Stem cell-derived hepatocyte, liver transplantation composition containing said stem cell-derived hepatocyte, method for producing pluripotent stem cell-derived hepatocyte having freezing resistance, use of molecular marker for screening for hepatocyte having freezing resistance, and method for purifying hepatic progenitor cell or hepatocyte

Abstract

A stem cell-derived hepatocyte according to one embodiment of the present disclosure expresses at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1.

Description

Stem cell-derived hepatocytes, a liver transplantation composition containing said stem cell-derived hepatocytes, a method for producing cryo-tolerant pluripotent stem cell-derived hepatocytes, the use of molecular markers for selecting cryo-tolerant hepatocytes, and a method for purifying hepatic progenitor cells or hepatocytes.

[0001] The present invention relates to stem cell-derived hepatocytes, a liver transplantation composition containing said stem cell-derived hepatocytes, a method for producing cryo-tolerant pluripotent stem cell-derived hepatocytes, the use of molecular markers for selecting cryo-tolerant hepatocytes, and a method for purifying hepatic progenitor cells or hepatocytes.

[0002] The liver plays a vital role in maintaining life, including nutrient storage, drug detoxification, and metabolism. While liver transplantation is the only definitive treatment for serious liver diseases, it faces numerous challenges, including a severe shortage of donors and significant physical and financial burdens. As an alternative to liver transplantation, transplantation therapy using hepatocytes, the main constituent cells of the liver, is gaining attention.

[0003] The therapeutic effect of hepatocyte transplantation therapy is achieved when engrafted hepatocytes exhibit high proliferative capacity within the liver, forming colonies and replacing diseased hepatocytes. Therefore, hepatocytes intended for medical use are required to not only engraft within the recipient liver but also to have the ability to regrow. Furthermore, hepatocytes are not only useful for medical purposes but are also in high demand in a wide range of fields, such as elucidating disease mechanisms and drug discovery research, and the shortage of their resources is a major challenge.

[0004] As a source for stably obtaining a wide variety of cells that meet high demand, pluripotent stem cells, which possess unlimited proliferative capacity, are attracting attention. On the other hand, in order to popularize pluripotent stem cell-derived hepatocytes, it is essential to establish efficient methods for preparing highly functional hepatocytes and a system that can supply them in large quantities and stably.

[0005] To date, culture systems have been established to efficiently obtain human hepatocytes by producing, selecting, and utilizing hepatic progenitor cells that possess both high proliferative capacity and differentiation ability into hepatocytes (Non-Patent Documents 1-6). The obtained cells have the following typical characteristics of hepatic progenitor cells: (1) high proliferative capacity, (2) positive for both liver markers and bile duct epithelial cell markers, and (3) the ability to reversibly acquire liver characteristics (liver maturation).

[0006] On the other hand, Non-Patent Document 7 describes the successful isolation and proliferation culture of hepatic progenitor cells, but their functionality is low, and they have not been applied to transplant therapy. The technologies described in Non-Patent Documents 8 and 9 describe the successful culture of hepatic progenitor cells and the acquisition of functional hepatocytes using them. However, Non-Patent Document 8 does not clarify the transplant therapy effect of the cultured hepatocytes, and the transplant effect of the hepatocytes developed in Non-Patent Document 9 is very low, and only short-term evaluations have been conducted.

[0007] Therefore, hepatocytes derived from pluripotent stem cells are a promising cell source, but it is necessary to address the low engraftment and regrowth capabilities required for medical applications. Currently, it is known that there are significant differences in engraftment and regrowth capabilities among hepatocyte lots, but useful cell markers have not yet been identified.

[0008] Furthermore, cryopreservation is essential for establishing a stable supply system because it enables the long-term storage of cells. However, hepatocytes derived from pluripotent stem cells generally have low cryopreservation tolerance, and it is known that their characteristics and functionality are greatly reduced after cryopreservation. For example, Non-Patent Documents 10 and 11 show that hepatocytes that have undergone cryopreservation exhibit reduced cell viability, cell adhesion efficiency, albumin production, liver marker gene expression, and cell transplantation efficiency.

[0009] International Publication No. 2019 / 131938

[0010] Pareja E, Gomez-Lechon MJ, Tolosa L. Induced pluripotent stem cells for the treatment of liver diseases: challenges and perspectives from a clinical viewpoint. Ann Transl Med. 2020;8: 566.Yao J, Yu Y, Nyberg SL. Induced Pluripotent Stem Cells for the Treatment of Liver Diseases: Novel Concepts. Cells Tissues Organs. 2022;211: 368-384.Wu H, Zhou X, Fu G-B, He Z-Y, Wu H-P, You P, et al. Reversible transition between hepatocytes and liver progenitors for in vitro hepatocyte expansion. Cell Res. 2017;27: 709-712.Zhang K, Zhang L, Liu W, Ma X, Cen J, Sun Z, et al. In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity. Cell Stem Cell. 2018;23: 806-819.e4.Katsuda T, Matsuzaki J, Yamaguchi T, Yamada Y, Prieto-Vila M, Hosaka K, et al. Generation of human hepatic progenitor cells with regenerative and metabolic capacities from primary hepatocytes. Elife. 2019;8. doi:10.7554 / eLife.47313Guo R, Jiang M, Wang G, Li B, Jia X, Ai Y, et al.IL6 supports long-term expansion of hepatocytes in vitro. Nat Commun. 2022;13: 7345.Yanagida A, Ito K, Chikada H, Nakauchi H, Kamiya A. An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential. PLoS One. 2013;8(7):e67541.Pan T, Tao J, Chen Y, et al. Robust expansion and functional maturation of human hepatoblasts by chemical strategy. Stem Cell Res Ther. 2021;12(1):151.Takayama K, Nagamoto Y, Mimura N, et al. Long-term self-renewal of human ES / iPS-derived hepatoblast-like cells on human laminin 111-coated dishes. Stem Cell Reports. 2013;1(4):322-335.Inui J, Ueyama-Toba Y, Mitani S, Mizuguchi H. Development of a method of passaging and freezing human iPS cell-derived hepatocytes to improve their functions. PLoS One. 2023;18(5):e0285783.Terry C, Dhawan A, Mitry RR, Hughes RD. Cryopreservation of isolated human hepatocytes for transplantation: State of the art. Cryobiology. 2006;53(2):149-159.

[0011] There is a need for technology to generate cryo-tolerant hepatocytes from stem cells. Furthermore, the identification of molecular markers for selecting cryo-tolerant hepatocytes is desirable.

[0012] One aspect of the present invention aims to realize a technique for producing cryo-resistant liver cells.

[0013] As a result of diligent research to solve the above problems, the inventors of the present invention have for the first time discovered that the expression of 20 specific genes, including CD44, is upregulated in cryo-resistant hepatocytes, and have completed the present invention.

[0014] In other words, in order to solve the above problems, a stem cell-derived hepatocyte having freeze resistance according to one aspect of the present invention expresses at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1.

[0015] Furthermore, a method for producing freeze-tolerant pluripotent stem cell-derived hepatocytes according to one aspect of the present invention includes the following steps (1) to (2): (1) a step of subculturing a population of pluripotent stem cell-derived hepatocyte-like cells differentiated from pluripotent stem cells on laminin-111; (2) a step of inducing liver maturation in the cells after step (1) on laminin-111.

[0016] Furthermore, the use of a molecular marker for selecting cryo-tolerant hepatocytes according to one aspect of the present invention is that the molecular marker consists of the following substances (a) or (b): (a) the translation product of at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1; (b) A transcript of at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1.

[0017] Furthermore, a method for purifying freeze-tolerant hepatic progenitor cells or hepatocytes according to one aspect of the present invention includes the step of culturing an isolated cell population containing hepatic progenitor cells or hepatocytes on laminin-111.

[0018] According to one aspect of the present invention, a technique for producing cryogenically resistant hepatocytes can be realized.

[0019] This is a schematic diagram illustrating the overall picture of a method for producing freeze-tolerant pluripotent stem cell-derived hepatocytes according to one aspect of the present invention. This is a diagram showing the results of an example, and shows the analysis results for cultured ES cell-derived hepatocytes and ES cell-derived hepatocytes after induction of liver maturation. (A) is a schematic diagram representing the overall picture of this experiment; (B) is a diagram showing phase-contrast microscope images over time; (C) is a diagram showing morphological changes over time; (D) and (E) are diagrams showing the results of gene expression analysis; (F) is a diagram showing human ALB secretion ability; (G) is a diagram showing the results of comparison of urea production ability; (H) is a diagram showing the results of gene expression analysis in ES cell-derived hepatocytes (passage 5) cultured on LN111 or LN511. This is a diagram showing the results of an example, and shows the results of transplantation of ES cell-derived hepatocytes into the liver injury model mouse TK-NOG. (A) is a schematic diagram showing the overall picture of this experiment; (B) is a diagram showing the results of transplanting hepatocytes into TK-NOG, a model mouse for liver injury; (C) is a diagram showing the results of transplanting cryopreserved cells into TK-NOG, a model mouse for liver injury. This is a diagram showing the results of the example, showing the results of transplanting ES cell-derived hepatocytes into TK-NOG, a model mouse for liver injury. (A) is a diagram showing the human ALB levels in plasma in TK-NOG mice; (B) is a diagram showing the results of transplanting human ES cell-derived hepatocytes into TK-NOG, a model mouse for liver injury; (C) is a diagram showing the results of immunostaining with human CYP2C in the liver of TK-NOG mice. This is a diagram showing the results of the example, showing the results of single-cell RNA-seq analysis. (A) is a UMAP showing the results of integrating and clustering single-cell RNA-seq analysis results; (B) is a UMAP showing the results of extracting cell populations with a high proportion of cells cultured with Laminin-111 from the single-cell RNA-seq analysis results and clustering them; (C) is a UMAP showing the gene expression levels of genes that are highly expressed in cell populations specific to hepatocytes [1].This figure shows the results of the example, the results of single-cell RNA-seq analysis, and the results of RT-qPCR analysis of the gene expression of CD44, RORC, ENG, NT5E, SPP1, and ENG in hepatocytes [1] or hepatocytes [2] (two lots each). This figure shows the results of the example, the verification results using human iPS cells. (A) is a phase-contrast microscope image of iPS cell-derived hepatocytes; (B) is a figure showing the gene expression analysis results. This figure shows the results of the example, the results of verifying the effectiveness of the subculture and liver maturation induction system in the produced iPS cell-derived hepatocytes. (A) is a schematic diagram of this experiment; (B) is a phase-contrast microscope image; (C) and (D) are figures showing the gene expression analysis results. This figure shows the results of the example, the results of the examination of the matrix used during subculture of ES cell-derived hepatocytes. (A) is a schematic diagram representing the overall structure of this experiment; (B) is a diagram showing a phase-contrast microscope image; and (C) and (D) are diagrams showing the results of analysis by quantitative real-time PCR (RT-qPCR).

[0020] The present invention will now be described in detail, but the present invention is not limited to the embodiments described herein. Unless otherwise specified herein, "A to B" representing a numerical range means "A or greater, and B or less."

[0021] [1. Stem cell-derived hepatocytes] Stem cell-derived hepatocytes according to one aspect of the present invention express at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1.

[0022] Stem cell-derived hepatocytes according to one aspect of the present invention express at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1, and therefore possess freeze resistance. In this specification, these 20 genes may be referred to as "hepatocyte freeze resistance marker genes" or "freeze resistance marker genes" for convenience.

[0023] Furthermore, stem cell-derived hepatocytes according to one aspect of the present invention express at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1, and therefore have long-term transplantability. For example, stem cell-derived hepatocytes according to one aspect of the present invention preferably engraft for more than one month after transplantation, and more preferably have long-term transplantability, engrafting for more than three months after transplantation.

[0024] Herein, in this specification, "freeze tolerance" means that cells have transplantability after cryopreservation.

[0025] In this specification, "having transplantability" means that the transplanted cells have the ability to engraft in the liver and to regrow in the liver. The engraftment and regrowth ability of transplanted cells can be confirmed by measuring the amount of albumin produced by the transplanted cells in the recipient's plasma at four weeks after cell transplantation. If albumin produced by the transplanted cells is present in the recipient's plasma in a detectable amount, the transplanted cells can be said to have transplantability.

[0026] Stem cell-derived hepatocytes according to one aspect of the present invention preferably exhibit freeze tolerance after being stored in liquid nitrogen for one month or more. Furthermore, while the degree of freeze tolerance is not particularly limited, it is preferable that stem cell-derived hepatocytes according to one aspect of the present invention exhibit freeze tolerance exceeding that of stem cell-derived hepatocytes cultured on laminin-511 and cryopreserved under the same conditions. Note that "stem cell-derived hepatocytes cultured on laminin-511" refers to stem cell-derived hepatocytes obtained under the same culture conditions as stem cell-derived hepatocytes according to one aspect of the present invention, except that laminin-511 is used instead of laminin-111.

[0027] Furthermore, in this specification, "gene expression" encompasses both the synthesis of mRNA from a specific nucleotide sequence of the gene (also known as transcription or mRNA expression) and the synthesis of a protein based on the information in the mRNA (also known as translation or protein expression).

[0028] In this specification, "gene expression" means that the mRNA or protein corresponding to the gene is expressed in an amount greater than or equal to the amount detectable by methods known in the art. mRNA expression can be confirmed using known nucleic acid amplification methods and / or nucleic acid detection methods, such as RT-PCR, quantitative RT-PCR (RT-qPCR), and comprehensive gene expression analysis (RNA-seq). Protein expression can be confirmed using known methods, such as Western blotting.

[0029] Furthermore, in this specification, "stem cell-derived hepatocytes" means hepatocytes differentiated from stem cells. Here, "stem cells" means cells that have the ability to self-renew and differentiate. Stem cells are classified into pluripotent stem cells, multipotent stem cells, unipotent stem cells, etc., according to their differentiation ability, but any type of stem cell is acceptable as long as it has the ability to differentiate into hepatocytes. In addition, even stem cells that originally differentiate into adipocytes, skeletal muscle cells, osteoblasts, etc., and do not differentiate into hepatocytes are included in the category of "stem cells" in this specification if they can acquire the ability to differentiate into hepatocytes through the differentiation induction procedure described later.

[0030] Stem cells may be derived from any vertebrate, but are preferably derived from mammals such as mice, rats, rabbits, sheep, pigs, cattle, goats, monkeys, and humans, and are particularly preferably derived from humans. Stem cells may also be prepared from blastocysts, fetal tissue, adult tissue, or umbilical cord blood. The tissue used to prepare stem cells is not particularly limited, and for example, stem cells can be prepared from bone marrow, muscle, brain, pancreas, liver, kidney, etc. Stem cells may be embryonic stem (ES) cells, somatic stem cells, or induced pluripotent stem (iPS) cells, but are preferably ES cells or iPS cells. iPS cells may be derived from healthy individuals or from patients with drug-induced liver injury.

[0031] Methods for preparing ES cells and iPS cells are well-established and can be prepared according to methods known in this field (Takahashi, K. et al., Cell 2007; 131(5): 861-72, doi: 10. 1016 / j. cell. 2007. 11. 019 (Reference 12)). Alternatively, already established ES cell lines or iPS cell lines may be obtained from sources such as the RIKEN BioResource Center (RIKEN BRC) or the ATCC (American Type Culture Collection).

[0032] Various culture conditions for inducing differentiation of stem cells into hepatocytes are known, and by appropriately selecting these conditions, it is possible to prepare an isolated cell population containing hepatocytes of a desired degree of differentiation or maturity. Since it is possible to induce high-performance pluripotent stem cell-derived hepatocytes that are closer to those of a living liver, it is preferable that the stem cell-derived hepatocytes according to one aspect of the present invention are produced (prepared) by the method for producing pluripotent stem cell-derived hepatocytes according to one aspect of the present invention, which will be described later.

[0033] Stem cell-derived hepatocytes according to one aspect of the present invention may include both mature and immature hepatocytes. The degree of hepatic maturity can be selected depending on the purpose for which the stem cell-derived hepatocytes according to one aspect of the present invention are used. In this specification, "immature hepatocytes" means "hepatic progenitor cells," which are hepatocytes that have not undergone maturation induction.

[0034] The induction of maturation of stem cell-derived hepatocytes according to one aspect of the present invention can be carried out using methods known in the art. In recent years, it has become clear that the transplantability of cultured hepatic progenitor cells is positively correlated with the degree of liver maturation (References 5, 7). For this reason, from the viewpoint of making stem cell-derived hepatocytes according to one aspect of the present invention clinically applicable and sufficiently inducing liver maturation, it is preferable that maturation is induced by a liver maturation induction method described later as one of the steps in the method for producing pluripotent stem cell-derived hepatocytes.

[0035] Furthermore, stem cell-derived hepatocytes according to one aspect of the present invention may have undergone one or more passaging cycles. Stem cell-derived hepatocytes according to one aspect of the present invention have sufficient cryotolerance and long-term transplantability even after passaging.

[0036] Furthermore, stem cell-derived hepatocytes according to one aspect of the present invention may have undergone one or more cryopreservations. Since stem cell-derived hepatocytes according to one aspect of the present invention are cryo-tolerant, they retain transplantability even after cryopreservation.

[0037] In addition, the stem cell-derived hepatocytes according to one aspect of the present invention may optionally be immortalized. Here, a cell being "immortalized" means that the cell maintains a state where it can still proliferate even after repeating a certain number of divisions, that is, the cell has the ability of infinite proliferation. Methods for immortalizing cells have already been established, and known techniques can be employed. For example, cells can be immortalized by introducing an immortalizing gene such as the telomerase reverse transcriptase (TERT) gene into cells using a retroviral vector.

[0038] The stem cell-derived hepatocytes according to one aspect of the present invention may be purified or may be unpurified. A purification method may be appropriately selected according to the functionality required for the stem cell-derived hepatocytes according to one aspect of the present invention. For example, a method of purifying only cells having drug-metabolizing ability by utilizing the cytotoxicity of a drug is known (International Publication No. 2019 / 131938). From the viewpoint of preparing cells having sufficient functionality such as drug-metabolizing ability with high purity, the stem cell-derived hepatocytes according to one aspect of the present invention are preferably purified by the purification method described as one of the steps of the method for producing pluripotent stem cell-derived hepatocytes to be described later.

[0039] (Freezing tolerance marker gene of hepatocytes) The 20 freezing tolerance marker genes expressed by the stem cell-derived hepatocytes according to one aspect of the present invention will be described below.

[0040] (1) The CD44 gene encodes the CD44 protein. The CD44 protein is a single-pass transmembrane glycoprotein, and multiple isoforms have been reported. CD44 is one type of hyaluronic acid-binding protein and is known to be involved in cell-cell interaction, cell adhesion, and migration. For example, the nucleotide sequence of the cDNA of human CD44 is Accession No. NM_000610, and the amino acid sequence of the protein is Accession No. NP_000601, which are registered in the database NCBI.

[0041] (2) The CD9 gene encodes the CD9 protein. The CD9 protein is a type of transmembrane protein tetraspanin (also known as Transmembrane 4 superfamily), and it is known to interact with the integrin family and other membrane proteins and be involved in cell adhesion and migration. For example, the nucleotide sequence of the cDNA of human CD9 is registered in the database NCBI as Accession No. NM_001769, and the amino acid sequence of the protein is registered as Accession No. NP_001760.

[0042] (3) The CXCL1 gene encodes the CXCL1 (C-X-C motif chemokine ligand 1) protein. The CXCL1 protein is a low molecular weight protein belonging to the CXCL chemokine family. It is known to bind to CXCR2, a CXCL chemokine receptor, and be involved in fibroplasia and angiogenesis. For example, the nucleotide sequence of the cDNA of human CXCL1 is registered in the database NCBI as Accession No. NM_001511, and the amino acid sequence of the protein is registered as Accession No. NP_001502.

[0043] (4) The CXCL6 gene encodes the CXCL6 (C-X-C motif chemokine ligand 6) protein. The CXCL6 protein is a low molecular weight protein belonging to the CXCL chemokine family. It is known to bind to CXCR1 and CXCR2, which are CXCL chemokine receptors, and be involved in angiogenesis. For example, the nucleotide sequence of the cDNA of human CXCL6 is registered in the database NCBI as Accession No. NM_002993, and the amino acid sequence of the protein is registered as Accession No. NP_2984.

[0044] (5) The EMP3 gene encodes the EMP3 (Epitis membrane protein 3) protein. The EMP3 protein is a multi-pass transmembrane protein belonging to the PMP-22 / EMP / MP20 family, and several isoforms have been reported. EMP3 is known to be involved in cell proliferation, intercellular interactions, and tumor suppression. For example, the nucleotide sequence of human EMP3 cDNA is registered in the NCBI database as Accession No. NM_001425, and the amino acid sequence of the protein is registered as Accession No. NP_001416.

[0045] (6) The ENG gene encodes the ENG (Endoglin) protein. The ENG protein is a homodimeric transmembrane protein, and several isoforms have been reported. ENG is known to act as a co-receptor for the Transforming Growth Factor (TGF)-β receptor. For example, the nucleotide sequence of human ENG cDNA is registered in the NCBI database as Accession No. NM_001114753, and the amino acid sequence of the protein is registered as Accession No. NP_001108225.

[0046] (7) The ITGA3 gene encodes the ITGA3 (Integrin subunit alpha 3) protein. The ITGA3 protein is a single-pass transmembrane glycoprotein that binds to ITGB1 to form an integrin and is known to interact with the extracellular matrix, including fibronectin, collagen, laminin 1, laminin V, and entactin, as cell surface adhesion molecules. Multiple isoforms of the ITGA3 protein have been reported. For example, the nucleotide sequence of human ITGA3 is registered in the NCBI database as Accession No. NM_002204, and the amino acid sequence of the protein is registered as Accession No. NP_002195.

[0047] (8) The ITGA6 gene encodes the ITGA6 (Integrin subunit alpha 6) protein. The ITGA6 protein is a single-pass transmembrane glycoprotein that binds to ITGB1 or ITGB4 to form an integrin and is known to interact with the extracellular matrix, including the laminin family, as an adhesion molecule on the cell surface. Multiple isoforms of the ITGA6 protein have been reported. For example, the nucleotide sequence of human ITGA6 is registered in the NCBI database as Accession No. NM_001079818, and the amino acid sequence of the protein is registered as Accession No. NP_001073286.

[0048] (9) The ITGB1 gene encodes the ITGB1 (Integrin subunit beta 1) protein. The ITGB1 protein is a single-pass transmembrane glycoprotein, and multiple isoforms have been reported. ITGB1 is known to bind to ITGA to form integrins and act as adhesion molecules on the cell surface. Multiple isoforms of the ITGB1 protein have been reported. For example, the nucleotide sequence of human ITGB1 cDNA is registered in the NCBI database as Accession No. NM_002211, and the amino acid sequence of the protein is registered as Accession No. NP_002202.

[0049] (10) The ITGB8 gene encodes the ITGB8 (Integrin subunit beta 8) protein. The ITGB8 protein is a single-pass transmembrane glycoprotein and is known to bind with ITGA to form integrins, acting as an adhesion molecule on the cell surface. Multiple isoforms of the ITGB8 protein have been reported. For example, the nucleotide sequence of human ITGB8 cDNA is registered in the NCBI database as Accession No. NM_002214, and the amino acid sequence of the protein is registered as Accession No. NP_002205.

[0050] (11) The KDR gene encodes the KDR (Kinase insert domain receptor) protein. The KDR protein is a receptor tyrosine kinase that is activated by vascular endothelial growth factor (VEGF) as a ligand and is known to be involved in angiogenesis and cell proliferation. For example, the nucleotide sequence of human KDR cDNA is registered in the NCBI database as Accession No. NM_002253, and the amino acid sequence of the protein is registered as Accession No. NP_002244.

[0051] (12) The MMP7 gene encodes the MMP7 (Matrix metallopeptidase 7) protein. The MMP7 protein is a proteolytic enzyme belonging to the Matrix Metalloproteinase family and has the function of degrading extracellular matrix such as proteoglycans and fibronectin. For example, the base sequence of human MMP7 cDNA is registered in the NCBI database as Accession No. NM_002423, and the amino acid sequence of the protein is registered as Accession No. NP_002414.

[0052] (13) The NT5E gene encodes the NT5E (5'-Nucleotide Ecto) protein. The NT5E protein is a GPI-anchored cell surface protein, and several isoforms have been reported. NT5E is known to catalyze the conversion of extracellular nucleotides to membrane-permeable nucleosides. For example, the base sequence of human SPP1 cDNA is registered in the NCBI database as Accession No. NM_002526, and the amino acid sequence of the protein is registered as Accession No. NP_002517.

[0053] (14) The PECAM1 gene encodes the PECAM1 (Platelet and Endothelial Cell Adhesion Molecule 1) protein. The PECAM1 protein is a single-pass transmembrane glycoprotein belonging to the immunoglobulin superfamily. PECAM1 is known to be involved in angiogenesis, integrin activation, and blood cell migration. Multiple isoforms of the PECAM1 protein have been reported. For example, the nucleotide sequence of human PECAM1 cDNA is registered in the NCBI database as Accession No. NM_00042, and the amino acid sequence of the protein is registered as Accession No. NP_00043.

[0054] (15) The PLXND1 gene encodes the PLXND1 (Plexin D1) protein. The PLXND1 protein is a single-pass transmembrane glycoprotein and a cell surface receptor for class 3 semaphorins. It is predicted to be involved in cell migration, nervous system development, and angiogenesis. Multiple isoforms of the PLXND1 protein have been reported. For example, the nucleotide sequence of human PLXND1 is registered in the NCBI database as Accession No. NM_015103, and the amino acid sequence of the protein is registered as Accession No. NP_055918.

[0055] (16) The RORC gene encodes the RORC (RAR-related orphan receptor C) protein. The RORC protein is a DNA-binding transcription factor belonging to the NR1 subfamily of nuclear receptors, and multiple isoforms have been reported. It is an important regulator of cell differentiation, immunity, and metabolism, and in the liver, it is known to be involved in the regulation of lipid, steroid, extracellular substance, and glucose metabolism. For example, the nucleotide sequence of human RORC cDNA is registered in the NCBI database as Accession No. NM_005060, and the amino acid sequence of the protein is registered as Accession No. NP_005051.

[0056] (17) The S1PR1 gene encodes the S1PR1 (Sphingosine-1-phosphate receptor 1) protein. The S1PR1 protein is a G protein-coupled receptor that uses sphingosine-1-phosphate as a ligand, and several isoforms have been reported. It is known to be involved in the differentiation, proliferation, migration, and cell-cell adhesion of vascular endothelial cells. For example, the nucleotide sequence of human S1PR1 cDNA is registered in the NCBI database as Accession No. NM_001400, and the amino acid sequence of the protein is registered as Accession No. NP_001391.

[0057] (18) The SDC3 gene encodes the SDC3 (Syndecan 3) protein. The SDC3 protein is a type I transmembrane proteoglycan that has a chondroitin sulfate group and a heparan sulfate group. Syndecans are known to be involved in extracellular matrix adhesion, growth factor binding, and cell morphology via cytoskeletal actin. For example, the nucleotide sequence of human SDC3 cDNA is registered in the NCBI database as Accession No. NM_014654, and the amino acid sequence of the protein is registered as Accession No. NP_055469.

[0058] (19) The SPP1 gene encodes the SPP1 (Secretized phosphoroprotein 1) protein. The SPP1 protein is a type of secreted phosphorylated glycoprotein, and several isoforms have been reported. It interacts with CD44 and integrins and is suggested to be involved in bone development and remodeling, inflammatory and immune responses, carcinogenesis, and cancer progression. For example, the nucleotide sequence of human SPP1 cDNA is registered in the NCBI database as Accession No. NM_001040058, and the amino acid sequence of the protein is registered as Accession No. NP_001035147.

[0059] (20) The VIPR1 gene encodes the VIPR1 (Vasoactive intestinal peptide receptor 1) protein. The VIPR1 protein is a G protein-coupled receptor that binds to VIP (vasoactive intestinal peptide) and PACAP (pituary adenylate cycle activating peptide), and multiple isoforms have been reported. VIPR1 is also known to be involved in the regulation of various physiological processes, including smooth muscle relaxation, water and electrolyte secretion, and immune response regulation. For example, the nucleotide sequence of human VIPR1 is Accession No. NM_004624, and the amino acid sequence of the protein is Accession No. It is registered in the NCBI database as NP_004615.

[0060] CD44, CD9, CXCL1, CXCL6, EMP3, ENG, FOSB, IFITM1, ITGA3, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1 are preferably endogenous genes.

[0061] Stem cell-derived hepatocytes according to one aspect of the present invention only need to express at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1. For example, any one of the above 20 freeze-tolerance marker genes may be expressed, any two or more freeze-tolerance marker genes may be expressed, any three or more freeze-tolerance marker genes may be expressed, any four freeze-tolerance marker genes may be expressed, any five or more freeze-tolerance marker genes may be expressed, any ten or more freeze-tolerance marker genes may be expressed, or all 20 freeze-tolerance marker genes may be expressed.

[0062] In one aspect of the present invention, it is particularly preferable that stem cell-derived hepatocytes express at least one gene selected from the group consisting of CD44, SPP1, NT5E, PECAM1, KDR, RORC, ENG, and S1PR1 (hereinafter referred to as the "first group of freeze-tolerance marker genes") among the above 20 freeze-tolerance marker genes.

[0063] Among the first group of freeze-tolerance marker genes, the expression of the RORC gene is presumed to be necessary for hepatocyte differentiation and acquisition of function, including drug metabolism. Therefore, it is preferable that stem cell-derived hepatocytes according to one aspect of the present invention express at least the RORC gene from the first group of freeze-tolerance marker genes.

[0064] Furthermore, in one aspect of the present invention, it is preferable that stem cell-derived hepatocytes express at least one gene selected from the group consisting of CD9, ITGA3, ITGB1, ITGA6, and ITGB8 (hereinafter referred to as the "second freeze-tolerance marker gene group"), together with the genes of the first freeze-tolerance marker gene group described above.

[0065] Furthermore, the expression levels of the above 20 freeze-resistance marker genes in stem cell-derived hepatocytes according to one aspect of the present invention are not particularly limited, but it is preferable that the gene expression levels be higher than those in stem cell-derived hepatocytes cultured on laminin-511. For example, when the expression level of the above freeze-resistance marker genes in stem cell-derived hepatocytes cultured on laminin-511 is set to 1.0, it is preferable that the expression level of the above freeze-resistance marker genes in stem cell-derived hepatocytes according to one aspect of the present invention be 1.5 times or more, and more preferably 2 times or more.

[0066] (Expression of other marker genes) In addition to the expression of the above-mentioned freeze-tolerance marker gene, it is preferable that the stem cell-derived hepatocytes according to one aspect of the present invention express liver marker genes. Examples of known liver marker genes include albumin (ALB), α-fetoprotein (AFP), α1-antitrypsin (AAT), and hepatocyte nuclear transcription factor 4α (HNF4A). In the stem cell-derived hepatocytes according to one aspect of the present invention, it is sufficient that at least one of the liver marker genes mentioned above is expressed, but since transplantability is evaluated by ALB secretion ability, it is more preferable that ALB is expressed among these.

[0067] In one aspect of the present invention, it is preferable that stem cell-derived hepatocytes further express liver function marker genes. Examples of known liver function marker genes include ornithine transcarbamylase (OTC), carbamoyl phosphate synthase 1 (CPS1), glucose-6-phosphatase (G6PC), cytochrome P450 (CYP) 2C9 (CYP2C9), and cytochrome P450 (CYP) 3A4 (CYP3A4). In one aspect of the present invention, it is sufficient that stem cell-derived hepatocytes express at least one of the liver marker genes described above. If the stem cell-derived hepatocytes in one aspect of the present invention are cells obtained through the selection of hepatic progenitor cells using a cytotoxic antibiotic (so-called selective antibiotic) commonly used to select stable transformants that retain exogenous genes, it is preferable that they express CYP3A4 among the liver function markers described above.

[0068] In one aspect of the present invention, stem cell-derived hepatocytes preferably have the function of adult hepatocytes. In one aspect of the present invention, stem cell-derived hepatocytes preferably have, for example, one or more hepatocyte functions, in addition to ALB secretion ability, such as cytochrome P450 activity, drug metabolism, glycogen accumulation, low-density lipoprotein (LDL) uptake, ammonia metabolism, and urea synthesis.

[0069] Furthermore, in stem cell-derived hepatocytes according to one aspect of the present invention, it is preferable that the expression levels of differentiation marker genes other than liver marker genes and liver function marker genes are low. Examples of such differentiation marker genes include COL1A1, a dermal fibrosis marker; ASMA, a myofibroblast marker; and cytokeratin (CK) 7 and SRY-Box9 (SOX9), which are bile duct markers. In stem cell-derived hepatocytes according to one aspect of the present invention, it is preferable that the expression levels of differentiation marker genes other than liver marker genes and liver function marker genes are low. For example, when the expression level of hepatic progenitor cells is set to 1.0, the expression level of bile duct markers in stem cell-derived hepatocytes according to one aspect of the present invention is preferably 0.75 or less, and more preferably 0.5 or less.

[0070] (Applications) Stem cell-derived hepatocytes according to one aspect of the present invention are cryo-tolerant. Furthermore, stem cell-derived hepatocytes according to one aspect of the present invention have properties similar to those of hepatocytes derived from living organisms, and also possess the following abilities: (i) the ability to maintain typical characteristics of hepatic progenitor cells in vitro, (ii) the ability to acquire hepatic characteristics, and (iii) the ability to engraft and regrow in the body after transplantation.

[0071] Stem cell-derived hepatocytes according to one aspect of the present invention are cryo-tolerant and can therefore be used in gene therapy, cell therapy by ex vivo genome editing, cell therapy in allogeneic transplantation (off the shelf), and the like.

[0072] Furthermore, stem cell-derived hepatocytes according to one aspect of the present invention can be prepared by a xeno-free culture method according to the manufacturing method described later, and are therefore suitable for clinical application. Thus, stem cell-derived hepatocytes according to one aspect of the present invention are suitable as transplantable hepatocytes for use in stem cell transplantation therapy. In addition, stem cell-derived hepatocytes according to one aspect of the present invention are also useful in the field of drug discovery research, such as as cells for screening candidate drug compounds with high reliability.

[0073] [2. Method for Producing Freeze-Resistant Pluripotent Stem Cell-Derived Hepatocytes] The method for producing the "pluripotent stem cell-derived hepatocytes" described above will be explained below. A method for producing freeze-resistant pluripotent stem cell-derived hepatocytes according to one aspect of the present invention includes the following steps (1) to (2): (1) A step of subculturing a population of pluripotent stem cell-derived hepatocyte-like cells differentiated from pluripotent stem cells on laminin-111; (2) A step of inducing liver maturation in the cells after step (1) on laminin-111.

[0074] According to one aspect of the present invention, a method for producing freeze-tolerant pluripotent stem cell-derived hepatocytes can be produced (prepared) as described above. In this specification, "a method for producing freeze-tolerant pluripotent stem cell-derived hepatocytes according to one aspect of the present invention" may be referred to as "a method for producing pluripotent stem cell-derived hepatocytes according to one aspect of the present invention" for convenience.

[0075] Figure 1 is a schematic diagram illustrating the overall method for producing freeze-tolerant pluripotent stem cell-derived hepatocytes according to one embodiment of the present invention. Steps (1) and (2) will be described in detail below.

[0076] (Step (1): Subculture Step) Step (1) is a step in which a population of pluripotent stem cell-derived hepatocyte-like cells differentiated from pluripotent stem cells is subcultured on laminin-111. This allows for the efficient proliferation of hepatic progenitor cells that have the ability to differentiate into hepatocytes with freeze tolerance and long-term transplantability.

[0077] Examples of culture media that can be used in step (1) include conventionally known culture media that can be used as hepatocyte proliferation media. Such hepatocyte proliferation media include media containing Wnt signaling promoters such as Wnt3A, FBS, TGFb inhibitors, Rock inhibitors, etc. (Katsuda T et. al., Elife. 2019 Aug 8;8:e47313. (Reference 5); Kun Zhang et. al., Cell Stem Cell. 2018 Dec 6;23(6):806-819.e4. (Reference 4); Gong-Bo Fu et. al., Cell Res. 2019 Jan;29(1):8-22. (Reference 13)); and media containing EGF, dexamethasone, IL6, HGF, etc. (Guo R et. al., Nat Commun. 2022 Nov 29;13(1):7345. (Reference 6)). Furthermore, commercially available hepatocyte growth media can be used as the culture medium in step (1). Examples of such hepatocyte growth media include EMUKK-05 (manufactured by EMUKK) and growth media from Promocell. The amount of each component contained in the hepatocyte growth media can be appropriately changed from the values ​​described in the literature to a range of 0.1% to 20%, preferably 0.1% to 10%.

[0078] From the viewpoint of maintaining the characteristics of liver progenitor cells, in step (1), adherent culture is performed using a plate coated with laminin-111 as the extracellular matrix. Laminin-111 may be full-length laminin or fragmented laminin, but from the viewpoint of stability and cost efficiency, purified laminin-E8 fragments are preferred.

[0079] Laminin (purified laminin-E8 fragment) may be pre-coated or pre-mixed into the culture medium. In the case of pre-coating, it can be used at a concentration that does not affect cell adhesion, for example, 0.03 μg / cm³. ２ ~25 μg / cm³ ２ This can be done. Also, in the case of a premix, the concentration should be 0.03 μg / cm³ relative to the volume of the culture medium.２ ~4 μg / cm³ ２ It can be done this way.

[0080] (Step (2): Liver Maturation Induction Step) Step (2) is a step in which the cells after Step (1) are subjected to liver maturation on laminin-111. The cell population after Step (1) contains highly purified liver progenitor cells that have the ability to differentiate into hepatocytes with freeze resistance and long-term transplantability. By inducing liver maturation in such a cell population in Step (2), hepatocytes with a high degree of liver maturation that have long-term transplantability, such as being able to engraft for more than one month after transplantation, preferably more than three months after transplantation, can be obtained.

[0081] Examples of culture media that can be used in step (2) include conventionally known culture media that can be used as liver maturation induction media. Such maturation media can be commercially available liver maturation induction media, such as Hepatocyte Basal Medium (Lonza, model number: CC-3199), Williams' Medium E (Thermo Fisher, model number: A1217601), HepatoZYME-SFM (Thermo Fisher, model number: 17705021), Cellartis Powertm Primary HEP Medium (Takara Bio Europe S.A.S., model number: Y20020), and AnaBios Hepatocyte Maintenance Media (Cell Examples include systems (model number: HEM-100). The liver maturation induction medium may contain, if necessary, transferrin, ascorbic acid, HEGF, insulin, hydrocortisone, BSA, antibiotics, etc.

[0082] In step (2), the culture is preferably carried out using the adherent culture method on laminin-111. This makes it possible to sufficiently induce liver maturation while maintaining the ability to differentiate into hepatocytes that have freeze tolerance and long-term transplantability. The method and concentration of laminin-111 are as described in step (1).

[0083] It is preferable to repeat the series of processes from steps (1) to (2) at least once on the cells after step (2). This makes it possible to provide cells of higher purity. From the viewpoint of obtaining cells of higher purity, it is preferable to repeat the series of processes from steps (1) to (2) two or more times, and more preferably three or more times. There is no particular upper limit to the number of repetitions, but the expected sufficient effect can be obtained by repeating it three times.

[0084] Cells may be cryopreserved after each of steps (1) and (2). According to one embodiment of the present invention, a method for producing pluripotent stem cell-derived hepatocytes can be produced that retains long-term transplantability even after cryopreservation. Cell cryopreservation can be carried out by known methods. For example, STEM-CELLBANKER (AMS Biotechnology, Massachusetts, USA) can be suitably used as the cryopreservation solution.

[0085] (Other steps) A method for producing pluripotent stem cell-derived hepatocytes according to one aspect of the present invention may include steps other than steps (1) and (2) described above. For example, before step (1), a differentiation induction step may be further included in which pluripotent stem cells are differentiated to obtain a population of pluripotent stem cell-derived hepatocyte-like cells. Also, for example, before step (2), a selection step may be further included in which the cells after step (1) are cultured in the presence of a selective antibiotic. Each step will be described below.

[0086] (Differentiation Induction Process) In the differentiation induction process, the method for differentiating pluripotent stem cells into hepatocyte-like cells is not particularly limited. Differentiation of pluripotent stem cells into hepatocyte-like cells can be performed according to conventionally known methods for differentiating hepatocytes, and for example, the methods described in International Publication No. 2019 / 131938, International Publication No. 2018 / 152120, etc., can be applied.

[0087] In step (1), differentiation induction is preferably carried out by adherent culture. For adherent culture, it is preferable to use plates coated with extracellular matrix such as laminin, collagen, gelatin, fibronectin, or Matrigel, or feeder cells such as mouse embryonic fibroblasts (MEF).

[0088] (Selection Step) The cell population after step (1) contains hepatic progenitor cells or hepatocytes. In the selection step, the cell population containing hepatic progenitor cells or hepatocytes is cultured in the presence of a selective antibiotic. Cells that cannot metabolize the selective antibiotic are killed, and only hepatic progenitor cells or hepatocytes that can metabolize the selective antibiotic can be selected. This allows for the efficient and highly purified preparation of hepatic progenitor cells or hepatocytes with drug-metabolizing ability.

[0089] As the selective antibiotic used in the selection process, cytotoxic antibiotics commonly used to select stable transformants that retain exogenous genes can be used. Examples of such selective antibiotics include puromycin. Among these selective antibiotics, puromycin is preferred because it is commonly used and is metabolized by CYP3A4, the major drug-metabolizing enzyme in hepatocytes.

[0090] In the selection process, drug selection can be carried out according to conventionally known drug selection methods for hepatic progenitor cells or hepatocytes. For example, the method described in International Publication No. 2019 / 131938; Akiyama, S., Saku, N., Miyata, S. et al. Stem Cell Res Ther 13, 104 (2022) (Reference 14) can be applied.

[0091] In the selection process, instead of drug selection using selective antibiotics, cell selection may be performed using a selection method that utilizes the ammonia metabolic capacity of the liver. This allows for the efficient and highly purified preparation of hepatic progenitor cells or hepatocytes that possess ammonia metabolic capacity. Selection methods utilizing the ammonia metabolic capacity of the liver can be applied as described in Tsuneishi, Ruri et al. Scientific reports vol. 11,1 11381. 31 May. 2021 (Reference 15); Tomotsune, Daihachiro et al. PloS one vol. 11,9 e0162693. 15 Sep. 2016 (Reference 16), etc.

[0092] [3. Use of Molecular Markers for Selecting Freeze-Resistant Hepatocytes] The use of a molecular marker for selecting freeze-resistant hepatocytes according to one aspect of the present invention (hereinafter sometimes referred to as the "hepatocyte freeze-resistant marker" or "freeze-resistant marker") is such that the molecular marker consists of the following substances (a) or (b): (a) the translation product of at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1; (b) A transcript of at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1.

[0093] According to one aspect of the present invention, hepatocytes possessing cryo-tolerance and long-term transplantability can be selected using the expression of the above molecular marker as an indicator. This makes it possible to reduce differences in engraftment and / or regrowth ability between hepatocyte lots.

[0094] The molecular marker genes mentioned above were explained in section [1. Stem Cell-Derived Hepatocytes] above, so we will not repeat the explanation here.

[0095] In this specification, the term "gene transcript" means mRNA transcribed from genomic DNA that codes for a gene. This mRNA may be unmodified mRNA or an unspliced ​​mRNA precursor.

[0096] In this specification, the term "translation product of a gene" refers to a polypeptide produced from genomic DNA encoding a gene through the processes of transcription and translation. This polypeptide may include oligopeptides and proteins, and may be unmodified or post-translationally modified.

[0097] Of the 20 freeze-tolerance markers listed above, CD44, CD9, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, NT5E, PECAM1, PLXND1, S1PR1, SDC3, and VIPR1 are genes that encode cell surface proteins. Therefore, molecular markers consisting of the translation products of these genes have the advantage of being easily detectable.

[0098] The hepatocytes selected by the 20 freeze-tolerance marker genes mentioned above may be stem cell-derived hepatocytes obtained by hepatocyte-like cells differentiated from pluripotent stem cells or other stem cells and then matured in the liver, or they may be hepatocytes of living origin isolated from liver tissue.

[0099] Of the 20 cryogenic resistance marker genes listed above, SPP1, NT5E, PECAM1, KDR, ENG, CD9, ITGA3, ITGB8, CXCL1, CXCL6, EMP3, MMP7, PLXND1, S1PR1, SDC3, and VIPR1 are usually not expressed, or if expressed, at low levels, in hepatocytes isolated from liver tissue. Therefore, these 16 cryogenic resistance marker genes can be considered molecular markers whose expression is specifically upregulated in stem cell-derived hepatocytes that possess cryogenic resistance and long-term transplantability. By using the significant positivity of at least one of these 16 cryogenic resistance marker genes as an indicator, it becomes possible to select stem cell-derived hepatocytes that possess cryogenic resistance and long-term transplantability.

[0100] Furthermore, RORC is presumed to be a molecule necessary for acquiring functions, including hepatocyte differentiation and drug metabolism. For this reason, among the 20 freeze-tolerance marker genes mentioned above, the RORC gene is particularly useful as a marker for freeze-tolerance and long-term transplantability. Therefore, it is preferable to use the RORC gene in combination with at least one of the remaining 19 freeze-tolerance marker genes as a freeze-tolerance marker.

[0101] Furthermore, among the 16 marker genes for stem cell-derived hepatocytes possessing cryo-tolerance and long-term transplantability mentioned above, SPP1 and S1PR1 are presumed to be particularly useful as markers for stem cell-derived hepatocytes possessing cryo-tolerance and long-term transplantability. Therefore, it is more preferable to select stem cell-derived hepatocytes possessing cryo-tolerance and long-term transplantability using the fact that at least three genes, including the RORC gene mentioned above and these two genes (SPP1 and S1PR1), are significantly positive as indicators.

[0102] A method for selecting cryogenically tolerant hepatocytes using a cryogenically tolerant hepatocyte marker according to one aspect of the present invention (hereinafter sometimes referred to as the "hepatocyte selection method") is also included in the scope of the present invention. According to the hepatocyte selection method according to one aspect of the present invention, hepatocytes possessing cryogenic tolerance and long-term transplantability can be selected. The hepatocytes to be selected by the hepatocyte selection method according to one aspect of the present invention may be stem cell-derived hepatocytes obtained by hepatic maturation of stem cell-derived hepatocyte-like cells obtained by differentiation induction from stem cells such as pluripotent stem cells, or they may be living-derived hepatocytes isolated from liver tissue.

[0103] The aforementioned "hepatocytes of living organism isolated from liver tissue" may be primary cultures derived from liver tissue, subcultures thereof, or established cell lines. The liver tissue may be derived from any vertebrate, preferably from mammals such as mice, rats, rabbits, sheep, pigs, cattle, goats, monkeys, and humans, and particularly preferably from humans. The liver tissue may be either fetal or adult tissue. Methods for preparing primary cultures from liver tissue are well established and can be prepared according to methods known in this field (Cole, KE et al., Cancer Res., 1986; 46(3): 1290-6; Adams RS et al., Proc. Natl. Acad. Sci. USA., 1992; 89(19): 8981-5). Primary cultures from small intestinal epithelial tissue can also be prepared according to methods known in this field (Wang et al., Nature, 2015; 522: 173-178).

[0104] Furthermore, the hepatocytes to be selected in the hepatocyte selection method according to one aspect of the present invention may be derived from any vertebrate. In the hepatocyte selection method according to one aspect of the present invention, the hepatocyte freeze-tolerance marker according to one aspect of the present invention may be used in combination with other known molecular markers.

[0105] [4. Composition for Liver Transplantation] A liver transplantation composition according to one aspect of the present invention contains stem cell-derived hepatocytes according to the above-described aspect of the present invention. The stem cell-derived hepatocytes according to one aspect of the present invention in the liver transplantation composition according to one aspect of the present invention have already been described, so they will not be described again here.

[0106] According to one aspect of the present invention, the liver transplant composition has freeze resistance and long-term transplantability, making it possible to distribute cells in a frozen state.

[0107] A liver transplantation composition according to one aspect of the present invention may contain other components of stem cell-derived hepatocytes according to the above-described aspect of the present invention. Such components may be components that are commonly added to transplantation compositions.

[0108] [5. Method for purifying freeze-tolerant hepatic progenitor cells or hepatocytes] A method for purifying freeze-tolerant hepatic progenitor cells or hepatocytes according to one aspect of the present invention includes the step of culturing an isolated cell population containing hepatic progenitor cells or hepatocytes on laminin-111.

[0109] The method for culturing an isolated cell population containing hepatic progenitor cells or hepatocytes on laminin-111 is as described in "Step (1)" in section [2. Method for producing freeze-tolerant pluripotent stem cell-derived hepatocytes] above, so the explanation will not be repeated here.

[0110] Here, the "isolated cell population containing hepatic progenitor cells or hepatocytes" is not particularly limited in its origin, as long as it is an isolated cell population containing hepatic progenitor cells or hepatocytes. For example, it may be a population of stem cell-derived hepatocyte-like cells obtained by differentiation induction from stem cells such as pluripotent stem cells, or it may be a population of cells isolated from liver tissue.

[0111] According to one aspect of the present invention, a method for purifying cryogenically tolerant hepatic progenitor cells or hepatocytes can be efficiently prepared with high purity, thereby enabling cryogenically tolerant and long-term transplantability.

[0112] [Summary] Stem cell-derived hepatocytes according to embodiment 1 of the present invention express at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1.

[0113] In the stem cell-derived hepatocytes according to aspect 2 of the present invention, the stem cells in aspect 1 above may be human embryonic stem cells or human induced pluripotent stem cells.

[0114] The liver transplantation composition according to embodiment 3 of the present invention contains stem cell-derived hepatocytes as described in embodiment 1 or 2 above.

[0115] A method for producing freeze-tolerant pluripotent stem cell-derived hepatocytes according to aspect 4 of the present invention comprises the following steps (1) to (2): (1) a step of subculturing a population of pluripotent stem cell-derived hepatocyte-like cells differentiated from pluripotent stem cells on laminin-111; (2) a step of inducing liver maturation in the cells after step (1) on laminin-111.

[0116] In the method for producing freeze-tolerant pluripotent stem cell-derived hepatocytes according to aspect 5 of the present invention, it is preferable that the series of processes from steps (1) to (2) be repeated at least once on the cells after step (2) in aspect 4.

[0117] The use of a molecular marker for selecting cryo-tolerant hepatocytes according to aspect 6 of the present invention is characterized in that the molecular marker consists of the following substances (a) or (b): (a) the translation product of at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1; (b) A transcript of at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1.

[0118] A method for purifying freeze-tolerant hepatic progenitor cells or hepatocytes according to aspect 7 of the present invention includes the step of culturing an isolated cell population containing hepatic progenitor cells or hepatocytes on laminin-111.

[0119] In the method for purifying hepatic progenitor cells or hepatocytes according to aspect 8 of the present invention, the isolated cell population in aspect 7 may be a cell population isolated from liver tissue or a cell population differentiated from stem cells.

[0120] The present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0121] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0122] <Cells> The cells used in the example are as follows: ・Human embryonic stem (ES) cell line: SEES2 (accession number: HES0007), a human embryonic stem (ES) cell line established by the National Center for Child Health and Development, was used. ・HAES (Human Embryonic Stem Cell-Derived Hepatocytes), a regenerative medicine product for congenital urea cycle disorders. ・Human liver cancer cell line HepG2: Purchased from Cellular Engineering Technologies. HepG2 Human Hepatocellular Carcinoma Expansion Media (manufactured by Cellular Engineering Technologies, model number: HEPG2.E.MEDIA-450) was used at 37°C and 5% CO2. ２ The cells were cultured in an incubator. The culture method followed the datasheet provided by the company. • Human liver cells: Human liver total RNA (Takara Bio, model number: 636531) • Human infant-derived liver progenitor cells: Human infant-derived liver cells were purchased from Veritas Corporation. The cryopreserved human liver cells were thawed and cultured on irradiated feeder cells (irrMEF) using hepatocyte growth medium EMUKK-05 (EMUKK) at 37°C and 5% CO2. ２ The cells were cultured in an incubator. Hepatic progenitor cells were selected by puromycin treatment, and after inducing hepatic maturation, they were subcultured using 0.25% Trypsin-EDTA at a substrate area ratio of 1:4 to 1:8. The culture medium was changed every three days.

[0123] <Primer Set> Table 1 shows the base sequences of the primer sets used in the examples.

[0124] A schematic diagram illustrating the overall structure of this experiment is shown in Figure 2(A). In this experiment, the following four steps were performed in this order: (1) differentiation induction step, (2) subculturing step, (3) drug selection step, and (4) liver maturation induction step.

[0125] [1. Differentiation induction step] According to the conventionally known method for inducing the differentiation of hepatocytes described in Reference 9 (Raggi, Claudia et al. Stem cell reports vol. 17, 3 (2022)), the human ES cell line SEES2 cells were induced to differentiate to obtain a population of ES cell-derived hepatocyte-like cells.

[0126] [2. Subculture step] The ES cell-derived hepatocyte-like cells obtained in the above 1. differentiation induction step were cryopreserved using the cryopreservation solution STEM-CELLBANKER (AMS Biotechnology, Massachusetts, USA) (-196°C for 30 days). Thereafter, the cells were thawed, and after confirming by microscopy that they were still culturable after cryopreservation, as shown in (A) of FIG. 2, they were subcultured in the hepatocyte growth medium EMUKK-05 and Laminin-511 (LN511) or Laminin-111 (LN111).

[0127] The culture conditions for the subculture (first passage) were as follows. A 6-well plate (manufactured by FALCON, model number 351146) for cell culture coated with Laminin-511 (LN511) (manufactured by Matrixome, model number 892021) or Laminin-111 (LN111) (manufactured by Matrixome, model number 892072) by the precoating method (0.5 μg / cm ２ ) was seeded with ES cell-derived hepatocyte-like cells at a seeding density of 3×10 ４ cells / cm ２ and cultured under the conditions of 37°C and 5% CO ２ using the hepatocyte growth medium EMUKK-05 (EMUKK, Japan). LN511 and LN111 were not full-length Laminin but purified products of Laminin-E8 fragment. EMUKK-05 is a medium containing Wnt3A and R-spondin 1.

[0128] Cell adhesion was confirmed the day after seeding, and the cells formed colonies and proliferated over time (first passage). Figure 2(B) shows phase-contrast microscope images of the subcultured ES cell-derived hepatocyte-like cells over time. The seeding day of the ES cell-derived hepatocyte-like cells was designated as Day 0, and the cells were observed up to Day 8 (8 days after seeding). As shown in Figure 2(B), cell adhesion was confirmed in the subcultured ES cell-derived hepatocyte-like cells the day after seeding, and the cells formed colonies and proliferated over time.

[0129] [3. Drug Selection Process] Next, the ES cell-derived hepatocyte-like cells proliferated by the subculture process described in 2. above were treated with puromycin to obtain hepatocytes and hepatic progenitor cells with drug-metabolizing ability. Specifically, the ES cell-derived hepatocyte-like cells proliferated in the subculture process were treated with hepatocyte proliferation medium EMUKK-05 (EMUKK, Japan) to which puromycin (Fujifilm, model no. 160-23151) was added to a final concentration of 1 μg / mL at 37°C and 5% CO2. ２ The cells were cultured for three days under the specified conditions.

[0130] [4. Liver Maturation Induction Process] After the cells had sufficiently proliferated following the drug selection process described in 3. above, they were washed once with PBS and then cultured for one week using liver maturation induction medium. The medium was changed every three days. The liver maturation induction medium used was Hepatocyte Basal Medium (Lonza, model number: CC-3199) with HCM SingleQuots Kit (Lonza, model number: CC-4182) added.

[0131] Figure 2(C) shows the morphological changes over time associated with the induction of maturation of cells (cultured on LN511) after puromycin treatment. When the cells had sufficiently proliferated after puromycin treatment, cultivation in liver maturation induction medium was started. The day on which the cells were changed to liver maturation induction medium is designated as Day 0, and phase-contrast microscope images from Day 0, Day 1, Day 3, Day 5, and Day 7 are shown.

[0132] As shown in Figure 2(C), morphological changes were observed from the day after the induction of liver maturation by culturing in a liver maturation induction medium, and clear, pale nuclei appeared on the fifth day. Similar results were obtained for cells cultured on LN111, although these are not shown in the figure.

[0133] (Gene Expression Analysis) Gene expression was analyzed by RT-qPCR in cells 7 days after liver maturation induction. Albumin (ALB), α1 antitrypsin (AAT), hepatocyte nuclear transcription factor 4α (HNF4A), glucose-6-phosphatase (G6PC), Arginase-1 (ARG1), ornithine transcarbamylase (OTC), carbamoyl phosphate synthase 1 (CPS1), cytochrome P450 (CYP) 2C9 (CYP2C9), cytochrome P450 (CYP) 3A4 (CYP3A4), cytokeratin (CK) 7, and SRY-Box9 (SOX9) were selected as target genes for analysis.

[0134] RT-qPCR was performed according to the following procedure. Using the RNeasy MicroKit (Qiagen, 74004), total RNA was extracted from ES cell-derived hepatocyte-like cells according to the kit's protocol. Using the SuperScript III First-Strand Synthesis System for rRT-PCR (Thermo Fisher Scientific), 1 μg of total RNA was reverse transcribed according to the kit's protocol to prepare cDNA. The obtained cDNA was stored at -20°C.

[0135] The obtained cDNA was used as a template, and RT-qPCR was performed using the primer set shown in Table 1. The RT-qPCR reaction was performed using Platinum SYBR Green qPCR SuperMix-UDG (Thermo Fisher Scientific) according to the protocol provided with the kit. The PCR reaction conditions were as follows: 50°C 2 min x 1 cycle, 95°C 2 min x 1 cycle, (95°C 15 sec, 60°C 30 sec) x 40 cycles, 95°C 15 sec x 1 cycle, 60°C 15 sec x 1 cycle, 95°C 15 sec x 1 cycle, 50°C 2 min x 1 cycle. The measurements were performed using Quant Studio 12K Flex (Applied Biosystems).

[0136] The results are shown in Figures 2(D) and (E). Figures 2(D) and (E) show the results of gene expression analysis in cells (cultured on LN511) on day 7 of liver maturation induction. For each marker, the relative value is shown with the expression in cells before liver maturation induction set to 1.0. The data were normalized by the housekeeping gene UBC. The results were calculated from independent experiments (n=2), and the error bars indicate the standard error.

[0137] As shown in Figure 2(D), gene expression was analyzed by RT-qPCR, and significant enhancements in the expression of liver markers ALB, AAT, and HNF4A, as well as liver function markers OTC, G6PC, CYP2C9, and CYP3A4, were observed in cells 7 days after liver maturation induction. Enhanced expression was also observed in the liver function markers ARG1 and CPS1, with the enhancement of ARG1 expression being particularly pronounced.

[0138] Furthermore, as shown in Figure 2(E), the expression of CK7 and SOX9, which are bile duct and hepatic progenitor cell markers, was decreased. Although not shown, similar results were obtained for cells cultured on LN111.

[0139] (Measurement of ALB protein secretion) ALB protein secretion was measured by the following procedure. The culture supernatant was collected after 24 hours of incubation, and the ALB concentration was measured using the Human Albumin ELISA Quantitation Set (Bethyl Laboratory, Texas, USA). ６ The amount secreted per cell was calculated. The measurement method followed the data sheet provided by the company.

[0140] The results are shown in Figure 2(F). Figure 2(F) shows the human ALB secretion capacity of cells (cultured on LN511) after the start of liver maturation induction. Human albumin secretion was measured by ELISA on the day the medium was changed to liver maturation induction medium ("Before induction" in Figure 2(F)) and after liver maturation induction ("After induction" in Figure 2(F)). The results were calculated from independent experiments (n=3), and the error bars indicate the standard error.

[0141] As shown in Figure 2(F), the secretion of human ALB in cells treated with puromycin increased significantly after induction of liver maturation. Similar results were obtained for cells cultured on LN111, although these are not shown.

[0142] (Measurement of Urea Production Capacity) Urea production capacity was measured by the following procedure. The culture supernatant was collected after 24 hours of incubation, and the urea concentration was measured using the QuantiChrome Urea Assay Kit (BioAssay System, California, USA). ６ The amount secreted per cell was calculated. The measurement method followed the data sheet provided by the company.

[0143] The results are shown in Figure 2(G). Figure 2(G) shows the results of comparing the urea production capacity of cells (cultured on LN511) before and after liver maturation induction. The results were calculated from independent experiments (n=3), and the error bars indicate the standard error (*p<0.05, **p<0.01, ***p<0.001).

[0144] As shown in Figure 2(G), urea production in cells treated with puromycin decreased after induction of liver maturation. Similar results were obtained for cells cultured on LN111, although these are not shown.

[0145] [5. Verification of the effects of repeatedly performing the series of processes from the subculturing process to the liver maturation induction process on cells after the liver maturation induction process] The cells after the liver maturation induction process described in 4. above were subjected to the series of processes from the subculturing process to the liver maturation induction process, and this was repeated four times to obtain ES cell-derived hepatocytes (5th passage) and gene expression analysis was performed on them. Albumin (ALB), α-fetoprotein (AFP), hepatocyte nuclear transcription factor 4α (HNF4A), cytochrome P450 (CYP) 3A4 (CYP3A4), and prominin 1 (PROM1) were selected as the target genes for analysis. RT-qPCR was performed according to the procedure and PCR reaction conditions described above.

[0146] The results are shown in Figure 2(H). Figure 2(H) shows the results of gene expression analysis in ES cell-derived hepatocytes (passage 5) cultured on LN111 or LN511. For each marker, the relative value is shown with the expression in ES cell-derived hepatocyte-like cells at the end of differentiation induction set to 1.0. The data were normalized by the housekeeping gene UBC. The results were calculated from independent experiments (n=3), and the error bars indicate the standard error.

[0147] As shown in Figure 2(H), analysis of gene expression by RT-qPCR revealed that ES cell-derived hepatocytes after the liver maturation induction process showed significantly higher ALB and CYP3A4 expression than cells immediately after differentiation induction, and furthermore, cells that had undergone the series of processes from the subculturing process to the liver maturation induction process repeated once or multiple times were subculturized. In addition, cells cultured on LN111 showed higher expression of hepatocyte markers (ALB, AFP, HNF4A) and liver function markers (CYP3A4), as well as lower expression of the hepatic progenitor cell marker (PROM1), compared to cells cultured on LN511.

[0148] [6. Evaluation of therapeutic usefulness] (Confirmation of cryo-tolerance: Evaluation of transplantability after cryopreservation) To evaluate whether the obtained ES cell-derived hepatocytes are useful for therapy, the transplantability of the cells in recipient livers, i.e., their ability to engraft and regrow in mouse livers, was evaluated. Specifically, to verify whether the obtained ES cell-derived hepatocytes retain their transplantability after several passages and after cryopreservation, the following five types of cells were transplanted and evaluated by plasma human albumin concentration: ・Hepatocytes [1]: ES cell-derived hepatocytes cultured on LN111 (3rd passage); ・Hepatocytes [2]: ES cell-derived hepatocytes cultured on LN511 (3rd passage); ・Hepatocytes [3]: Cryopreserved hepatocytes [1]; ・Hepatocytes [4]: ​​Cryopreserved hepatocytes [2]; ・Hepatocytes [5]: Cryopreserved human liver tissue-derived hepatocytes (2 lots).

[0149] Here, as shown in Figure 3(A), all hepatocytes [1] to [4] were cultured in maturation medium for 7 days. All cryopreserved hepatocytes [3] to [5] were stored in liquid nitrogen for more than one month. Furthermore, the cryopreserved hepatocytes [3] to [5] were transplanted into mice immediately after freeze-thawing (they were not cultured after freeze-thawing).

[0150] First, the transplantability of hepatocytes [1] and hepatocytes [2] was compared. Both hepatocytes [1] and hepatocytes [2] were transplanted, and their engraftment and regrowth ability were evaluated from the human albumin concentration in the plasma. The human albumin concentration in mouse plasma was measured using the procedure described above.

[0151] The results are shown in Figure 3(B). Figure 3(B) shows the results of transplanting hepatocytes into TK-NOG, a mouse model of liver damage. In Figure 3, "[1] LN111" represents the results for hepatocytes [1], and "[1] LN511" represents the results for hepatocytes [2]. The results were calculated from independent experiments (n=3), and the error bars indicate the standard error.

[0152] As shown in Figure 3(B), human albumin in mouse plasma was highest in mice transplanted with hepatocytes [1], and this level was maintained until week 8 after transplantation. On the other hand, in mice transplanted with hepatocytes [2], the level decreased to 1 / 10 of that in mice transplanted with hepatocytes [1] at week 4, and to 1 / 100 at week 8.

[0153] (Confirmation of cryo-tolerance: Evaluation of albumin production capacity after cryopreservation) Next, the transplantability of the cryopreserved cells [3] to [5] was evaluated at 4 weeks post-transplantation based on the human albumin concentration in the plasma. The human albumin concentration in mouse plasma was measured using the procedure described above.

[0154] The results are shown in Figure 3(C). Figure 3(C) shows the results of transplanting human ES cell-derived hepatocytes into TK-NOG, a mouse model of liver damage. The results were calculated from independent experiments (n numbers are listed below), and the error bars represent the standard error. In the figure, "[1] LN111" represents the results for hepatocytes [1] (n=3), "[2] LN511" represents the results for hepatocytes [2] (n=3), "[3] Frozen LN111" represents the results for hepatocytes [3] (n=4), "[4] Frozen LN511" represents the results for hepatocytes [4] (n=5), and "[5] Frozen human hepatocytes" represents the results for hepatocytes [5] (n=5).

[0155] As shown in Figure 3(C), when comparing hepatocytes cultured in LN511 before cryopreservation [2] and hepatocytes after cryopreservation [4], the transplantation efficiency of hepatocytes after cryopreservation [4] decreased to less than 1 / 100 of that of hepatocytes before cryopreservation [2] at 4 weeks (2 out of 5 cells were below the detection limit).

[0156] In contrast, when comparing cells cultured in LN111 with hepatocytes before cryopreservation [1] and hepatocytes after cryopreservation [3], the transplantation efficiency of hepatocytes after cryopreservation [3] was about 1 / 10 of that of hepatocytes before cryopreservation [1] at 4 weeks. Furthermore, the transplantation efficiency of hepatocytes [3] was about the same as that of hepatocytes [5] at 4 weeks.

[0157] (Confirmation of cryo-tolerance: Evaluation of long-term transplantability after cryopreservation) Figure 4(A) shows the plasma human ALB levels in TK-NOG mice (n=15) transplanted with mature ES cell-derived hepatocytes prepared by culturing ES cell-derived hepatocytes (passage 3) on LN511 using a liver maturation induction medium for 7 days. Each line in the graph shows the value for an individual mouse.

[0158] Figure 4(B) shows the results of transplanting human ES cell-derived hepatocytes into TK-NOG, a mouse model of liver damage (n=4). Hepatocytes [3] were transplanted, and long-term transplantability was evaluated based on plasma human albumin concentration.

[0159] As shown in Figure 4(B), human albumin in the plasma of mice transplanted with hepatocytes [3] increased over 12 weeks, indicating that cells cultured in LN111 maintain their long-term transplantability even after cryopreservation.

[0160] Figure 4(C) shows the results of immunohistochemical staining with human CYP2C in the liver of TK-NOG mice transplanted with mature hepatocytes derived from ES cells, which were cultured on LN111 for 7 days using a liver maturation induction medium. The image shows the stained image 8 weeks after transplantation.

[0161] The results above demonstrate that cells with high transplant efficiency can be selectively cultured and proliferated on LN111. ES cell-derived hepatocytes cultured on LN111 showed resistance to cryopreservation and retained their long-term transplantability even after freeze-thawing.

[0162] [7. Comprehensive Gene Expression Analysis] Gene expression was analyzed using single-cell RNA-seq analysis for the following two types of cells: • Hepatocytes [1]: ES cell-derived hepatocytes (passage 3) cultured on LN111; • Hepatocytes [2]: ES cell-derived hepatocytes (passage 3) cultured on LN511.

[0163] Figure 5 shows the results of single-cell RNA-seq analysis. Figure 5(A) is a UMAP showing the results of integrating and clustering the single-cell RNA-seq analysis results in hepatocytes [1] or hepatocytes [2]. Figure 5(B) is a UMAP showing the results of extracting cell populations with a high proportion of cells cultured with Laminin-111 from the single-cell RNA-seq analysis results in hepatocytes [1] or hepatocytes [2] and clustering them.

[0164] Figure 5(C) shows the UMAP of genes that are highly expressed only in hepatocytes [1] and extracted from a specific cell population. When genes that are highly expressed only in the identified cell population were extracted, 20 marker genes were identified: CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1.

[0165] Figure 6 shows the results of RT-qPCR analysis of the gene expression of CD44, RORC, ENG, NT5E, and SPP1 in hepatocytes [1] or hepatocytes [2] (two lots each). The relative values ​​are shown in the heatmap, with the expression in hepatocytes [1] set to 1.0. The data were normalized by the housekeeping gene UBC. The results were calculated from independent experiments (n=3 per lot). LN511: hepatocytes [2], LN111: hepatocytes [1].

[0166] As shown in Figure 6, bulk RT-qPCR analysis was performed on CD44, RORC, ENG, NT5E, and SPP1 among the identified genes, and these genes showed elevated levels in hepatocytes [1].

[0167] [Conclusion] Single-cell RNA-seq analysis was performed to search for marker genes specific to hepatocytes [1] that showed long-term transplantability and freeze tolerance. CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1 were identified. CD44, RORC, ENG, NT5E, and SPP1 also showed high levels in bulk RT-qPCR analysis of cells cultured from hepatocytes [1], suggesting that the 20 marker genes listed above can serve as indicators of transplantability and freeze tolerance.

[0168] [8. Application of proliferation and maturation protocols to human iPS cells]

[0169] We used human iPS cells to verify the established subculturing and liver maturation induction system for human ES cell-derived hepatocyte-like cells. Figure 5 shows the verification results using human iPS cells.

[0170] Figure 7(A) shows a phase-contrast microscope image of iPS cell-derived hepatocyte-like cells created from human iPS cells, specifically the phase-contrast microscope image at the point where differentiation into hepatocytes is complete (day 30).

[0171] Figure 7(B) shows the results of gene expression analysis at the end of differentiation (day 30) of iPS cell-derived hepatocyte-like cells. The expression of liver progenitor marker genes and liver marker genes was analyzed by RT-qPCR. The relative values ​​are shown with the expression in ES cell-derived hepatocytes (HAES - a regenerative medicine product for congenital urea cycle disorders) set to 1.0. The data were normalized by the housekeeping gene UBC. The results were calculated from independent experiments (n=3), and the error bars indicate the standard error. iPSC-derived: iPS cell-derived hepatocytes, ESC-derived: ES cell-derived hepatocytes.

[0172] In this experiment, human iPS cells were differentiated using the same method as that used for the human ES cell line SEES2. As shown in Figure 7(A), some of the generated iPS cell-derived hepatocyte-like cells showed the presence of round, white nuclei characteristic of hepatocytes.

[0173] Using RT-qPCR, gene expression in iPS cell-derived hepatocyte-like cells and ES cell-derived hepatocyte-like cells at the differentiation completion site (30 days from the start of differentiation) was compared with that of HAES cells. As shown in Figure 7(B), in both iPS cell-derived and ES cell-derived hepatocyte-like cells, liver-related genes ALB, HNF4A, AFP, CYP3A4, and CPS1 showed expression levels equivalent to or higher than those of HAES cells. OTC levels were higher than those of HAES cells only in ES cell-derived hepatocyte-like cells. On the other hand, the expression of the liver progenitor cell marker SOX9 was lower than that of HAES cells in both cell types.

[0174] Next, we verified the effectiveness of the subculture and liver maturation induction system in iPS cell-derived hepatocyte-like cells. Figure 8 shows the results of verifying the effectiveness of the subculture and liver maturation induction system in iPS cell-derived hepatocyte-like cells.

[0175] Figure 8(A) is a schematic diagram of this experiment. Hepatocyte-like cells (HLCs) were generated from iPS cells, and these iPS cell-derived HLCs were passaged and cultured in hepatocyte proliferation medium EMUKK-05. After treatment with puromycin for 3 days, iPS cell-derived liver progenitor cells were selected. After the iPS cell-derived liver progenitor cells were grown, they were cultured for 7 days in the liver maturation induction medium described above to induce maturation.

[0176] Figure 8(B) shows a phase-contrast microscope image of mature hepatocytes (passage 2) generated from human iPS cells.

[0177] Figures 8(C) and (D) show the results of gene expression analysis of iPS cell-derived hepatocytes (2 lots) and ES cell-derived hepatocytes (2 lots) in which maturation was induced. Hepatic maturation was induced in iPS cell-derived hepatic progenitor cells and ES cell-derived hepatic progenitor cells using a liver maturation induction medium, and the gene expression changes of liver-related genes (C) and bile duct / hepatic progenitor cell markers (D) after induction were analyzed by RT-qPCR. The relative values ​​are shown with the expression in ES cell-derived hepatocytes HAES (regenerative medicine product for congenital urea cycle disorders) set to 1.0. The data were normalized by the housekeeping gene UBC. The results were calculated from independent experiments (n=3 per lot), and the error bars indicate the standard error. iPS-derived: iPS cell-derived hepatic progenitor cells in which maturation was induced (iPS cell-derived mature hepatocytes).

[0178] As shown in Figure 8(B), when hepatocyte-like cells derived from iPS cells that had been subcultured were cultured in liver maturation medium for 7 days, hepatocyte-like morphology with cobblestone-like structures and distinct nuclei was observed. As shown in Figure 7(C), the expression of liver-related marker genes in iPS cell-derived hepatocytes was higher or equivalent to that of HAES (hepatocytes derived from ES cells, a regenerative medicine product). Furthermore, as shown in Figure 8(D), when compared with hepatocytes derived from ES cells that were subcultured and matured using the same method, the gene expression was equivalent or higher in all cases. On the other hand, the expression of liver progenitor cell markers was lower than that of HAES (hepatocytes derived from ES cells, a regenerative medicine product) and lower or equivalent to that of ES cell-derived hepatocytes.

[0179] [Conclusion] The resulting population of iPS cell-derived hepatocyte-like cells was equivalent to or better than the population of ES cell-derived hepatocyte-like cells. The iPS cell-derived hepatocyte-like cell population could be cultured using the established passaging and liver maturation induction system, and its gene expression levels were equivalent to or better than those of the ES cell-derived hepatocyte-like cell population. These values ​​were higher than those of the regenerative medicine product HAES, and therefore, the established passaging and liver maturation induction system for pluripotent stem cell-derived hepatocyte-like cells is considered to be the optimal protocol for selecting pluripotent stem cell-derived hepatocytes with cryotolerance and long-term transplantability.

[0180] [9. Investigation of conditions for subculture of ES cell-derived hepatocytes] We investigated the conditions for subculture of ES cell-derived hepatocytes. The experiment was the same as the one shown in Figure 2 (A) (Figure 9 (A)), except that the following three types of matrix were investigated for use during subculture of ES cell-derived hepatocytes: ・Mouse embryonic fibroblast (MEF) feeder ・Matrigel (Corning, model number: 354234) ・LN511 (iMatrix-511).

[0181] The results are shown in Figure 9. Figure 9(B) shows phase-contrast micrographs of hepatocytes derived from subcultured ES cells, specifically the phase-contrast micrographs immediately after differentiation induction into hepatocytes (day 0) and after subculturing (after induction of liver maturation).

[0182] Figures 9(C) and (D) show the results of analyzing gene expression in hepatocytes derived from subcultured ES cells using quantitative real-time PCR (RT-qPCR).

[0183] As shown in Figure 9, the hepatocyte morphology of subcultured ES cell-derived hepatocytes was not maintained when the matrix was MEF, but was maintained when the matrix was Matrigel and LN511 (Figure 9(B)). Hepatic gene expression was increased in the order of MEF << Matrigel = LN511, and gene expression of ALB, CPS1, and OTC was particularly pronounced when the matrix was Matrigel or LN511 (Figure 9(C)).

[0184] Furthermore, the expression of hepatic progenitor and bile duct genes decreased in the order of MEF >> Matrigel = LN511, and no decrease in SOX9 expression was observed when the matrix was MEF. This indicates that differentiation from hepatic progenitor cells to hepatocytes does not proceed when the matrix is ​​MEF. From these results, it was found that the effect of maintaining hepatocyte morphology of subcultured ES cell-derived hepatocytes is highest in the order of MEF << Matrigel = LN511. Matrigel is not suitable for clinical application because it is of biological origin, but by selecting LN511 as the matrix, ES cell-derived hepatocytes can be subcultured under xeno-free culture conditions.

[0185] [References] 1. Pareja E, Gomez-Lechon MJ, Tolosa L. Induced pluripotent stem cells for the treatment of liver diseases: challenges and perspectives from a clinical viewpoint. Ann Transl Med. 2020;8: 566. 2. Yao J, Yu Y, Nyberg SL. Induced Pluripotent Stem Cells for the Treatment of Liver Diseases: Novel Concepts. Cells Tissues Organs. 2022;211: 368-384. 3. Wu H, Zhou X, Fu G-B, He Z-Y, Wu H-P, You P, et al. Reversible transition between hepatocytes and liver progenitors for in vitro hepatocyte expansion. Cell Res. 2017;27: 709-712. 4. Zhang K, Zhang L, Liu W, Ma X, Cen J, Sun Z, et al. In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity. Cell Stem Cell. 2018;23: 806-819.e4. 5. Katsuda T, Matsuzaki J, Yamaguchi T, Yamada Y, Prieto-Vila M, Hosaka K, et al. Generation of human hepatic progenitor cells with regenerative and metabolic capacities from primary hepatocytes. Elife. 2019;8. doi:10.7554 / eLife.47313 6.Guo R, Jiang M, Wang G, Li B, Jia X, Ai Y, et al. IL6 supports long-term expansion of hepatocytes in vitro. Nat Commun. 2022;13: 7345. 7. Ishii M, Kino J, Ichinohe N, Tanimizu N, Ninomiya T, Suzuki H, et al. Hepatocytic parental progenitor cells of rat small hepatocytes maintain self-renewal capability after long-term culture. Sci Rep. 2017;7: 46177. 8. Pan T, Tao J, Chen Y, Zhang J, Getachew A, Zhuang Y, et al. Robust expansion and functional maturation of human hepatoblasts by chemical strategy. Stem Cell Res Ther. 2021;12: 151. 9. Raggi C, M’Callum M-A, Pham QT, Gaub P, Selleri S, Baratang NV, et al. Leveraging interacting signaling pathways to robustly improve the quality and yield of human pluripotent stem cell-derived hepatoblasts and hepatocytes. Stem Cell Reports. 2022;17: 584-598. 10. Inui J, Ueyama-Toba Y, Mitani S, Mizuguchi H. Development of a method of passaging and freezing human iPS cell-derived hepatocytes to improve their functions. PLoS One. 2023;18(5):e0285783.11. Terry C, Dhawan A, Mitry RR, Hughes RD. Cryopreservation of isolated human hepatocytes for transplantation: State of the art. Cryobiology. 2006;53(2):149-159. 12. Takahashi, K. et al., Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007 Nov 30;131(5):861-72. doi: 10.1016 / j.cell.2007.11.019. 13. Gong-Bo Fu et. al., Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens. Cell Res. 2019 Jan;29(1):8-22. doi: 10.1038 / s41422-018-0103-x. Epub 2018 Oct 25. 14. Akiyama, S., Saku, N., Miyata, S. et al. Drug metabolic activity is a critical cell-intrinsic determinant for selection of hepatocytes during long-term culture. Stem Cell Res Ther 13, 104 (2022). 15. Tsuneishi, Ruri et al. Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells. Scientific reports vol. 11,1 11381. 31 May. 2021, doi:10.1038 / s41598-021-90708-3. 16.Tomotsune, Daihachiro et al. Enrichment of Pluripotent Stem Cell-Derived Hepatocyte-Like Cells by Ammonia Treatment. PloS one vol. 11,9 e0162693. 15 Sep. 2016, doi:10.1371 / journal.pone.0162693.

[0186] This invention can be used in the fields of drug discovery research and medicine.

[0187] HES0007

Claims

1. Stem cell-derived hepatocytes expressing at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1.

2. The stem cell-derived hepatocyte according to claim 1, wherein the stem cell is a human embryonic stem cell or a human induced pluripotent stem cell.

3. A liver transplantation composition containing stem cell-derived hepatocytes as described in claim 1 or 2.

4. A method for producing freeze-tolerant pluripotent stem cell-derived hepatocytes, comprising the following steps (1) to (2): (1) a step of subculturing a population of pluripotent stem cell-derived hepatocyte-like cells differentiated from pluripotent stem cells on laminin-111; (2) a step of inducing liver maturation in the cells after step (1) on laminin-111.

5. A method for producing freeze-tolerant pluripotent stem cell-derived hepatocytes according to claim 4, wherein the series of processes from steps (1) to (2) are repeated at least once on the cells after step (2).

6. Use of a molecular marker for selecting cryo-tolerant hepatocytes, wherein the molecular marker consists of the following substances (a) or (b): (a) the translation product of at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1; (b) A transcript of at least one gene selected from the group consisting of CD44, CD9, CXCL1, CXCL6, EMP3, ENG, ITGA3, ITGA6, ITGB1, ITGB8, KDR, MMP7, NT5E, PECAM1, PLXND1, RORC, S1PR1, SDC3, SPP1, and VIPR1.

7. A method for purifying freeze-tolerant hepatic progenitor cells or hepatic cells, comprising the step of culturing an isolated cell population containing hepatic progenitor cells or hepatic cells on laminin-111.

8. The method for purifying freeze-tolerant liver progenitor cells or hepatocytes according to claim 7, wherein the isolated cell population is a cell population isolated from liver tissue or a cell population differentiated from stem cells.

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