medium that does not contain a ROCK inhibitor.

[0034] The Wnt signaling promoter used in step (A) is not particularly limited to any of the above-mentioned substances. Preferably, the Wnt signaling promoter is CHIR99021.

[0035] The concentration of the Wnt signaling promoter in the medium can be appropriately set depending on the substance used. For example, when CHIR99021, a type of GSK3 inhibitor, is used as the Wnt signaling promoter, its concentration is usually 0.1 μM to 50 μM, preferably 0.5 μM to 10 μM, and more preferably 1 μM to 3 μM (in one embodiment, 2.5 μM). When a Wnt signaling promoter other than CHIR99021 is used, the concentration of the Wnt signaling promoter in the medium is appropriately selected.

[0036] The Activin A used in step (A) may be commercially available from Nacalai Tesque, etc. The concentration of Activin A in the medium in step (A) is not particularly limited, but is typically 20 to 100 ng / ml, preferably 50 ng / ml.

[0037] The culture period in step (A) is not particularly limited as long as it is a period during which mesendodermal cells can be obtained, but is typically 6 to 48 hours, preferably 12 to 36 hours, and more preferably 24 hours.

[0038] Mesendoderm cells are obtained by step (A). Typically, such mesendoderm cells are capable of differentiating into definitive endoderm cells and are characterized by expressing Goosecoid, Mixl1, and Brachyury T but not expressing CXCR4. The mesendoderm cells obtained in step (A) can be used directly in step (B) after medium replacement.

[0039] The Activin A used in step (B) is not particularly limited to those described above. The concentration of Activin A in the medium in step (B) is not particularly limited, but is typically 20 to 100 ng / ml, preferably 50 ng / ml.

[0040] The culture period in step (B) is not particularly limited as long as it is a period during which definitive endoderm cells can be obtained, but is typically 1 to 5 days, preferably 2 to 4 days, and more preferably 3 days.

[0041] Definitive endoderm cells are obtained by step (B). Typically, such definitive endoderm cells are capable of differentiating into hepatic endoderm cells and are characterized by expressing CXCR4, SOX17, and FOXA2, but not expressing Goosecoid, Mixl1, Brachyury T, NANOG, OCT4, SOX2, HNF4α, AFP, or albumin. The definitive endoderm cells obtained in step (B) can be used directly in step (C) after medium replacement.

[0042] The BMP signaling promoter used in step (C) is a substance capable of enhancing the signaling pathway mediated by bone morphogenetic protein (BMP). Examples of substances capable of enhancing the signaling pathway mediated by BMP include substances that stabilize BMP ligands in the culture environment and increase their potency, substances that bind to type I BMP receptors ALK-1, ALK-2, ALK-3, and ALK-6 and activate or induce intracellular signaling downstream of the receptor, substances that induce phosphorylation of Smad-1, Smad-5, Smad-8, and Smad-9 involved in intracellular BMP signaling, and substances that induce or enhance functions such as activation or repression of gene transcription by Smad-1 / 5 / 8 / 9. Examples of BMP signaling promoters include proteins such as BMP2, BMP4, BMP7, BMP13, and GDF7 (among which BMP2, BMP4, and BMP7 are preferred, with BMP4 being particularly preferred), GDF proteins such as GDF5, 6, and 7, anti-BMP receptor antibodies, and BMP partial peptides. These substances may be used alone or in combination.

[0043] The concentration of the BMP signaling promoter in the medium can be appropriately set depending on the substance used. For example, when BMP4 is used as the BMP signaling promoter, it is usually used at a concentration of 1 ng / ml to 200 ng / ml, preferably 5 ng / ml to 40 ng / ml, and more preferably 10 ng / ml to 20 ng / ml. When a BMP signaling promoter other than BMP4 is used, the concentration of the BMP signaling promoter in the medium is appropriately selected.

[0044] Compounds well known to those skilled in the art can also be used as BMP signaling promoters. Examples of BMP signaling promoters include Smurf1 inhibitors, Chk1 inhibitors, and phosphorylated Smad stabilizers. Examples of compounds having the above-mentioned activity include A-01 ([4-[[4-Chloro-3-(trifluoromethyl)phenyl]sulfonyl]-1-piperazinyl][4-(5-methyl-1H-pyrazol-1-yl)phenyl]methanone), PD407824 (9-Hydroxy-4-phenyl-pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione), SB4 (2-[[(4-Bromophenyl)methyl]thio]benzoxazole), SJ000291942 (2-(4-Ethylphenoxy)-N-(4-fluoro-3-nitrophenyl)-acetamide), and derivatives thereof.

[0045] The FGF signaling promoter used in step (C) is not particularly limited and may be any of those mentioned above. The FGF signaling promoter is preferably bFGF.

[0046] The concentration of the FGF signaling promoter in the medium can be appropriately set depending on the substance used. For example, when bFGF is used as the FGF signaling promoter, the concentration is usually 1 ng / ml to 1 μg / ml, preferably 5 ng / ml to 200 ng / ml, and more preferably 10 ng / ml to 100 ng / ml (10 ng / ml in one embodiment). When an FGF other than bFGF is used, the concentration of FGF in the medium is appropriately selected.

[0047] The culture period in step (C) is not particularly limited as long as it is a period during which hepatic endoderm cells can be obtained, but is typically 1 to 7 days, preferably 3 to 5 days, and more preferably 4 days.

[0048] Hepatic endoderm cells are obtained by step (C). Typically, such hepatic endoderm cells are capable of differentiating into hepatocyte-like cells and are characterized by expressing GATA4, HNF4α, and AFP, but not expressing OCT4, SOX17, or albumin. The hepatic endoderm cells obtained in step (C) can be used directly in step (D) after medium replacement.

[0049] The HGF (Hepatocyte Growth Factor) used in step (D) may be commercially available from Peprotech, etc. The concentration of HGF in the medium in step (D) is not particularly limited, but is typically 10 to 100 ng / ml, preferably 20 ng / ml.

[0050] The culture period in step (D) is not particularly limited as long as it is a period during which hepatocyte-like cells can be obtained, but is typically 1 to 7 days, preferably 3 to 5 days, and more preferably 4 days.

[0051] Hepatocyte-like cells are obtained by step (D). Typically, such hepatocyte-like cells are capable of differentiating into mature hepatocytes and are characterized by expressing FOXA2, HNF4α, AFP, ASGPR1, and albumin. In one embodiment, the hepatocyte-like cells are further characterized by not expressing OCT4, SOX17, and GATA4. The hepatocyte-like cells obtained in step (D) can be used as they are in production method (1) of the present invention by changing the medium, or can be recovered and reseeded to be used as hepatocytes in production method (1) of the present invention.

[0052] As described above, hepatic stem cells can be produced by the production method (1) of the present invention. As described in the Examples below, when produced hepatic stem cells are passaged, TGF-β signaling is activated, which can lead to cell death due to senescence. To address this issue, the present inventors cultured hepatic stem cells in a medium containing a Wnt signaling promoter, an FGF signaling promoter, and an ALK inhibitor. This suppressed hepatic stem cell death and maintained the cell's proliferation ability even after passage. Therefore, the present invention also provides a method for producing expandable hepatic stem cells from hepatic stem cells (or a method for expanding hepatic stem cells) (hereinafter referred to as "production method (2) of the present invention" or "expansion method of the present invention"). Specifically, the present invention provides a method for producing hepatic stem cells, comprising culturing hepatic stem cells in a medium containing a Wnt signaling promoter, an FGF signaling promoter, and an ALK inhibitor (or a method for expanding hepatic stem cells, comprising culturing hepatic stem cells in a medium containing a Wnt signaling promoter, an FGF signaling promoter, and an ALK inhibitor). Hereinafter, unless otherwise specified, "production method (2) of the present invention" may be interpreted as "expansion method of the present invention."

[0053] The hepatic stem cells used in production method (2) of the present invention may be selected, for example, from a portion of the liver isolated from a living body using a cell marker as an indicator, by a method using flow cytometry or mass cytometry, a magnetic cell separation method, an affinity column on which a desired antigen is immobilized, etc. Preferably, the hepatic stem cells used in production method (2) of the present invention may be produced from hepatocytes by production method (1) of the present invention.

[0054] The Wnt signaling promoter used in the production method (2) of the present invention is not particularly limited to those mentioned above, and CHIR99021 is preferred.

[0055] The concentration of the Wnt signaling promoter in the medium can be appropriately set depending on the substance used. For example, when CHIR99021, a type of GSK3 inhibitor, is used as the Wnt signaling promoter, its concentration is usually 0.1 μM to 50 μM, preferably 0.5 μM to 10 μM, and more preferably 1 μM to 2 μM (in one embodiment, 1 μM or 2 μM). When a Wnt signaling promoter other than CHIR99021 is used, the concentration of the Wnt signaling promoter in the medium is appropriately selected.

[0056] The FGF signaling promoter used in the production method (2) of the present invention is not particularly limited to those mentioned above, and is preferably bFGF.

[0057] The concentration of the FGF signaling promoter in the medium can be appropriately set depending on the substance used. For example, when bFGF is used as the FGF signaling promoter, the concentration is usually 1 ng / ml to 1 μg / ml, preferably 5 ng / ml to 200 ng / ml, and more preferably 10 ng / ml to 100 ng / ml (50 ng / ml in one embodiment). When an FGF other than bFGF is used, the concentration of FGF in the medium is appropriately selected.

[0058] As used herein, the term "ALK inhibitor" refers to a substance that has inhibitory activity against receptors belonging to the ALK (activin receptor-like kinase) family. ALK, also known as the type I TGFβ receptor, controls cell proliferation, cell differentiation, cell death, and other conditions through signal transduction primarily via activation of Smad (R-Smad). A type II TGFβ receptor forms a dimer, and this dimer associates with two molecules of type I TGFβ receptor to form a heterotetramer. Upon heterotetramer formation, the type II TGFβ receptor phosphorylates the type I TGFβ receptor, which induces the kinase activity of the type I TGFβ receptor and phosphorylates downstream Smads.

[0059] The "ALK inhibitor" used in production method (2) of the present invention is not particularly limited as long as it can inhibit any of the above-mentioned stages of signal transduction, and examples thereof include substances that inhibit the binding of TGFβ to its receptor, substances that inhibit the phosphorylation of type I TGFβ receptor by type II TGFβ receptor, and substances that inhibit the phosphorylation of Smads (e.g., Smad2, Smad3, etc.) by phosphorylated type I TGFβ receptor. In humans, ALK-1, ALK-2, ALK-3, ALK-4, ALK-5, ALK-6, and ALK-7 are known, and as the ALK inhibitor used in production method (2) of the present invention, an inhibitor of ALK-5 (also referred to as a "TGFβ inhibitor") is particularly preferred.

[0060] Examples of the ALK inhibitor used in step (2) of the production method of the present invention include SB431542 (4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide, 4-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide), A83-01 (3-(6-methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole), LDN193189 (4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), GW788388 (4-[4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl]-N-(tetrahydro-2H-pyran-4-yl)benzamide), SM16 (4-[4-(1,3-Benzodioxol-5-yl)-5-(6- Examples include 3-[[5-(6-Methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]-bicyclo[2.2.2]octane-1-carboxamide], IN-1130 (3-[[5-(6-Methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]-benzamide), GW6604 (2-Phenyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine), and SB505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine), with SB431542 being preferred. These may be used in combination of two or more.

[0061] The concentration of the ALK inhibitor in the medium can be appropriately set depending on the substance used. For example, when SB431542 is used as the ALK inhibitor, its concentration is usually 5 μM to 15 μM (in one embodiment, 10 μM). When an ALK inhibitor other than SB431542 is used, the concentration of the ALK inhibitor in the medium can be appropriately selected.

[0062] The culture period for one culture in the production method (2) of the present invention is not particularly limited as long as it is a period during which hepatic stem cells can be expanded, but is typically 1 to 14 days, preferably 5 to 10 days (in one embodiment, 7 days).

[0063] In the production method (2) of the present invention, hepatic stem cells may be passaged after one culture period, and then the production method (2) of the present invention may be carried out again. The number of times hepatic stem cells can be passaged is not limited as long as hepatic stem cells can proliferate using the production method (2) of the present invention, but is typically 1 to 10 times. Furthermore, as shown in the Examples, no decrease in cell proliferation rate was observed even after 10 passages using the production method (2) of the present invention. Therefore, the number of times hepatic stem cells can be passaged may be 10 or more times (e.g., about 20 times).

[0064] The cell density at the time of seeding hepatic stem cells is not particularly limited as long as the hepatic stem cells can proliferate. Typically, the cell density is 1.0 × 10 4 ~5.0×10 4 cells / cm 2 is.

[0065] In the present invention, the culture method is not particularly limited as long as it is possible to produce hepatic stem cells from hepatocytes in production method (1), and as long as it is possible to proliferate hepatic stem cells in the present invention, but adherent culture is preferred. As used herein, "adherent culture" refers to culture under conditions that allow strong cell-substrate bonds to form between cells or cell aggregates and cultureware, etc.

[0066] Culture vessels used in adhesion culture include those whose surfaces have been artificially treated to improve cell adhesion (e.g., coating with basement membrane preparations, extracellular matrices such as fibronectin, laminin or fragments thereof, entactin, collagen, gelatin, synthemax, vitronectin, etc., or polymers such as polylysine or polyornithine, or surface treatments such as positive charge treatment). Of these, culture vessels coated with laminin or fragments thereof are preferred.

[0067] Examples of laminin or fragments thereof include laminin-111 or fragments containing its E8 region, laminin-211 or fragments containing its E8 region (e.g., iMatrix-211), laminin-121 or fragments containing its E8 region, laminin-221 or fragments containing its E8 region, laminin-332 or fragments containing its E8 region, laminin-3A11 or fragments containing its E8 region, laminin-411 or fragments containing its E8 region (e.g., iMatrix-411), laminin-421 or fragments containing its E8 region, and laminin-511 or fragments containing its E8 region (e.g., iMatrix-511, iMatrix-511 silk), laminin-521 or a fragment thereof containing its E8 region, laminin-213 or a fragment thereof containing its E8 region, laminin-423 or a fragment thereof containing its E8 region, laminin-523 or a fragment thereof containing its E8 region, laminin-212 / 222 or a fragment thereof containing its E8 region, and laminin-522 or a fragment thereof containing its E8 region.

[0068] The medium used in the production method (1) or (2) of the present invention can be prepared using a medium used for culturing mammalian cells as a basal medium. Examples of basal media include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham's medium, Ham's F-12 medium, RPMI1640 medium, Fischer's medium, Neurobasal medium, Essential 8 (manufactured by Thermo Fisher Scientific), Essential 6 (manufactured by Thermo Fisher Scientific), S-medium (manufactured by DS Pharma Biomedical), StemPro (manufactured by Thermo Fisher Scientific), hESF9, mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), mTeSR Plus (manufactured by STEMCELL Technologies), and StemFit. Examples of such media include AK02N (Ajinomoto Co., Inc.), StemFit AK03N (Ajinomoto Co., Inc.), ReproMed iPSC Medium (ReproCELL, Inc.), NutriStem XF (Biological Industries, Inc.), NutriStem V9 (Biological Industries, Inc.), Cellartis DEF-CS Xeno-Free Culture Medium (Takara Bio Inc.), Stem-Partner SF (Kyokuto Pharmaceuticals Co., Ltd.), PluriSTEM Human ES / iPS Cell Medium (Merck & Co., Inc.), StemSure hPSC MediumΔ (Fujifilm Wako Pure Chemical Industries, Inc.), and mixed media thereof (e.g., DMEM / F-12 medium (a 1:1 mixed medium of DMEM medium and Ham's F-12 medium)).

[0069] The medium used in the production method (1) or (2) of the present invention may be either a serum-containing medium or a serum-free medium, but a serum-free medium is preferred. A serum-free medium refers to a medium that does not contain any serum, or a medium that does not contain unprepared or unpurified serum. A medium containing purified blood-derived components or animal tissue-derived components (e.g., growth factors) is considered to be serum-free. The medium used in the production method of the present invention may also contain a serum substitute. The serum substitute may contain, for example, albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof, as appropriate. Such a serum substitute can be prepared, for example, by the method described in WO98 / 30679. To more easily carry out the production method of the present invention, commercially available serum substitutes can be used. Examples of such commercially available serum substitutes include KSR (knockout serum replacement) (manufactured by Invitrogen), Chemically-defined Lipid concentrated (manufactured by Gibco), and Glutamax (manufactured by Gibco).

[0070] The medium may contain other additives, such as, but not limited to, insulin, an iron source (e.g., transferrin), minerals (e.g., sodium selenate), sugars (e.g., glucose), organic acids (e.g., pyruvic acid, lactic acid), serum proteins (e.g., albumin), amino acids (e.g., L-glutamine), reducing agents (e.g., 2-mercaptoethanol), vitamins (e.g., ascorbic acid, d-biotin), antibiotics (e.g., streptomycin, penicillin, gentamicin), and buffers (e.g., HEPES).

[0071] The culture temperature is not particularly limited, but is usually 30°C to 40°C, preferably 37°C, and culture is carried out in an atmosphere of CO2-containing air, with the CO2 concentration preferably being 2% to 5%.

[0072] As described above, hepatic stem cells can be obtained by production method (1) or (2) of the present invention. Therefore, as another aspect of the present invention, hepatic stem cells obtained by production method (1) or (2) are also provided. Such hepatic stem cells typically express one or more genes selected from SOX17 and HNF4α, or SOX17 and Axin2 (Axis inhibition protein 2), and have the ability to differentiate into hepatocytes. Furthermore, the hepatic stem cells may be characterized by not expressing AFP and albumin. Furthermore, the hepatic stem cells may be characterized by maintaining their proliferation ability after passage.

[0073] The present invention further provides hepatic stem cells (hereinafter, sometimes referred to as "hepatic stem cells (1) of the present invention") having all of the following properties (A) to (C): (A) derived from pluripotent stem cells; (B) expressing SOX17 and Axin2; and (C) having the ability to differentiate into hepatocytes.

[0074] In another aspect, the present invention provides hepatic stem cells (hereinafter, sometimes referred to as "hepatic stem cells (2) of the present invention") having all of the following properties (A') to (C'): (A') Derived from pluripotent stem cells. (B') Expressing one or more genes selected from SOX17 and HNF4α (preferably, all two genes). (C') Possessing the ability to differentiate into hepatocytes. Hereinafter, unless otherwise specified, the term "hepatic stem cells of the present invention" will be used to collectively refer to hepatic stem cells (1) of the present invention and hepatic stem cells (2) of the present invention.

[0075] The hepatic stem cells of the present invention preferably further have the following characteristic (D): (D) they are positive for at least one (i.e., one, two, three, or four) selected from the group consisting of LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5), HNF4α, phosphorylated retinoblastoma protein, and non-phosphorylated β-catenin, and preferably are positive for at least LGR5.

[0076] The hepatic stem cells of the present invention preferably further have the following characteristic (E): (E) they do not express one or more genes selected from AFP and albumin (preferably, both of these genes).

[0077] The hepatic stem cells of the present invention preferably further have the following characteristic (F): (F) Proliferative capacity is maintained after passaging. In a preferred embodiment, the hepatic stem cells of the present invention have all of the above characteristics (A) to (F) (characteristic (B) can also be read as characteristic (B')).

[0078] As used herein, "derived from pluripotent stem cells" means that the cells are produced from pluripotent stem cells. The pluripotent stem cells may be any of the cells described above, but are preferably iPS cells. The species of origin of the pluripotent stem cells is not particularly limited, with humans being a preferred species of origin. Methods for producing the hepatic stem cells of the present invention from pluripotent stem cells include production methods (1) and (2) of the present invention.

[0079] As used herein, unless otherwise specified, the phrase "expressing (or not expressing) a gene" is used to mean at least "producing (or not producing) mRNA from the gene," but preferably also means "producing (or not producing) a protein encoded by the gene."

[0080] Detection of mRNA produced from a gene can be performed by means known in the art, such as quantitative RT-PCR. Quantitative RT-PCR can be performed, for example, as follows: Total RNA is extracted from cells using ISOGEN II (Nippon Gene Co. Ltd.). cDNA is prepared by reverse transcription of mRNA contained in 0.5 μg of total RNA using ReverTra Ace qPCR RT Master Mix (TOYOBO). Quantitative RT-PCR analysis is performed using THUNDERBIRD Next SYBR qPCR Mix (TOYOBO) on a Rotor-Gene Q 2plex HRM system (QIAGEN). Herein, detection of mRNA by quantitative RT-PCR indicates gene expression. On the other hand, detection of no mRNA (i.e., below the detection limit) or at background levels by quantitative RT-PCR indicates no gene expression. Herein, mRNA also includes pre-mRNA.

[0081] The protein encoded by the gene can be detected by means known in the art, such as enzyme immunoassay (EIA), fluorescence immunoassay (FIA), immunochromatography, Western blotting, radioimmunoassay, flow cytometry analysis, etc. When it is necessary to distinguish between phosphorylated and non-phosphorylated forms of a protein, for example, antibodies specific to the phosphorylated or non-phosphorylated form of the protein can also be used.

[0082] As used herein, "having the ability to differentiate into hepatocytes" means that the hepatic stem cells of the present invention have the ability to differentiate back into hepatocytes. Methods for producing hepatocytes from the hepatic stem cells of the present invention include a method comprising culturing the hepatic stem cells in a medium containing an ALK inhibitor but not containing a Wnt signaling promoter. As used herein, the ALK inhibitor may be as described above, but is preferably SB431542.

[0083] As used herein, "maintaining proliferation ability after passaging" means that the hepatic stem cells of the present invention maintain the proliferation ability prior to passaging even after being passaged during culture. In one embodiment, when the hepatic stem cells of the present invention have the property of maintaining proliferation ability after passaging, the hepatic stem cells of the present invention are cells produced by production method (2) of the present invention.

[0084] 2. Method for Producing Hepatocytes As described above, the hepatic stem cells of the present invention have the ability to differentiate into hepatocytes. Therefore, the present invention further provides a method for producing hepatocytes (hereinafter, sometimes referred to as "production method (3) of the present invention"), which comprises the step of inducing the differentiation of the hepatic stem cells of the present invention into hepatocytes.

[0085] The step of inducing differentiation of hepatic stem cells into hepatocytes of the present invention may include a step of culturing the hepatic stem cells in a medium containing an ALK inhibitor but not containing a Wnt signaling promoter. In production method (3) of the present invention, the ALK inhibitor contained in the medium may be as described above, but is preferably SB431542.

[0086] The concentration of the ALK inhibitor in the medium can be appropriately set depending on the substance used. For example, when SB431542 is used as the ALK inhibitor, the concentration is usually 1 μM to 50 μM, preferably 2 μM to 40 μM, and more preferably 5 μM to 20 μM (10 μM in one embodiment). When an ALK inhibitor other than SB431542 is used, the concentration of the ALK inhibitor in the medium is appropriately selected.

[0087] In the production method (3) of the present invention, the Wnt signaling promoter not contained in the culture medium is at least one of the substances mentioned above (e.g., CHIR99021), preferably all of the substances mentioned.

[0088] Furthermore, since the FGF signaling promoter can promote the differentiation of hepatic stem cells into hepatocytes, the medium in production method (3) of the present invention may further contain an FGF signaling promoter.

[0089] In the production method (3) of the present invention, the FGF signaling promoter contained in the medium is not particularly limited to those mentioned above, and is preferably bFGF.

[0090] The concentration of the FGF signaling promoter in the medium can be appropriately set depending on the substance used. For example, when bFGF is used as the FGF signaling promoter, the concentration is usually 1 ng / ml to 1 μg / ml, preferably 5 ng / ml to 200 ng / ml, and more preferably 10 ng / ml to 100 ng / ml (50 ng / ml in one embodiment). When an FGF other than bFGF is used, the concentration of FGF in the medium is appropriately selected.

[0091] The culture period for production method (3) of the present invention is not particularly limited as long as it is a period during which hepatic stem cells can be induced to differentiate into hepatocytes, but is typically 10 to 35 days (in one embodiment, 25 to 35 days).

[0092] The cell density at the time of seeding hepatic stem cells is not particularly limited as long as the hepatic stem cells can be induced to differentiate into hepatocytes. Typically, the cell density is 0.75 × 10 5 ~1.0×10 5 cells / cm 2 is.

[0093] 3. Cell Transplantation Therapy Conventional hepatocyte transplantation has the drawback that high-quality hepatocytes isolated from donor livers are difficult to maintain and survive in vitro. Furthermore, due to their low proliferation capacity, even successful transplants fail to engraft in the liver over the long term, resulting in limited therapeutic efficacy. This problem is also true for hepatocytes obtained by differentiation from pluripotent stem cells. Furthermore, when transplanting cells into damaged livers, the transplanted hepatocytes tend to engraft easily because of the favorable environment for hepatocyte regeneration. However, in livers with liver dysfunction due to congenital disease but without damage, such an environment is not formed, making it difficult for transplanted cells to engraft. However, the hepatic stem cells of the present invention can engraft even in undamaged livers. Therefore, the hepatic stem cells of the present invention can be suitably used as transplant cells not only for damaged livers but also for undamaged livers of patients with liver dysfunction due to congenital disease. Therefore, in another aspect of the present invention, a cell transplantation therapy comprising the hepatic stem cells of the present invention (hereinafter sometimes referred to as the "cell transplantation therapy of the present invention") is provided. The present invention also encompasses a method for treating liver damage (including liver deficiency) or disease, in which an effective amount of the hepatic stem cells of the present invention is administered or transplanted into a target mammal (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.) at a site requiring transplantation of hepatocytes (the damaged site or a site requiring functional normal hepatocytes). Furthermore, "treatment of liver damage" also encompasses regeneration of the damaged liver.

[0094] The hepatic stem cells of the present invention can be transplanted into a living body for the purpose of directly regenerating a damaged liver or replacing hepatocytes that have lost function due to disease with normal hepatocytes. Examples of such diseases include pathological conditions without cirrhosis. Among these pathological conditions, preferred are congenital metabolic liver diseases in which some metabolic enzymes in hepatocytes are congenitally deficient, or acute liver failure (fulminant hepatitis) caused by drugs or viruses. Examples of congenital metabolic liver diseases include urea cycle disorders (e.g., ornithine transcarbamylase deficiency, citrullinemia), bilirubin metabolism disorders (e.g., Crigler-Najjar syndrome), familial hypercholesterolemia, hemophilia, and glycogen storage diseases. Transplanting the hepatic stem cells of the present invention into the patient's liver can supplement the deficient enzymes, potentially improving the condition, similar to insulin supplementation for diabetes patients. For acute liver failure, transplantation of the hepatic stem cells of the present invention into the patient's liver can support the metabolism until necrotic and shed hepatocytes regenerate and recover, and can serve as a supplementary cell transplantation therapy. Therefore, the cell transplantation therapy of the present invention is preferably applied to liver diseases.

[0095] When the hepatic stem cells of the present invention are used in cell transplantation therapy, it is desirable to use cells derived from iPS cells established from somatic cells with the same or substantially the same HLA genotype as the recipient individual, in order to avoid rejection. Here, "substantially the same" means that the HLA genotype is identical to the transplanted cells to an extent that immune responses can be suppressed with immunosuppressants, e.g., somatic cells with an HLA type that matches the three HLA loci (HLA-A, HLA-B, and HLA-DR) or the four HLA loci (HLA-C). However, when the purpose is to treat congenital metabolic disorders, cells derived from iPS cells established from patient-derived somatic cells are typically not used in cell transplantation therapy.

[0096] The hepatic stem cells of the present invention can be prepared as parenteral formulations such as injections, suspensions, and infusions by mixing with a pharmaceutically acceptable carrier according to conventional methods. Therefore, in one embodiment, a method for producing a cell transplantation therapy agent is provided, which includes a step of formulating the hepatic stem cells of the present invention. Such a method may also include a step of preparing the hepatic stem cells of the present invention. It may also further include a step of preserving the hepatic stem cells of the present invention.

[0097] Pharmaceutically acceptable carriers that can be included in the parenteral formulation include aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). The cell transplantation therapeutic agent of the present invention may be formulated with, for example, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc. When the cell transplantation therapeutic agent of the present invention is formulated as an aqueous suspension, for example, about 1 × 10 cells are added to the aqueous solution. 6 ~Approx. 1×10 8 The cells may be suspended to a concentration of 1000 cells / mL. The dosage or transplantation amount and the number of administrations or transplantations of the cell transplantation therapeutic agent of the present invention can be appropriately determined depending on the age, body weight, symptoms, etc. of the mammal to be treated.

[0098] The cell transplantation therapeutic agent of the present invention is provided in a cryopreserved state under conditions typically used for cryopreserving cells, and can be thawed immediately before use. In this case, it may further contain serum or a serum substitute, an organic solvent (e.g., DMSO), etc. In this case, the concentration of the serum or serum substitute is not particularly limited, but may be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, but may be about 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0099] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0100] Materials and Methods 1. Human iPS Cell Culture Three human iPSC lines were used. RIKEN-2F and 585A1 were both provided by the RIKEN BioResource Center (Tsukuba, Japan), and Fuji-5 was established in the present inventors' laboratory. Human iPSCs were cultured in NutriStem hESC XF (Biological Industries) medium on 45x diluted Matrigel (Growth Factor Reduced, BD Biosciences). iPSC colonies were passaged using TrypLE express (Thermo Fisher Scientific) and supplemented with 5 μM Y-27632 (FUJIFILM Wako) for the first few days.

[0101] 2. Superfolder GFP knock-in into the albumin gene locus using the CRISPR-Cas9 system. To target the albumin stop codon, a single guide RNA (sgRNA) was constructed using the online CRISPR design service CRISPRdirect (http: / / crispr.dbcls.jp). The sgRNA with the lowest off-target effects was selected and inserted into a lentiCRISPRv2 plasmid (addgene) prepared with Esp3I. The LITMUS29 vector was used as the donor for homologous recombination (HR) and prepared with SpeI and XhoI. The human ALB gene was amplified by PCR using genomic DNA extracted from iPSC 585A1. The 5' and 3' homology arms targeted 803 bp upstream and 879 bp downstream of the ALB stop codon, respectively. The 5' and 3' homology arms, P2A, and Superfolder GFP (sfGFP) fragments were integrated and amplified by PCR. The final fragment was inserted into a vector using the In-Fusion HD Cloning Kit (Takara). For transfection, iPSC 585A1 cells were prepared as single cells in Opti-MEM (Thermo Fisher Scientific). Both the donor plasmid and the Cas9 vector were added to the cell suspension and electroporated. Transfected cells (1.25 × 10 cells per well) were added to each well. 6hESC XF cells were seeded onto iMatrix-precoated 6-well plates containing NutriStem hESC XF containing 5 μM Y-27632. The following day, 5 μg / mL puromycin was added to enrich for correctly targeted cells for one day. Targeted cells were then isolated using single-cell cloning. Successful HR was confirmed by genomic PCR.

[0102] 3. Hepatocyte-like cell (HLC) differentiation The differentiation protocol was based on a previous report (Si-Tayeb K, Noto FK, Nagaoka M et al. Hepatology 2010; 51: 297-305.) with some modifications. iPS cells were dissociated with TrypLE express and suspended in NutriStem hESC XF supplemented with 5 μM Y-27632. Cells were cultured at 1 × 10 in 6-well plates pre-coated with iMatrix (Reprocell). 5 cells / cm 2Cells were seeded at a density of 100 μM and cultured for only one day before differentiation. Definitive endoderm induction was initiated with 2.5 μM CHIR99021 (FUJIFILM Wako) and 50 ng / ml Activin A (Nacalai tesque) in RPMI 1640 (Thermo Fisher Scientific) containing 0.25% B27 Supplement (Thermo Fisher Scientific). After 24 hours of treatment, CHIR99021 was removed, and cells were treated with RPMI 1640 containing 50 ng / ml Activin A and 0.5% B27 Supplement. After 24 hours, the B27 supplement was changed to 1.0%, and cells were cultured with 50 ng / ml Activin A for an additional two days. After 4 days of differentiation, the medium was changed to RPMI 1640 containing 1.0% B27 supplement with 10 ng / ml bFGF (FUJIFILM Wako) and 20 ng / ml recombinant human BMP4 (Peprotech), followed by treatment with 20 ng / ml hepatocyte growth factor (HGF, Peprotech) in 1.0% B27 supplement in RPMI 1640 for 4 days. After 12 days of differentiation, the cells were transferred to hepatocyte growth medium containing DMEM / F12 containing 2% ITS-G supplement and 64 mg / L L-ascorbic acid 2-phosphate, supplemented with 1 μM CHIR99021 and 50 ng / ml bFGF. After an additional 8 days of culture, the cells were plated at 1.0 × 10 cells per well onto 12-well plates pre-coated with iMatrix. 5 cells / cm 2 The cells were passaged at 0.5 × 10 and 10 μM SB431542 (Tokyo Chemical Industry) was added to hepatocyte growth medium containing 1 μM CHIR99021 and 50 ng / ml bFGF. The cells were then passaged once a week, dissociated using TrypLE express, and plated at 0.25–0.5 × 10 cells per well onto 12-well plates precoated with iMatrix. 5 cells / cm 2CHIR99021 was used at a concentration of 1 μM or 2 μM depending on the human iPSC line used. To generate mature HLCs after long-term culture, cells were dissociated with TrypLE and plated onto 24-well plates pre-coated with iMatrix at a density of 0.8 × 10 5 cells / cm 2 The cells were seeded at a density of 100 μg / ml. The cells were cultured in hepatocyte growth medium lacking both CHIR99021 and bFGF. The degree of hepatocyte differentiation was monitored by observing the fluorescence emitted by iPSCs expressing sfGFP-conjugated albumin (ALB) after hepatocyte differentiation.

[0103] 4. Cell viability assay Cells were plated at 0.25 × 10 cells / well onto a 96-well plate pre-coated with iMatrix. 5 cells / cm 2 The cells were seeded at a density of 100 μL / well. To compare cell viability, Cell Counting Kit-8 (CCK-8, Dojindo) was used according to the manufacturer's instructions. After 4 days of culture, 200 μL of medium containing 10% CCK-8 was added to each well. The absorbance at 450 nm was measured using a plate reader to determine the cell density in each well.

[0104] 5. RNA Extraction, Reverse Transcription (RT), and Real-Time Quantitative RT-Polymerase Chain Reaction (PCR). Total RNA was extracted from cultured cells using ISOGEN II (Nippon Gene Co. Ltd.) according to the manufacturer's protocol. Messenger RNA was reverse-transcribed using ReverTra Ace qPCR RT Master Mix (Toyobo). cDNA was prepared from 0.5 μg of total RNA for each sample. PCR amplification was performed using TaKaRa Ex Taq (Takarabitec Co. Ltd.) on a T100 Thermal Cycler (Bio-Rad). Amplified cDNA was separated by electrophoresis on a 1.5% agarose gel (Nippon Gene Co. Ltd.) supplemented with 0.1 μg / ml ethidium bromide (Nacalai Tesque). Real-time quantitative PCR analysis was performed using THUNDERBIRD Next SYBR qPCR Mix (Toyobo) on a Rotor-Gene Q 2plex HRM system (Qiagen). The cDNA was amplified with the following cycling parameters: 95°C for 1 minute, followed by 40 cycles of 95°C for 5 seconds, 58°C for 25 seconds, and 72°C for 30 seconds. The primer information used for RT-PCR is shown in Table 1.

[0105]

[0106] 6. Immunocytochemistry. Differentiated cells were washed with PBS and fixed with 4% paraformaldehyde (FUJIFILM Wako) for 30 minutes. Then, cells were incubated with 0.25% Triton X (Nacalai Tesque) for 10 minutes at room temperature. After 30 minutes of incubation with Blocking One (Nacalai Tesque), cells were incubated with diluted primary antibodies overnight at 4°C. After washing three times with PBS, cells were incubated with diluted secondary antibodies for 1 hour at room temperature. Goat anti-rabbit IgG-FITC (Abcam) and donkey anti-mouse IgG-Alexa Fluor 647 (Abcam) were used as secondary antibodies. Cells were washed three times more with PBS, and nuclei were stained with Hoechst 33342 (Dojindo). Information on the antibodies used for immunocytochemistry (ICC) is shown in Table 2.

[0107] 7. Flow cytometry Differentiated cells were dissociated with TrypLE express and resuspended in phosphate-buffered saline (PBS, Nacalai tesque) containing 2% FBS. Cell fixation and permeabilization were performed using a Fixation / Permeabilization Kit (BD Biosciences). Antibody-labeled cells were analyzed using a FACSCalibur or FACSCanto II (BD Biosciences). A negative control in which the primary antibody was omitted was also included in each experiment. Data analysis was performed using FlowJo (BD Biosciences). Information on the antibodies used for flow cytometry (FCM) is shown in Table 2.

[0108] 8. Western Blotting. Differentiated cells were lysed in ice-cold RIPA buffer (Nacalai tesque) supplemented with Protease Inhibitor Cocktail (Nacalai tesque) and Phosphatase Inhibitor Cocktail (Nacalai tesque). Equal amounts of protein were separated on Bolt Bis-Tris Plus Gels (Thermo Fisher Scientific) under reducing conditions and then transferred to a polyvinylidene difluoride membrane (Thermo Fisher Scientific). After transfer, the membrane was incubated for 10 minutes in Tris-buffered saline (TBS) supplemented with 0.1% Tween-20 (Nacalai tesque) and blocked for 20 minutes with Blocking One. Primary antibodies were added to the membrane and incubated overnight at 4°C. After washing with TBS supplemented with 0.1% Tween-20, the membrane was incubated for 60 minutes at room temperature with horseradish peroxidase-conjugated species-specific secondary antibodies (Abcam) diluted in Blocking One. Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) was used according to the manufacturer's instructions, and chemiluminescence was recorded using a FUSION Solo S (M&S Instruments Inc.). Information on the antibodies used for Western blotting is shown in Table 2.

[0109]

[0110]

[0111] 9. Cytochrome P450 (CYP) Induction. Hepatocyte-like cells (HLCs) were incubated with 0.1% DMSO, 1000 μM phenobarbital, or 50 μM rifampicin for 24 hours. After incubation, mRNA and protein expression of CYP2C19 and CYP3A4 were analyzed.

[0112] 10. Cell Transplantation and Analysis Immunodeficient NOD / ShiJic-scidJcl mice (CLEA Japan, Inc.) were used as recipients of human cells. Unconditioned mice aged 6-8 weeks underwent cell transplantation. HLCs were transplanted via splenic injection after a left midline laparotomy. Human iPSCs and mature hepatocyte-like cells (MHLCs), which were differentiated for 12 days as usual and then treated with HGF for an additional 8 days, were also used as donor cells. 1-2 × 10 cells were placed in 0.1 ml of phosphate-buffered saline (PBS). 5 The cells were injected using a 27-gauge needle, and hemostasis was achieved by compressing the injection site. Livers were harvested 1–3 months after transplantation, and Hoechst 33342-treated frozen sections (6 μm) were examined by fluorescence microscopy to detect sfGFP-positive cells derived from iPSCs bearing sfGFP-conjugated ALBs.

[0113] 11. Statistical analysis All data were expressed as mean ± SE. Differences between groups were evaluated by one-way analysis of variance or Kruskal-Wallis test. A p value of <0.05 was considered significant.

[0114] 12. Targeting stem cell-related signals involved in proliferating hepatocyte-like cells (pHLC) using the CRISPR-Cas9 system. Single guide RNA (sgRNA) and related products were purchased from Integrated DNA Technologies, Inc. (https: / / sg.idtdna.com / jp / site / index.html). Prior to CRISPR / Cas9 electroporation, expanded proliferating hepatocyte-like cells (pHLC) were harvested and washed with PBS. To prepare the RNP (ribonucleoprotein) complex, 1.2 μl of sgRNA and 1.7 μl of Cas9 enzyme were mixed in 2.1 μl of PBS and incubated at room temperature for 15 minutes. For transfection, 1.25 × 10 6pHLCs were prepared as single cells in 100 μl of Opti-MEM (Thermo Fisher Scientific). RNP complexes and 1 μl of electroporation enhancer were added to the cell suspension, followed by electroporation. Transfected HLCs were plated at 1.0 × 10 cells onto iMatrix-precoated 24- or 96-well plates in hepatocyte growth medium supplemented with 2 μM CHIR99021, 50 ng / ml bFGF, and 10 μM SB431542. 5 cells / cm 2 HLCs were harvested and evaluated 2 or 6 days after electroporation.

[0115] Example 1: Establishment of Hepatocyte-Like Cells (HLCs) from iPSCs. Three iPSC lines were efficiently differentiated into HLCs using established methods (Si-Tayeb K, Noto FK, Nagaoka M et al. Hepatology 2010; 51: 297-305.) with minor modifications. The differentiation process consisted of three stages (Figure 1). Flow cytometry analysis revealed that approximately 90% of differentiated cells expressed CXCR4 after the first stage. CXCR4-positive cells were then efficiently induced into the hepatocyte lineage, with approximately 90% expressing hepatocyte nuclear factor 4α (HNF4α) by the end of the second stage and approximately 60% expressing asialoglycoprotein receptor 1 (ASGR1) by the end of the third stage. A conventional 12-day HLC differentiation protocol efficiently produced HLCs from iPSCs (Figure 2). However, as HLC differentiation progressed, Western blot analysis showed a time-dependent decrease in the expression of Ki67 protein, a cellular marker of proliferation (Fig. 3).

[0116] Example 2: Establishment of iPSC lines with sfGFP gene inserted into the albumin locus using the CRISPR-Cas9 system. To monitor differentiation into hepatocyte lineages, we established iPSC line 585A1, which carries an albumin allele linked to the sfGFP gene. Using the CRISPR-Cas9 system, we performed HR-mediated knock-in at the targeted albumin locus. The sfGFP cDNA was inserted together with the P2A fragment immediately before the ALB stop codon (Figure 4). The transfected human iPSC line 585A1 was selected by culture using puromycin, followed by expansion of single-cell clones. To confirm successful HR at the designated site, we sequenced the binding site. Finally, we established three 585A1 clones carrying sfGFP-conjugated ALB. To examine whether this method can be used to monitor hepatocyte lineage differentiation, iPSCs carrying sfGFP-conjugated ALBs were differentiated into HLCs for 12 days as described in Example 1 and then treated with HGF for an additional 8 days. Analysis by flow cytometry and immunocytochemistry showed that sfGFP expression correlated with endogenous ALB expression (Figure 5).

[0117] Example 3: Effects of GSK-3β Inhibitor and bFGF on iPSC-Derived HLCs. To evaluate the effects of Wnt / β-catenin signaling on iPSC-derived HLCs after 12 days of differentiation, we primed HLCs in the presence of CHIR99021, a potent GSK-3β inhibitor. Inhibition of GSK-3β promotes the nuclear accumulation of β-catenin. Increased β-catenin levels allow β-catenin to freely bind to DNA-binding members of the T-cell factor / lymphocyte-enhancing factor (TCF / LEF) family of proteins, activating the transcription of Wnt target genes (Mosimann C, Hausmann G, Basler K. Nature Reviews Molecular Cell Biology 2009; 10: 276-286). Daily administration of CHIR99021 increased the expression of Tcf7 and Lef1 in HLCs after 12 days of differentiation (Figure 6). Activation of Wnt / β-catenin signaling in iPSC-derived HLCs suppressed albumin expression, characteristic of mature hepatocytes, while inducing the expression of Sox17, a definitive endoderm marker. However, the cells also showed increased expression of HNF4α, suggesting that their hepatocyte identity was maintained (Figure 8). However, Western blotting analysis showed that expression of genes related to cell cycle progression, such as cyclin D1 and c-Myc, was not significantly increased compared to growth factor- or compound-free culture conditions (NGC), although Wnt / β-catenin signaling induced phosphorylation of retinoblastoma protein (Rb) (Figures 9 and 10). iPSCs expressing sfGFP-conjugated ALB after 12 days of differentiation were treated with HGF or CHIR99021 for an additional 8 days. HGF treatment induced slightly more ALB-producing cells than culture conditions without growth factors or compounds. Although HGF treatment did not result in Sox17-expressing cells, CHIR99021 treatment induced Sox17-expressing cells in a dose-dependent manner. ALB-expressing cells, in contrast to Sox17 expression, appeared to be reduced in a dose-dependent manner by CHIR99021 (Fig. 11). Treatment with bFGF for 1 day activated the MAPK pathway, but not the Akt pathway (Fig. 7).bFGF activated the expression of genes related to cell cycle progression, such as cyclin D1 and c-Myc (Fig. 9). Furthermore, Western blotting revealed that bFGF treatment induced Rb phosphorylation and Ki67 expression (Fig. 10). However, bFGF-treated HLCs decreased the expression of both Foxa2 and HNF4α, suggesting a shift away from the hepatic lineage (Figs. 8 and 10).

[0118] Example 4: Synergistic effect of GSK3β inhibitor and bFGF on iPSC-derived HLCs. To examine the effect of both CHIR99021 and bFGF on the cell proliferation of iPSC-derived HLCs, HLCs differentiated for 10 days were plated on a 96-well plate pre-coated with iMatrix at 0.25 × 10 5 cells / cm 2The HLCs were seeded at a density of 100 μg / ml and further incubated with 20 ng / ml HGF for 2 days. The cells were then treated with either CHIR99021 or bFGF, or both, at different concentrations for 4 days, after which a CCK-8 assay was performed to assess cell viability. The results showed that CHIR99021 and bFGF cooperated to maintain a significantly higher cell density than NGCs. Treatment with either CHIR99021 or bFGF alone had no significant effect on increasing cell density (Figure 12). After 12 days of differentiation, HLCs were treated with both CHIR99021 and bFGF for an additional 2 days. While CHIR99021 alone failed to induce cyclin D and c-Myc, the addition of CHIR99021 and bFGF enabled the induction of proteins related to cell cycle progression. Furthermore, bFGF treatment appeared to induce cell cycle-related proteins in a dose-dependent manner (Figures 13 and 15). Immunocytochemistry showed that most of the Sox17-expressing cells induced by CHIR99021 coexpressed cyclin D or phosphorylated retinoblastoma (phospho-RB) upon bFGF addition (Figures 16 and 17). However, the addition of bFGF to CHIR99021 appeared to inhibit the expression of definitive endoderm- and hepatic endoderm-related genes. Immunocytochemistry showed that CHIR99021 treatment maintained much of the Sox17 expression even after the addition of 50 ng / ml bFGF, but the addition of bFGF to CHIR99021 appeared to attenuate the expression of both hepatic endoderm- and hepatocyte-related genes, likely due to a moderate inhibition of CHIR99021-induced Sox17 expression (Figures 14 and 15).

[0119] Example 5: Activation of TGF-β Signaling by Passaging of HLCs. HLCs differentiated for 12 days were further differentiated for 8 days with a combination of 1 μM CHIR99021 and 50 ng / ml bFGF. These cells were dissociated and analyzed by flow cytometry. Results showed that over 80% of cells were positive for both ASGP1 and HNF4α. Furthermore, over 50% of cells expressed Sox17 (Figure 18). Dissociated cells were replated onto iMatrix-coated plates, and the next day they maintained their polarity and adhered. However, the cells subsequently began to detach from the plates. Under the same culture conditions with both CHIR99021 and bFGF, the number of cells was reduced to 2.0 × 10 5 cells / cm 2 Even when passaged at a higher density than 1000, HLCs failed to survive (Figure 19). Even HLCs that barely survived on plates became frail after 4 days of passage, when many cells were lost. This led us to suspect the involvement of TGF-β signaling, which is thought to be involved in hepatocyte-specific processes, including growth arrest (Bird TG, Muller M, Boulter L et al. Sci Transl Med 2018;10:eaan1230). TGF-β expression remained unchanged when HLCs were not passaged, but increased after passage on day 20. The results confirmed activation of the Smad pathway in passaged hepatocytes, revealing phosphorylation of Smad2 and induction of p15INK4b and p21 expression. In other words, passaging activated TGF-β signaling and induced the expression of senescence-related genes in HLCs (Figures 20 and 21).

[0120] Example 6: Inhibition of TGF-β signaling enabled the passage of HLCs. Considering the activation of TGF-β signaling after passaging, we employed SB431542, which inhibits activin receptor-like kinase (ALK) 4, ALK5, and ALK7, leading to the inhibition of activin and TGF-β signaling. When SB431542 was used together with CHIR99021 and bFGF after passaging, HLCs remained adherent. A CCK-8 assay performed on day 3 after passaging showed that the addition of SB431542 successfully produced significantly higher cell density than the absence of SB431542 (Figure 22). Western blotting analysis confirmed that SB431542 reversed the phosphorylation of Smad2 that occurred immediately after passaging HLCs. Blockade of the Smad pathway inhibited the activation of p15INK4b and p21 genes, subsequently resulting in the activation of Ki67 and c-Myc genes in post-passage HLCs (Figures 23 and 24). Despite the activation of genes related to cell cycle progression, the expression of definitive endoderm, hepatic endoderm, and hepatocyte-related genes was maintained. Western blot analysis showed that these genes increased in a time-dependent manner after passaging, whereas the expression of both Ki67 and c-Myc appeared to decrease over time (Figure 26). Immunocytochemistry showed that clusters of cells expressing Sox17 and HNF4α were produced over time after passaging (Figure 25).

[0121] Example 7: Long-Term Passaging of HLCs. The addition of SB431542 to CHIR99021 and bFGF treatment enabled the passage of HLCs. HLCs were maintained for more than 10 passages (Figure 27). Immunocytochemistry of cells after 21 passages revealed expression of Sox17, HNF4α, and ASGR1, maintaining a mixed phenotype of definitive endoderm, hepatic endoderm, and hepatocyte lineages. Most cells expressed either Sox17 or HNF4α, and some HNF4α-expressing cells also expressed ASGPR1 (Figure 28). When 1 μM CHIR99021 was used with 50 ng / ml bFGF and 10 μM SB431542, HLCs significantly increased expression of hepatocyte-related genes, including albumin and ASGR1. However, 2 μM CHIR99021 appeared to induce faster proliferation of HLCs, resulting in significantly more Sox17-expressing cells (data not shown).

[0122] Example 8: Maturation of HLCs after long-term passage HLCs after long-term passage were plated at 1 × 10 in a 24-well plate pre-coated with iMatrix. 5 cells / cm 2Cells were seeded at a density of 10 μM and preincubated with 10 μM SB431542 alone for 3 days. They were then cultured for an additional 7 days with or without 10 μM SB431542, along with CHIR99021 or bFGF. When HLCs were maintained without SB431542, regardless of the presence or absence of CHIR99021 or bFGF, phosphorylation of Smad2 and p15INK4b were induced. Activation of the Smad pathway led to a significant decrease in mRNA levels of Sox17, HNF4α, α-fetoprotein (AFP), and ALB, indicating that under activated TGF-β signaling, HLCs are unable to maintain phenotypes characteristic of definitive endoderm or hepatic endoderm (Figure 29). While CHIR99021 treatment alone resulted in HLCs remaining more immature, bFGF treatment did not appear to inhibit hepatic maturation. Long-term maintenance of HLCs in culture conditions containing CHIR99021, bFGF, and SB431542 after removal of CHIR99021 reduced Sox17 expression and significantly induced both AFP and ALB expression, but not HNF4α expression. In other words, SB431542 treatment appeared sufficient to redifferentiate HLCs cultured for long periods with CHIR99021, bFGF, and SB431542 into hepatocytes, whereas bFGF treatment only slightly promoted hepatocyte differentiation (Figure 30). Expression of sfGFP, which correlated with endogenous ALB expression, began to appear after approximately 1 week of culture in the absence of both CHIR99021 and bFGF after long-term passage. Expression plateaued after 20 days of maturation, with over 80% of HLCs expressing sfGFP (Figure 31). HLCs differentiated with 10 μM SB431542 for 20 days or more lost Sox17 expression but showed expression of proteins characteristic of mature hepatocytes (Fig. 32).Furthermore, treatment with 50 μM rifampicin for 1 day induced the expression of CYP2C19 and CYP3A4 (Fig. 33).

[0123] Example 9: Repopulation after HLC transplantation 1-2 x 10 cells derived from iPSCs with sfGFP-conjugated ALBs 5 We transplanted HLCs into unconditionally immunodeficient mice to evaluate their ability to repopulate intact livers. In addition to HLCs, we also used human iPSCs and MHLCs as donor cells. iPSCs carrying sfGFP-conjugated ALB were expected to fluoresce if they remained in the recipient liver as mature hepatocyte-like cells expressing ALB. Livers were harvested 1–3 months after transplantation and analyzed for the presence of fluorescent cells. Only HLCs successfully repopulated the recipient liver, exhibiting numerous fluorescent areas under a native fluorescence microscope (Figure 34). Notably, the cell population spontaneously expressing sfGFP appeared to be concentrated around the central vein (Figure 35).

[0124] Example 10: Identification of a stem cell population in proliferating hepatocyte-like cells (pHLCs) Wnt-responsive genes, including leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) and Axin2, are thought to identify actively dividing stem cells driven by the Wnt / β-catenin pathway in the small intestine, large intestine, stomach, hair follicles, and liver (Wang B. et al. (2015), Nature 524: 180-185; Ang C. H. et al. (2019), Proc Natl Acad Sci USA 116: 19530-19540; Clevers H, Loh KM, Nusse R. (2014), Science 346: 1248012). Current proliferating hepatocyte-like cells (pHLCs) can maintain hepatic characteristics even after long-term subculture. Therefore, we attempted to identify a hepatic stem cell population within these cells. pHLCs were able to grow and passage for long periods in hepatocyte growth medium supplemented with 2 μM CHIR99021, 50 ng / ml bFGF, and 10 μM SB431542. These cells maintained Wnt-responsive gene expression, including LGR5 and Axin2, in a GSK-3β inhibitor-dependent manner, while Sox17 expression also showed Wnt responsiveness (Figure 36). Reduction of GSK-3β inhibitor for just 3 days significantly reduced both cell cycle-related gene expression and cell density (Figure 37). Flow cytometry analysis demonstrated that GSK-3β inhibitor significantly maintained a population of pHLCs expressing both Axin2 and Sox17, or both pRb (phosphorylated Rb) and Sox17. Furthermore, all Sox17-expressing cells were positive for non-phosphorylated (active) β-catenin (Figure 38).

[0125] The role of β-catenin was confirmed using the CRISPR-Cas9 system. To verify the effectiveness of CRISPR-Cas9, we assessed the expression of β-catenin-related genes (Figures 39 and 40). Transfection with a CRISPR-Cas system targeting β-catenin (CRISPR-β-catenin) significantly suppressed β-catenin expression, along with the corresponding reductions in Tcf7 and Lef1 expression. As expected, β-catenin suppression also significantly reduced Sox17 expression. This also reduced the expression of cell cycle-related genes and led to the maturation of the pHLC (proliferative hepatocyte-like cell) population. Furthermore, cell viability assays demonstrated the suppression of pHLC proliferation. We then performed transfection with a CRISPR-Cas system targeting Sox17 (CRISPR-Sox17), which significantly suppressed Sox17 expression (Figure 41). Furthermore, the pHLC population significantly matured, and similar to transfection with CRISPR-β-catenin, cell proliferation was suppressed, accompanied by the downregulation of cell cycle-related genes.

[0126] The present invention makes it possible to provide hepatic stem cells with proliferation capacity, which is expected to be applicable to hepatic cell transplantation, which has hitherto been problematic in terms of long-term engraftment. In particular, the hepatic stem cells of the present invention are highly useful in hepatic cell transplantation for patients with liver disease who are difficult to transplant from a living donor, particularly neonatal patients.

[0127] This application is based on patent application No. 2023-210649 filed in Japan (filing date: December 13, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing hepatic stem cells, comprising the step of culturing hepatocytes in a medium containing a Wnt signaling promoter and an FGF signaling promoter.

2. The method of claim 1, wherein at least one of the Wnt signaling promoters is CHIR99021.

3. The method of claim 1 or 2, wherein at least one of the FGF signaling promoters is bFGF.

4. A method for producing hepatic stem cells, comprising the step of culturing hepatic stem cells in a medium containing a Wnt signaling promoter, an FGF signaling promoter, and an ALK inhibitor.

5. The method of claim 4, wherein at least one of the ALK inhibitors is SB431542.

6. The method according to claim 4 or 5, wherein the starting hepatic stem cells are cells obtainable by the method according to any one of claims 1 to 3.

7. The method according to any one of claims 1 to 6, wherein the hepatocytes or hepatic stem cells are derived from pluripotent stem cells.

8. The method according to claim 7, wherein the pluripotent stem cells are of human origin.

9. Hepatic stem cells obtained by the method according to any one of claims 1 to 8.

10. Hepatic stem cells that have all of the following characteristics (A) to (C). (A) Derived from pluripotent stem cells (B) Express SOX17 and Axin2 (C) Have the ability to differentiate into hepatic cells.

11. The cell according to claim 10, further having the characteristic (D) of being positive for at least one selected from the group consisting of LGR5, HNF4α, phosphorylated retinoblastoma protein, and non-phosphorylated β-catenin.

12. A method for producing hepatocytes, comprising the step of inducing differentiation of the hepatic stem cells according to any one of claims 9 to 11 into hepatocytes.

13. The method according to claim 12, wherein the step of inducing differentiation of hepatic stem cells into hepatocytes comprises a step of culturing the hepatic stem cells in a medium containing an ALK inhibitor and not containing a Wnt signaling promoter.

14. A cell transplantation therapeutic agent comprising the cells according to any one of claims 9 to 11 or the cells obtained by the method according to claim 12 or 13.

15. The agent according to claim 14 for treating liver disease.

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