Plasticity-inducing method of hepatocyte

Abstract

To provide a method for manufacturing a hepatoblast, a hepatoblast, and a method for manufacturing a hepatocyte.SOLUTION: A method for manufacturing a hepatoblast includes a step of culturing an iPS cell-derived entodermal cell serum-free medium containing FGF2, HGF, oncostatin M and dexamethasone and differentiating the cell into a hepatoblast. As one aspect, the medium further contains nicotinamide, or the serum-free medium is serum-free differentiation medium, or an iPS cell is derived from a human, or a culture period of the iPS cell-derived entodermal cell is 5 days or more and less than 10 days.SELECTED DRAWING: Figure 2-1

Description

Technical Field

[0001] The present invention relates to a method for inducing hepatocyte plasticity.

Background Art

[0002] Approximately 70% of liver tissue consists of hepatocytes. Hepatocyte plasticity plays an important role in maintaining liver regeneration capacity (Nat Cell Biol. 18(3):238 - 45(2016) (Non - Patent Document 1); Hepatology. 64(6):2244 - 6 (2016) (Non - Patent Document 2)). That is, it has been found that when liver injury occurs in mice, mature hepatocytes convert into proliferative hepatic progenitor cells and repair damaged hepatocytes and bile duct cells (Cell Stem Cell. 15(3):340 - 9(2014) (Non - Patent Document 3); Cell Stem Cell. 15(5):605 - 18(2014) (Non - Patent Document 4); Cell. 157,1324 - 38(2014) (Non - Patent Document 5); Genes Dev. 27(7):719 - 24(2013) (Non - Patent Document 6)). Also, similar hepatic progenitor cells have been detected during human liver cirrhosis (Cell Stem Cell. 23(1):114 - 22(2018) (Non - Patent Document 7)). Furthermore, liver regeneration capacity is related to the aging state of hepatocytes, and it has also been shown that liver grafts from elderly donors have significantly poor engraftment rates (J Hepatol. 70(4):745 - 58 (2019) (Non - Patent Document 8); J Hepatol. 57(2):288 - 96 (2012) (Non - Patent Document 9)).

[0003] Ochiya, T. et al. induced hepatocyte progenitor cells with proliferative ability from mature hepatocytes by exposing mouse and rat hepatocytes to small molecule compounds (YAC, Y-27632, A83-01, CHIR99021) in vitro, and showed that they could be cultured for 20 passages or more (Cell Stem Cell. 20(1):41-55 (2017) (Non-Patent Document 10)). Subsequently, many research groups attempted to apply this culture technique to human hepatocytes. However, the properties of human hepatocytes and rodent-derived hepatocytes are quite different, and it was impossible to culture them for more than 3 passages even when attempts were made to induce human hepatocyte progenitor cells (Cell Stem Cell. 23(6):806-819, 2018 (Non-Patent Document 11); Cell Research. 29, 8-22, 2019 (Non-Patent Document 12); J Hepatol. 70(1):97-107, 2019 (Non-Patent Document 13)). Also, not only was the proliferative ability not maintained, but the pluripotency to differentiate into both human hepatocytes and cholangiocytes could not be maintained, and the induction of hepatocyte progenitor cells based on plasticity control targeting human hepatocytes had not been achieved.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non - Patent Document 8

Non - Patent Document 9

Non - Patent Document 10

Non - Patent Document 11

Non - Patent Document 12

Non - Patent Document 13

Summary of the Invention

Problems to be Solved by the Invention

[0005] The prior art has the following drawbacks. 1. The method for inducing hepatic progenitor cells from rodent - derived hepatocytes cannot be applied to human cells. 2. Hepatic progenitor cells induced by the conventional method do not have high proliferative ability and multi - differentiation ability. 3. There is no technology for controlling the plasticity and aging of human hepatocytes.

[0006] The present invention aims to overcome these drawbacks.

Means for Solving the Problems

[0007] The inventors of the present invention have found a novel method for inducing hepatocytes derived from iPS cells and succeeded in producing hepatocytes that can maintain liver function over a long period while gradually accumulating aging markers and characteristics.

[0008] In addition, the present inventors newly discovered that, unlike rodent-derived hepatocytes, the plasticity of human hepatocytes is regulated by the FGF2-MAPK-EZH2 axis. That is, by increasing the transcriptional activity of EZH2 by adding FGF2, it is possible to convert human hepatocytes into hepatic progenitor cells having proliferative ability and pluripotency (the ability to differentiate into both hepatocytes and cholangiocytes), while in senescent hepatocytes, it was found that this plasticity is impaired.

[0009] Furthermore, the present inventors clarified that the decrease in histone acetylation is a factor inhibiting the acquisition of plasticity in these senescent cells. And by selectively inhibiting histone deacetylase (HDAC), it was found that plasticity can also be improved in senescent human hepatocytes derived from human iPS cells and primary human hepatocytes derived from the elderly (78 years old). The hepatic progenitor cells artificially induced from these senescent cells can be subcultured more than 20 times in an in vitro culture system and can be applied to the mass production of hepatocytes.

[0010] The gist of the present invention is as follows. (1) A method for producing hepatocytes, comprising differentiating endodermal cells into hepatocytes in the presence of a member of the FGF family, HGF, a member of the IL6 family, and dexamethasone. (2) The method according to (1), wherein the member of the FGF family is at least one selected from the group consisting of FGF2, FGF1, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. (3) The method according to (1) or (2), wherein the member of the IL6 family is at least one selected from the group consisting of oncostatin, IL-11, IL-27, IL-35, IL-39, LIF, CT-1, CNTF, and CLCF1. (4) The method according to any one of (1) to (3), wherein the endodermal cells are cells differentiated from pluripotent stem cells. (5) The method according to any one of (1) to (4), wherein the endoderm cells are of human origin. (6) Hepatocytes produced by the method according to any one of (1) to (5), capable of maintaining the ability to secrete albumin for 12 days or more in in vitro monolayer culture and surviving. (7) The hepatocytes according to (6), capable of surviving and having functions as hepatocytes other than the ability to secrete albumin for 12 days or more. (8) The hepatocytes according to (7), wherein the function as hepatocytes other than the ability to secrete albumin is at least one selected from the group consisting of drug metabolism ability, uptake and excretion of indocyanine green, glycogen storage, uptake of low-density lipoprotein, and gene expression. (9) The hepatocytes according to any one of (6) to (8), showing characteristics of aging. (10) The hepatocytes according to (9), wherein the characteristics of aging are at least one selected from the group consisting of an increase in cell volume, expression of aging-related genes, an increase in inflammatory response, DNA damage, an increase in intracellular reactive oxygen species level, an increase in cell senescence-related β-galactosidase level, epigenetic changes, shortening of telomere length, and a decrease in mitochondrial function. (11) A method for producing hepatic progenitor cells, comprising culturing hepatocytes in the presence of a member of the FGF family. (12) The method according to (11), comprising culturing hepatocytes in the presence of a member of the FGF family and an agent that brings about histone hyperacetylation. (13) The method according to (11) or (12), wherein the member of the FGF family is at least one selected from the group consisting of FGF2, FGF1, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. (14) The method according to (12) or (13), wherein the agent that brings about histone hyperacetylation is a histone deacetylase inhibitor. (15) The histone deacetylase inhibitor is at least one selected from the group consisting of Trichostatin A, Tacedinaline (CI-994), M344, ITSA-1, Sodium valproate, Sodium 4-phenylbutuyrate, Sodium Butyrate (NaB), valproic acid (VPA), Abexinostat (PCI-24781), Belinostat (PXD101), Citarinostat (ACY-241), Dacinostat (LAQ824), Depudecin, Domatinostat (4SC-202), Droxinostat, Entinostat (MS-275, SNDX-275), Fimepinostat (CUDC-907), Givinostat (ITF2357), Mocetinostat (MGCD0103), Nexturastat A, Panobinostat (LBH-589, NVP-LBH589), Pracinostat (SB939), Quisinostat (JNJ-26481585) 2HCl, Resminostat, Ricolinostat (ACY-1215), Tucidinostat (Chidamide), Vorinostat (SAHA), ACY-738, Apicidin, AR-42, BG45, BML-210, BRD73954, CAY10603, CUDC-101, Curcumin, Depudecin, H1388, HC Toxin, HPOB, LMK-235, MC1568, Oxamflatin, (-)-Parthenolide, PCI-34051, RG2833 (RGFP109), RGFP966, Romidepsin (FK228, Depsipeptide), Santacruzamate A (CAY10683), Scriptaid, SKLB-23bb, Splitomicin, Suberoyl bis-hydroxamic acid, Tasquinimod, TH34, Tinostamustine (EDO-S101), TMP195, TMP269, Tubacin and Tubastatin A, and is the method according to any one of (12) to (14). (16) The method according to any one of (11) to (15), wherein the hepatocytes are cells differentiated from pluripotent stem cells, cells obtained by subculturing cells differentiated from pluripotent stem cells, primary cultured cells isolated from a biological tissue, cells obtained by subculturing primary cultured cells isolated from a biological tissue, or a combination thereof. (17) The method according to any one of (11) to (16), wherein the hepatocytes are of human origin. (18) An alpha-fetoprotein (AFP)-negative hepatic progenitor cell having a proliferation ability and a bidirectional differentiation ability into hepatocytes and cholangiocytes, prepared by the method according to any one of (11) to (17). (19) The hepatic progenitor cell according to (18), which is capable of differentiating into hepatocytes and / or cholangiocytes. (20) A method for producing hepatocytes, comprising inducing differentiation of the hepatic progenitor cell according to (18) or (19) into hepatocytes. (21) A method for producing cholangiocytes, comprising inducing differentiation of the hepatic progenitor cell according to (18) or (19) into cholangiocytes. (22) A method for suppressing hepatocyte senescence with an agent that brings about histone hyperacetylation. (23) The method according to (22), wherein the agent that brings about histone hyperacetylation is used in combination with a member of the FGF family. (24) A hepatocyte senescence inhibitor containing, as an active ingredient, an agent that brings about histone hyperacetylation. (25) The agent according to (24), wherein the agent that brings about histone hyperacetylation is a histone deacetylase inhibitor. (26) The histone deacetylase inhibitor is at least one selected from the group consisting of Trichostatin A, Tacedinaline (CI-994), M344, ITSA-1, Sodium valproate, Sodium 4-phenylbutuyrate, Sodium Butyrate (NaB), valproic acid (VPA), Abexinostat (PCI-24781), Belinostat (PXD101), Citarinostat (ACY-241), Dacinostat (LAQ824), Depudecin, Domatinostat (4SC-202), Droxinostat, Entinostat (MS-275, SNDX-275), Fimepinostat (CUDC-907), Givinostat (ITF2357), Mocetinostat (MGCD0103), Nexturastat A, Panobinostat (LBH-589, NVP-LBH589), Pracinostat (SB939), Quisinostat (JNJ-26481585) 2HCl, Resminostat, Ricolinostat (ACY-1215), Tucidinostat (Chidamide), Vorinostat (SAHA), ACY-738, Apicidin, AR-42, BG45, BML-210, BRD73954, CAY10603, CUDC-101, Curcumin, Depudecin, H1388, HC Toxin, HPOB, LMK-235, MC1568, Oxamflatin, (-)-Parthenolide, PCI-34051, RG2833 (RGFP109), RGFP966, Romidepsin (FK228, Depsipeptide), Santacruzamate A (CAY10683), Scriptaid, SKLB-23bb, Splitomicin, Suberoyl bis-hydroxamic acid, Tasquinimod, TH34, Tinostamustine (EDO-S101), TMP195, TMP269, Tubacin and Tubastatin A, which is the agent described in (25). An agent according to any one of (24) to (26), wherein an agent that brings about high histone acetylation is used in combination with a member of the FGF family. (28) A method for increasing the plasticity of hepatocytes with an agent that brings about high histone acetylation. (29) The method according to (28), wherein an agent that brings about high histone acetylation is used in combination with a member of the FGF family. (30) An agent for increasing the plasticity of hepatocytes, comprising an agent that brings about high histone acetylation as an active ingredient. (31) The agent according to (30), wherein the agent that brings about high histone acetylation is a histone deacetylase inhibitor. (32) The agent according to (31), wherein the histone deacetylase inhibitor is at least one selected from the group consisting of Trichostatin A, Tacedinaline (CI-994), M344, ITSA-1, Sodium valproate, Sodium 4-phenylbutuyrate, Sodium Butyrate (NaB), valproic acid (VPA), Abexinostat (PCI-24781), Belinostat (PXD101), Citarinostat (ACY-241), Dacinostat (LAQ824), Depudecin, Domatinostat (4SC-202), Droxinostat, Entinostat (MS-275, SNDX-275), Fimepinostat (CUDC-907), Givinostat (ITF2357), Mocetinostat (MGCD0103), Nexturastat A, Panobinostat (LBH-589, NVP-LBH589), Pracinostat (SB939), Quisinostat (JNJ-26481585) 2HCl, Resminostat, Ricolinostat (ACY-1215), Tucidinostat (Chidamide), Vorinostat (SAHA), ACY-738, Apicidin, AR-42, BG45, BML-210, BRD73954, CAY10603, CUDC-101, Curcumin, Depudecin, H1388, HC Toxin, HPOB, LMK-235, MC1568, Oxamflatin, (-)-Parthenolide, PCI-34051, RG2833 (RGFP109), RGFP966, Romidepsin (FK228, Depsipeptide), Santacruzamate A (CAY10683), Scriptaid, SKLB-23bb, Splitomicin, Suberoyl bis-hydroxamic acid, Tasquinimod, TH34, Tinostamustine (EDO-S101), TMP195, TMP269, Tubacin and Tubastatin A. An agent according to any one of (30) to (32), wherein an agent that brings about high histone acetylation is used in combination with a member of the FGF family. (34) A method for producing a chimeric animal, comprising transplanting at least one cell selected from the group consisting of the hepatocytes according to (6), the hepatic progenitor cells according to (18), and cells differentiated from the hepatic progenitor cells according to (18) into a non-human animal. (35) A method for producing a chimeric animal according to (34), comprising transplanting at least one cell selected from the group consisting of the hepatocytes according to (6), the hepatic progenitor cells according to (18), and cells differentiated from the hepatic progenitor cells according to (18) into a non-human animal with liver failure. (36) A method for producing a chimeric animal according to (34) or (35), wherein the transplanted hepatic progenitor cells proliferate and / or differentiate. (37) A method for producing a chimeric animal according to any one of (34) to (36), wherein liver regeneration is promoted. (38) A composition for transplantation, comprising at least one cell selected from the group consisting of the hepatocytes according to (6), the hepatic progenitor cells according to (18), and cells differentiated from the hepatic progenitor cells according to (18). (39) An agent containing FGF10, retinoic acid, and forskolin for use in a medium used for inducing differentiation of the hepatic progenitor cells according to (18) or (19) into bile duct cells; and instructions for using the agent in the medium used for the differentiation induction; A kit for the medium in the differentiation induction. (40) A medium containing FGF10, retinoic acid, and forskolin for inducing differentiation of the hepatic progenitor cells according to (18) or (19) into bile duct cells. (41) A liver regeneration promoter containing an agent that brings about high histone acetylation as an active ingredient. (42) A method for promoting liver regeneration, comprising administering an agent that brings about high histone acetylation to a subject in a pharmaceutically effective amount. [Effect of the Invention]

[0011] According to the present invention, hepatocytes serving as an aging model were obtained.

[0012] According to the present invention, it has become possible to induce plasticity in hepatocytes. The hepatocytes (hepatic progenitor cells) in which plasticity has been induced can be differentiated into hepatocytes and bile duct cells.

[0013] According to the present invention, it has also become possible to induce plasticity in aged hepatocytes. This specification includes the content described in the specification and / or drawings of Japanese Patent Application No. 2019-177843, which is the basis of the priority of this application.

Brief Description of Drawings

[0014]

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Mode for Carrying Out the Invention

[0015] Hereinafter, the present invention will be described in detail. 1. Novel method for inducing hepatocytes and establishment of a hepatocyte aging model The present invention provides a method for producing hepatocytes, which includes differentiating endodermal cells into hepatocytes in the presence of a member of the FGF family, HGF, a member of the IL6 family, and dexamethasone. The method of the present invention is a simple method for producing hepatocytes, which can differentiate endodermal cells into hepatocytes in one step.

[0016] Fibroblast growth factors (FGF) are a type of growth factor and are involved in angiogenesis, wound healing, and embryonic development. In the present invention, examples of members of the FGF family include FGF2, FGF1, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, and combinations thereof, etc., and FGF2 is preferred.

[0017] Hepatocyte Growth Factor (HGF) was discovered as a substance that promotes the proliferation of hepatocytes in the blood of rats with partial hepatectomy and is a cytokine purified as a factor that strongly promotes the proliferation of primary cultured hepatocytes. It is a strong candidate for a liver regeneration factor that supports the robust regenerative capacity of the liver. As a growth factor, HGF is large. HGF mRNA is a single-chain precursor of 728 amino acid residues that is translated as Pro HGF (94kD) without biological activity. However, after removal of the N-terminal signal peptide and secretion into the extracellular space, it undergoes specific cleavage (processing) between Arg and Val by proteases such as HGF activator (HGFA), coagulation factor (XIa), urokinase (u-PA), and matriptase, and then is produced as Mature (active form) HGF with a heterodimer structure in which the α-chain (62kD) and β-chain (34kD) are disulfide-bonded. HGF promotes the proliferation of primary cultured rat hepatocytes and also promotes the proliferation of not only hepatocytes but also various epithelial cells, endothelial cells, and some mesenchymal cells. In addition, it has versatile physiological activities responsible for the regeneration and protection of tissues and organs, such as promoting cell proliferation, promoting cell dispersion and cell movement, anti-apoptosis (cell death), inducing morphogenesis (such as lumen formation), angiogenesis, anti-fibrotic effects, and regulating immune responses. The receptor for HGF is the product of the oncogene c-met and has tyrosine kinase activity, and the diverse biological actions of HGF are exerted through c-Met. During liver regeneration, an increase in the expression of the HGF gene in the liver, spleen, and lung, as well as an increase in the levels of HGF in the blood and liver, are observed prior to the induction of liver DNA synthesis, and liver regeneration is suppressed by the administration of anti-HGF antibodies. The producing cells of HGF are mainly produced and secreted from mesenchymal cells such as fibroblasts, adipocytes, neutrophils, macrophages, and vascular endothelial cells, and act paracrinally on normal epithelial cells or cancer cells. On the other hand, it has also been reported that cancer cells produce HGF and have an autocrine effect of activating their own c-MET. The cells reported to produce HGF in the liver are stellate cells and sinusoidal endothelial cells of the hepatic sinusoids. Taking liver regeneration as an example, with various acute injuries such as partial hepatectomy, hepatitis, and hepatic ischemia, the expression of HGF increases not only in the injured liver but also in uninjured organs such as the distant lung and kidney, thereby increasing the blood level of HGF.In fact, when an antibody that neutralizes the activity of HGF is administered to rats or mice that have caused liver injury, the liver injury significantly expands and liver regeneration fails. Similar results have also been observed in other organ injuries, and it has been clarified that HGF is an endogenous factor responsible for the regeneration and protection of various tissues and organs, including the liver, kidney, lung, cardiovascular system, and nervous system. HGF production in cultured cells is induced by PKC activators, PKA activators, cAMP elevators, various growth factors, inflammatory cytokines (such as IL-1 and TNF-α), etc., and is suppressed by TGF-β, glucocorticoids, active vitamin D, retinoic acid, etc. There have been numerous reports indicating the possibility of applying HGF as a therapeutic agent for various intractable organ diseases such as liver cirrhosis, chronic renal failure, pulmonary fibrosis, myocardial infarction, and obstructive arteriosclerosis by utilizing its strong growth-promoting effect on various cells. In addition, serum and plasma HGF quantitative ELISA kits are used for predicting the exacerbation of hepatitis.

[0018] Interleukin-6 (IL-6) is a lectin produced by cells such as T cells and macrophages, and is one of the cytokines that regulate humoral immunity. In the present invention, examples of members of the IL6 family include oncostatin (OSM), IL-6, IL-11, IL-27, IL-35, IL-39, LIF, CT-1, CNTF, CLCF1, and combinations thereof, and oncostatin is preferred.

[0019] Dexamethasone is a synthetic corticosteroid agent that exhibits an anti-inflammatory effect through the same mechanism as natural glucocorticoids and is used for acute inflammation, chronic inflammation, autoimmune diseases, allergic diseases, etc.

[0020] Endodermal cells express endodermal markers and are cells capable of differentiating into various endodermal cells (such as cells of the lung, liver, pancreas, stomach, small intestine, and large intestine).

[0021] Examples of endoderm markers include SOX17, FOXA2, CXCR4, EpCAM, C-KIT, CER1, etc. Examples of endoderm cells include cells such as the lung, liver, pancreas, stomach, small intestine, and large intestine.

[0022] Endoderm cells can be induced to differentiate from pluripotent stem cells. Examples of pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc. The method of differentiating iPS cells into endoderm cells is described in the examples below. That is, using a medium consisting of RPMI 1640, 1% B27, 50 ng / mL WNT3A, and 100 ng / mL activin A, cultured on a plate coated with GFR Matrigel or laminin 511 for 7 days to differentiate hiPSC clones into endoderm cells. 10 μM Y-27632 was added on day 0, and 0.5 mM NaB was added from day 1 to day 3. This method can be appropriately modified.

[0023] Also, the endoderm cells may be collected from a living body.

[0024] The endoderm cells are preferably of human origin, but are not necessarily limited to humans and may be derived from mammals such as mice, rats, guinea pigs, hamsters, rabbits, pigs, cats, dogs, sheep, cows, horses, goats, and monkeys.

[0025] By culturing endoderm cells in a medium containing a member of the FGF family, HGF, a member of the IL6 family, and dexamethasone, they can be induced to differentiate into hepatocytes.

[0026] As the basal medium, SFD medium (described in WO2016093222), DMEM / F12, DMEM, IMDM, RPMI1640, Williams' Medium E, etc. can be used, and it is preferable to add a member of the FGF family, HGF, a member of the IL6 family, and dexamethasone to the basal medium.

[0027] The concentrations of a member of the FGF family, HGF, a member of the IL6 family, and dexamethasone in the medium may be appropriately adjusted. For example, when using FGF2 as a member of the FGF family, the concentration of FGF2 is usually preferably 0.1 to 100 ng / mL, more preferably 1 to 50 ng / mL. The concentration of HGF is usually preferably 5 to 100 ng / mL, more preferably 5 to 50 ng / mL. When using oncostatin (OSM) as a member of the IL6 family, the concentration of OSM is usually preferably 5 to 100 ng / mL, more preferably 10 to 50 ng / mL. The concentration of dexamethasone is usually preferably 10 to 10000 nM, more preferably 50 to 500 nM.

[0028] Nicotinamide may be added to the medium. The concentration of nicotinamide in the medium is usually preferably 0.1 to 100 mM, more preferably 1 to 10 mM. Nicotinamide is preferably added after the 8th day of culture.

[0029] The medium may be a serum-containing medium or a serum-free medium, but in the examples described below, a serum-free medium was used.

[0030] It is preferable to seed and culture the endoderm cells on a gel. The gel to be used is not particularly limited, and GFR Matrigel (manufactured by Corning) etc. can be used.

[0031] Endoderm cells may be cultured on a container coated with a main component of the tissue basement membrane such as laminin.

[0032] The temperature during culture is not particularly limited, but it is preferably 30 to 40°C, more preferably 37°C.

[0033] The culture period is not particularly limited, but it is preferably 1 to 150 days, more preferably 1 to 90 days.

[0034] To confirm that endoderm cells have differentiated into hepatocytes, the expression of liver-specific genes and liver function can be examined, or the cell morphology of hepatocyte-like cells can be observed. Examples of hepatocyte-specific genes include TBX3 and TTR, DLK, CK19, EPCAM (hepatic progenitor cells), and A1AT, ALB, G6PC, ASGR1, TAT, TDO2, CYP2C9, CYP2C19, CYP3A4, and CYP7A1 (hepatocytes). In the examples described below, during the process of differentiation of endoderm cells into hepatocytes, at the initial stage of differentiation (for example, on the 5th day of differentiation), they are in the state of hepatic progenitor cells, and when the culture is continued further, they come to express liver function. Liver function will be described later.

[0035] According to the present invention, hepatocytes can be induced from pluripotent stem cells by a simple method.

[0036] The hepatocytes produced by the method of the present invention can retain their function as hepatocytes and survive over a long period of time, and can realize long-term culture of hepatocytes and imitation of the aging process in vitro.

[0037] Hepatocytes prepared by the method of the present invention can survive with hepatocyte functions in in vitro monolayer culture for a period of 12 days or more, 15 days or more, 17 days or more, 22 days or more, 26 days or more, 31 days or more, 32 days or more, 42 days or more, 52 days or more, 72 days or more, 82 days or more, 90 days or more, 100 days or more, or longer. The upper limit of the period during which hepatocytes prepared by the method of the present invention can survive with hepatocyte functions is not particularly limited. The inventors have observed that hepatocytes prepared by the method of the present invention can survive with the ability to secrete albumin for 82 days.

[0038] Functions as hepatocytes retained by hepatocytes prepared by the method of the present invention include, for example, albumin secretion ability, drug metabolism ability, uptake and excretion of indocyanine green (ICG), glycogen storage, uptake of low-density lipoprotein, gene expression, and the like. In one embodiment of the present invention, hepatocytes prepared by the method of the present invention can survive with the ability to secrete albumin for 12 days or more, and can further survive with functions as hepatocytes other than the ability to secrete albumin for 12 days or more.

[0039] The albumin secretion ability can be measured by a commercially available albumin ELISA quantification kit.

[0040] The drug metabolism ability can be measured by a commercially available Cytochrome P450-related assay kit.

[0041] Uptake and excretion of ICG can be examined by seeding cells in a medium supplemented with ICG, incubating for an appropriate time, and then performing microscopic observation.

[0042] Glycogen storage can be examined by detecting glycogen using periodic acid Schiff (PAS) staining.

[0043] The uptake of low-density lipoprotein can be examined by incubating the cells with Dil-Ac-LDL and Hoechst 33342 standard dilution solution, then washing the cells with PBS and analyzing the photographs using a fluorescence microscope.

[0044] Gene expression assays can be performed as follows. PureLink TM Isolate total RNA using the RNA Mini Kit. Use RNA (< 2 μg) as a template to synthesize single-stranded cDNA using a high-performance cDNA reverse transcription kit according to the manufacturer's instructions. Perform Q-PCR using the cDNA, specific primers, and probes from the Universal Probe Library.

[0045] Hepatocytes produced by the method of the present invention may exhibit characteristics of aging. Examples of characteristics of aging include an increase in cell volume, expression of aging-related genes, an increase in inflammatory response, DNA damage, an increase in intracellular reactive oxygen species levels, an increase in cell senescence-related β-galactosidase levels, epigenetic changes, shortening of telomere length, a decrease in mitochondrial function, metabolic abnormalities, and low responsiveness to growth factors.

[0046] Cell volume can be examined by incubating the cells with Hoechst 33342 standard dilution solution, then washing the cells with PBS and analyzing the photographs using a fluorescence microscope.

[0047] Examples of aging-related genes include CCL2, CDNK2A, CST1, CXCL1, GDF15, ID1, LIMCH1, LMO2, MAP2, MMP24, MYC, RCAN2, S100A8, S100A9, SERPINE1, TGFB1, etc. The expression of aging-related genes can be measured using the commercially available SurePrint G3 Human Gene Expression 8x60K (Agilent Technologies).

[0048] As an inflammatory response, an increase in intracellular inflammation-related factors can be exemplified, and the inflammatory response can be measured using the commercially available SurePrint G3 Human Gene Expression 8x60K (Agilent Technologies).

[0049] DNA damage can be measured using the commercially available SurePrint G3 Human Gene Expression 8x60K (Agilent Technologies).

[0050] The intracellular reactive oxygen species level can be measured by adding reagents such as CellROX TM Deep Red Reagent (Thermo Fisher Scientific) to cells and then observing under a microscope or counting the number of fluorescent cells.

[0051] The level of cell senescence-related β-galactosidase can be measured using a β-galactosidase detection kit (Dojindo).

[0052] Epigenetic changes can be measured using the commercially available Acetyl-Histone H3 Antibody Sampler kit (CST).

[0053] The telomere length can be measured using the commercially available telomere length qPCR kit (ScienCell Research Laboratories).

[0054] As mitochondrial function, the production of ATP (phosphorylation of ADP) by oxidative phosphorylation via the electron transport system can be exemplified, and a decrease in mitochondrial function can be confirmed by measuring the decrease in genes related to ATP production using the commercially available SurePrint G3 Human Gene Expression 8x60K (Agilent Technologies).

[0055] Examples of metabolic abnormalities include metabolic abnormalities such as organic acids, amino acids, the uric acid cycle, carbohydrates, fatty acids, lysosomes, lipoproteins, nucleic acids, and membrane transport proteins. Metabolic abnormalities can be confirmed by measuring changes in the expression of metabolism-related genes using the commercially available SurePrint G3 Human Gene Expression 8x60K (Agilent Technologies).

[0056] Low responsiveness to growth factors can be examined by adding growth factors, culturing for several days, incubating the cells with a Hoechst 33342 standard dilution solution, washing the cells with PBS, and analyzing the photographs taken using a fluorescence microscope.

[0057] The hepatocytes produced by the method of the present invention are different from the source of hepatocytes derived from a living body. Also, compared with the hepatocytes induced by the conventional method, the hepatocytes produced by the method of the present invention have significantly upregulated expression of liver-specific genes (ALB, G6PC, ASGR1, TAT, TDO2, CYP2C9, CYP2C19, CYP3A4, and CYP7A1), secretion of ALB, and activity of CYP3A4. Hepatocytes induced by the conventional method cannot be cultured in vitro for more than 30 days, while the hepatocytes produced by the method of the present invention can be cultured in vitro for 50 days or more and can reproduce the aging process of hepatocytes.

[0058] Regarding "expression of liver-specific genes, secretion of ALB, and activity of CYP3A4", an example of the numerical values upregulated in the hepatocytes (N-Hep on the 22nd day of culture in the following examples) produced by the method of the present invention compared with the hepatocytes induced by the conventional method is described below. TIFF2025094074000002.tif57135

[0059] TIFF2025094074000003.tif21135

[0060] TIFF2025094074000004.tif21141

[0061] Conventional method A: Si-Tayeb, K., Noto, F.K., Nagaoka, M., Li, J., Battle, M.A., Duris, C., North, P.E., Dalton, S., and Duncan, S.A. (2010). Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51, 297-305. Conventional method B: Kajiwara, M., Aoi, T., Okita, K., Takahashi, R., Inoue, H., Takayama, N., Endo, H., Eto, K., Toguchida, J., Uemoto, S., et al. (2012). Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America 109, 12538-12543. Each cell in the table has not been passaged. Method for measuring liver-specific genes: Q-PCR (see Examples below). Method for measuring ALB secretion: Measured by ELISA (see Examples below). Method for measuring CYP3A4 activity: Commercially available P450-Glo TM Measured using the CYP3A4 Assay kit (Promega, V8801).

[0062] When compared with hepatocytes induced by the conventional method A, the hepatocytes produced by the method of the present invention can have their expression increased by more than 10-fold, preferably more than 15-fold, more preferably more than 19-fold for ALB, more than 100-fold, preferably more than 500-fold, more preferably more than 1000-fold for G6PC, more than 2-fold, preferably more than 3-fold, more preferably more than 4-fold for ASGR1, more than 10-fold, preferably more than 20-fold, more preferably more than 25-fold for TAT, more than 10-fold, preferably more than 15-fold, more preferably more than 17-fold for TDO2, more than 2-fold, preferably more than 4-fold, more preferably more than 6-fold for CYP2C9, more than 2-fold, preferably more than 3-fold, more preferably more than 4-fold for CYP2C19, more than 2-fold, preferably more than 4-fold, more preferably more than 5-fold for CYP3A4, and more than 3-fold, preferably more than 5-fold, more preferably more than 8-fold for CYP7A1.

[0063] Also, with respect to the secretion of ALB, the hepatocytes produced by the method of the present invention can increase by more than 2-fold, preferably more than 3-fold, more preferably more than 4-fold compared with the hepatocytes induced by the conventional method A.

[0064] Furthermore, the hepatocytes produced by the method of the present invention have the activity of CYP3A4, while the activity of CYP3A4 is not detected in the hepatocytes induced by the conventional method A.

[0065] When compared with hepatocytes induced by the conventional method B, the hepatocytes produced by the method of the present invention show an increased expression of more than 2-fold, preferably more than 4-fold, more preferably more than 5-fold for ALB; more than 20-fold, preferably more than 25-fold, more preferably more than 29-fold for G6PC; about the same for ASGR1; more than 30-fold, preferably more than 40-fold, more preferably more than 50-fold for TAT; more than 2-fold, preferably more than 4-fold, more preferably more than 5-fold for TDO2; about the same for CYP2C9; more than 1.2-fold, preferably more than 1.5-fold, more preferably more than 2-fold for CYP2C19; more than 3-fold, preferably more than 5-fold, more preferably more than 8-fold for CYP3A4; and more than 2-fold, preferably more than 4-fold, more preferably more than 5-fold for CYP7A1.

[0066] Regarding the secretion of ALB, the hepatocytes produced by the method of the present invention can be increased by more than 1.2-fold, preferably more than 1.5-fold, more preferably more than 2-fold as compared with the hepatocytes induced by the conventional method B.

[0067] Furthermore, regarding the activity of CYP3A4, the hepatocytes produced by the method of the present invention can be increased by more than 30-fold, preferably more than 40-fold, more preferably more than 47-fold as compared with the hepatocytes induced by the conventional method B.

[0068] Examples of the measured values of the expression of liver-specific genes, the secretion of ALB, and the activity of CYP3A4 for the hepatocytes produced by the method of the present invention (N-Hep on the 22nd day of culture in the following examples), the hepatocytes induced by the conventional method A, and the hepatocytes induced by the conventional method B are described below. JPEG2025094074000005.jpg250111

[0069] TIFF2025094074000006.tif62132

[0070] TIFF2025094074000007.tif62149

[0071] N-Hep: Hepatocytes prepared by the method of the present invention (N-Hep on the 22nd day of culture in the examples described below) Human hepatocytes: IVT-F00995-P-AKB of BioreclamationIVT (donor is a 39-year-old female) Each cell in the table has not been passaged. Method for measuring liver-specific genes: Q-PCR (see the examples described below) Method for measuring ALB secretion: Measured by ELISA (see the examples described below) Method for measuring CYP3A4 activity: Commercially available P450-Glo TM Measured using the CYP3A4 Assay kit (Promega, V8801).

[0072] For the hepatocytes prepared by the method of the present invention, the expression levels measured by Q-PCR are: for ALB, 3 or more, preferably 5 or more, more preferably 9 or more; for G6PC, 0.05 or more, preferably 0.1 or more, more preferably 0.14 or more; for ASGR1, 0.05 or more, preferably 0.1 or more, more preferably 0.16 or more; for TAT, 0.00001 or more, preferably 0.00005 or more, more preferably 0.0001 or more; for TDO2, 0.01 or more, preferably 0.05 or more, more preferably 0.1 or more; for CYP2C9, 0.05 or more, preferably 0.01 or more, more preferably 0.02 or more; for CYP2C19, 0.00005 or more, preferably 0.0001 or more, more preferably 0.0006 or more; for CYP3A4, 0.00001 or more, preferably 0.00005 or more, more preferably 0.00009 or more; for CYP7A1, 0.00001 or more, preferably 0.00005 or more, more preferably 0.00007 or more.

[0073] Hepatocytes prepared by the method of the present invention may have an ALB secretion quantified by ELISA of 2 μg / ml / 24h / 1 million or more, preferably 4 μg / ml / 24h / 1 million or more, more preferably 5 μg / ml / 24h / 1 million or more.

[0074] Hepatocytes prepared by the method of the present invention have a CYP3A4 activity measured by a commercially available P450-Glo TM CYP3A4 Assay kit (Promega, V8801) of 50000 RLU / 4h / ml / million cells or more, preferably 100000 RLU / 4h / ml / million cells or more, more preferably 200000 RLU / 4h / ml / million cells or more.

[0075] 2. Induction of hepatic progenitor cells, induction of differentiation of hepatic progenitor cells into hepatocytes and bile duct cells The present invention also provides a method for producing hepatic progenitor cells, which includes culturing hepatocytes in the presence of a member of the FGF family.

[0076] In the present invention, examples of the member of the FGF family include FGF2, FGF1, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, etc., and FGF2 is preferred.

[0077] The hepatic progenitor cells or pHCs or hepatocytes used for induction of plasticity may be those that express liver-specific genes and exhibit liver functions. The hepatocytes may be hepatocytes differentiated from pluripotent stem cells, cells obtained by subculturing hepatocytes differentiated from pluripotent stem cells, primary cultured hepatocytes isolated from biological tissues, cells obtained by subculturing primary cultured hepatocytes isolated from biological tissues, or any combination thereof.

[0078] Examples of pluripotent stem cells include ES cells and iPS cells.

[0079] Examples of hepatocytes differentiated from pluripotent stem cells include the hepatocytes described in 1 above, i.e., hepatocytes induced to differentiate from endoderm cells in the presence of a member of the FGF family, HGF, a member of the IL6 family, and dexamethasone. In the examples described below, endoderm cells were seeded on a plate coated with GFR Matrigel or laminin 511 using a hepatic differentiation medium (HDM) that does not contain nicotinamide, and the medium was replaced with HDM every day from day 8 to day 10, and from day 11 onwards, the medium was replaced every two days to induce differentiation into hepatocytes. This HDM consisted of SFD containing 10 ng / mL FGF2, 20 ng / mL HGF, 10 ng / mL OSM, 100 nM dexamethasone, and 10 mM nicotinamide. This method may be modified as appropriate. To subculture the hepatocytes, they can be seeded on GFR Matrigel or Laminin 511 coated plates in HDM containing nicotinamide, changing the medium every 2 days, and subculture the hepatocytes. The HDM consisted of SFD containing 10 ng / mL FGF2, 20 ng / mL HGF, 10 ng / mL OSM, 100 nM dexamethasone, and 10 mM nicotinamide. This method can be modified as appropriate.

[0080] When hepatic progenitor cells are induced using hepatocytes differentiated from pluripotent stem cells, the hepatocytes are passaged typically 1 to 5 times, preferably 1 to 4 times, and more preferably 1 to 3 times, and the number of days for which the hepatocytes are cultured is typically 1 to 100 days, preferably 1 to 80 days, and more preferably 5 to 60 days.

[0081] The hepatocytes may be primary cultured hepatocytes isolated from biological tissues or cells obtained by subculturing the same. In the examples described below, primary human hepatocytes derived from donors aged 2 months, 39 years, and 78 years were used. To culture or subculture primary human hepatocytes, primary human hepatocytes may be seeded on plates coated with GFR Matrigel or laminin 511 using a human hepatocyte culture medium, and the medium may be replaced every two days to culture or subculture the primary human hepatocytes. There are two types of human hepatocyte culture media. One consisted of SFD containing HDM, 10 ng / mL FGF2, 20 ng / mL HGF, 10 ng / mL OSM, 100 nM dexamethasone, and 10 mM nicotinamide. The other consisted of Williams E medium containing 5% FBS, 1 μM dexamethasone, 4 μg / mL human recombinant insulin, 2 mM Glutamax, and 15 mM HEPES. This method can be appropriately modified.

[0082] When inducing hepatic progenitor cells from primary cultured cells isolated from human biological tissues, cells obtained by subculturing the same, or combinations thereof, the age of the human is usually 0 to 120 years old, preferably 0 to 100 years old, and more preferably 0 to 80 years old. When using primary cultured cells isolated from biological tissues of animals other than humans, cells obtained by subculturing the same, or combinations thereof, appropriate weeks, months, and ages can be estimated from the human case. The number of passages of the cultured cells is usually 1 to 5 times, preferably 1 to 4 times, and more preferably 1 to 3 times. The number of days of culturing the cells is usually 1 to 30 days, preferably 1 to 20 days, and more preferably 1 to 10 days.

[0083] The hepatocytes may be a combination of hepatocytes differentiated from pluripotent stem cells, cells obtained by subculturing hepatocytes differentiated from pluripotent stem cells, primary cultured hepatocytes isolated from biological tissues, and cells obtained by subculturing primary cultured hepatocytes isolated from biological tissues.

[0084] The hepatocytes are preferably of human origin, but are not limited to humans and may also be derived from mammals such as mice, rats, guinea pigs, hamsters, rabbits, pigs, cats, dogs, sheep, cows, horses, goats, and monkeys.

[0085] By culturing hepatocytes in a medium containing a member of the FGF family, hepatic progenitor cells can be induced (hereinafter sometimes referred to as "reprogrammed").

[0086] As the basal medium, SFD medium (described in WO2016093222), DMEM / F12, DMEM, IMDM, RPMI1640, Williams' Medium E, etc. can be used, and a member of the FGF family may be added to the basal medium.

[0087] The concentration of the member of the FGF family in the medium may be adjusted as appropriate. For example, when using FGF2 as a member of the FGF family, the concentration of FGF2 is usually preferably 0.01 - 1000 ng / mL, more preferably 0.1 - 100 ng / mL, and still more preferably 1 - 50 ng / mL.

[0088] EGF, HGF, a ROCK inhibitor, a selective inhibitor of ALK4, ALK5, ALK7 (TGF-β receptor inhibitor), and a GSK-3β inhibitor may be added to the medium.

[0089] Epidermal Growth Factor (EGF) is a 6045 Da protein consisting of 53 amino acid residues and three intramolecular disulfide bonds. It binds as a ligand to the epidermal growth factor receptor (EGFR) present on the cell surface and plays an important role in regulating cell growth and proliferation. The concentration of EGF in the medium is usually preferably 0.1 - 1000 ng / mL, more preferably 1 - 100 ng / mL, and still more preferably 1 - 50 ng / mL.

[0090] HGF has been described above. The concentration of HGF is usually preferably 1 to 1000 ng / mL, more preferably 5 to 100 ng / mL, and still more preferably 5 to 50 ng / mL.

[0091] Rho kinase (Rho-associated protein kinase: ROCK) is a serine-threonine protein phosphorylase identified as a target protein of the low molecular weight GTP-binding protein Rho. Examples of ROCK inhibitors include Y-27632, AT13148, Fasudil, Hydroxyfasudil, GSK269962A, GSK180736A, GSK429286A, KD025, Netarsudil, RKI-1447, Thiazovivin, Y-39983, and the like. When Y-27632 is used as the ROCK inhibitor, the concentration of Y-27632 is usually preferably 1 to 1000 μM, more preferably 1 to 100 μM, and still more preferably 1 to 50 μM.

[0092] ALK4, ALK5, and ALK7 are TGF-β1 activin receptor-like kinases (ALKs). Examples of selective inhibitors of ALK4, ALK5, and ALK7 (TGF-β receptor inhibitors) include A83-01, DMH1, GW788388, Galunisertib, K02288, LDN-193189, LDN-212854, LDN-214117, LY2109761, LY364947, ML347, SB431542, SB505124, SB525334, SD-208, RepSox, Vactosertib, and the like. When A83-01 is used as the selective inhibitor of ALK4, ALK5, and ALK7 (TGF-β inhibitor), the concentration of A83-01 is usually preferably 0.01 to 100 μM, more preferably 0.1 to 10 μM, and still more preferably 0.1 to 5 μM.

[0093] GSK3β is a serine / threonine protein kinase. Examples of GSK-3β inhibitors include CHIR99021, 1-Azakenpaullone, 2-D08, AR-A014418, AZD1080, AZD2858, Bikinin, BIO, BIO-acetoxime, CHIR-98014, IM-12, Indirubin, LY2090314, SB216763, SB415286, Tideglusib, TDZD-8, TWS119, etc. When using CHIR99021 as a GSK-3β inhibitor, the concentration of CHIR99021 is usually preferably 0.01 - 100 μM, more preferably 0.01 - 20 μM, and still more preferably 0.1 - 20 μM.

[0094] The medium may be a serum-containing medium or a serum-free medium. In the examples described below, a serum-free medium was used.

[0095] It is preferable to seed and culture hepatocytes on a gel. The gel to be used is not particularly limited, and GFR Matrigel (manufactured by Corning) etc. can be used.

[0096] Hepatocytes may be cultured on a container coated with a main component of the tissue basement membrane such as laminin.

[0097] The temperature during culture is not particularly limited, but is preferably 30 - 40°C, and more preferably 37°C.

[0098] The culture period is not particularly limited, but is preferably 4 - 15 days, and more preferably 5 - 10 days.

[0099] By the method of the present invention, hepatocyte-derived hepatic progenitor cells are proliferative and have differentiation ability, for example, alpha-fetoprotein (AFP)-negative hepatic progenitor cells having proliferation ability and bidirectional differentiation ability into hepatocytes and cholangiocytes.

[0100] The hepatic progenitor cells of the present invention have characteristics such as high (positive) expression of cell cycle genes, high (positive) expression of hepatic stem / progenitor cell-related genes, low (negative) expression of hepatic function-related genes, positive staining for HNF4A, SOX9, and CK19, but no staining for ALB and AFP (see the examples described below).

[0101] Hepatic progenitor cells can be passaged by seeding them on Matrigel or laminin coating using a medium containing a member of the FGF family and replacing it with a medium without a ROCK inhibitor at an appropriate time.

[0102] As described in the examples below, the proliferative ability of hepatic progenitor cells can be examined by calculating the population doubling time. Hepatic progenitor cells can have a high proliferative ability with a population doubling time of 14 to 36 hours.

[0103] By the method of the present invention, hepatic progenitor cells induced from hepatocytes can differentiate into hepatocytes, cholangiocytes, etc.

[0104] To differentiate hepatic progenitor cells into hepatocytes, they may be cultured in a hepatic differentiation medium. As the hepatic differentiation medium, HDM (described above in 1) can be used. A vitamin A metabolite (e.g., retinoic acid (RA)) may be added to the hepatic differentiation medium at an appropriate time for an appropriate period. In the examples described below, the medium was replaced with HDM containing RA for 8 days and then with HDM without RA for the next 7 days. The medium was replaced every 2 days. The total hepatocyte differentiation period was 15 days. This method can be appropriately modified. Hepatocytes differentiated from hepatic progenitor cells can show increased expression of functional genes (e.g., AAT, CTP2C9, CYP2C19, etc.) and increased ALB secretion. Furthermore, hepatocytes differentiated from hepatic progenitor cells can form tight junctions between their own cells and acquire ammonia excretion ability and hepatic function (ICG uptake and excretion, glycogen storage, and low-density lipoprotein uptake).

[0105] To differentiate hepatic progenitor cells into cholangiocytes, hepatic progenitor cells may be cultured in cholangiocyte differentiation medium (CDM) (see Francis et al., 2004; Sampaziotis et al., 2015; Sampaziotis et al., 2017). In the examples described below, hepatic progenitor cells mixed with GFR-Matrigel were seeded as a mount drop onto an untreated 24-well plate and incubated at 37 °C in a 5% CO2 incubator for 30 minutes. Next, the wells were filled with cholangiocyte differentiation medium (CDM). This CDM consisted of SFD containing 10 ng / mL EGF, 20 ng / mL HGF, 50 ng / mL WNT3A, 100 ng / mL R-spondin-1, 50 ng / mL FGF10, 3 μM RA, 10 μM Y-27632, and 10 μM forskolin. The medium was changed every 3 days. This method can be modified as appropriate. Cholangiocytes differentiated from hepatic progenitor cells can develop into cyst structures via ring-shaped structures. According to Q-PCR analysis, these cyst structures may be characterized by upregulated transcription of cholangiocyte-specific genes (Figs. 3-1 G and 3-2 H). These cyst cholangiocytes may be positive for CK19, F-ACTIN, and SOX9 staining and negative for AFP, ALB, and HNF4A staining. Furthermore, these cysts may have characteristics of mature bile ducts (including lumens with apical and basolateral cell membranes, microvilli, primary cilia on the apical membrane, tight junctions, and multivesicular bodies). These cholangiocytes may have cholangiocyte functions: 1) transport of rhodamine 123 into the lumen and 2) specific excretion of bile acids from the lumen.

[0106] Therefore, the present invention provides a method for producing hepatocytes, which includes inducing differentiation of hepatocyte progenitor cells obtained by a method for producing hepatocyte progenitor cells, which includes culturing hepatocytes in the presence of a member of the FGF family, into hepatocytes. The present invention also provides a method for producing bile duct cells, which includes inducing differentiation of hepatocyte progenitor cells obtained by a method for producing hepatocyte progenitor cells, which includes culturing hepatocytes in the presence of a member of the FGF family, into bile duct cells. The present invention also provides a medium containing FGF10, retinoic acid, and forskolin for inducing differentiation of the hepatocyte progenitor cells into bile duct cells. The medium of the present invention may contain other components included in the above-mentioned bile duct cell differentiation medium (CDM). Furthermore, the present invention provides an agent containing FGF10, retinoic acid, and forskolin for use in a medium used for inducing differentiation of the hepatocyte progenitor cells into bile duct cells; and an instruction manual for using the agent in the medium used for the differentiation induction; and provides a kit for the medium in the differentiation induction, which includes the above. The agent containing FGF10, retinoic acid, and forskolin may contain other components included in the above-mentioned bile duct cell differentiation medium (CDM).

[0107] By the method of the present invention, hepatocyte progenitor cells induced from hepatocytes can proliferate over a long period, for example, they can be passaged at least 20 times or more. Therefore, by the present invention, it becomes possible to mass-produce hepatocytes and bile duct cells.

[0108] 3. Improvement of the plasticity of senescent hepatocytes In the above 2, by adding an agent that brings about high histone acetylation to a medium containing a member of the FGF family, the plasticity of senescent hepatocytes can be improved, and hepatocyte progenitor cells can be induced.

[0109] The present invention provides a method for producing hepatocyte progenitor cells, which includes culturing hepatocytes in the presence of a member of the FGF family and an agent that brings about high histone acetylation.

[0110] Hepatocytes can have their senescence suppressed and plasticity induced by agents that bring about high histone acetylation. As used herein, the plasticity of hepatocytes refers to the ability to convert from hepatocytes into proliferative hepatic progenitor cells with the ability to differentiate into both hepatocytes and cholangiocytes. By improving the plasticity of hepatocytes, the responsiveness of hepatocytes to proliferation induction increases.

[0111] Therefore, the present invention provides a method for suppressing the senescence of hepatocytes with an agent that brings about high histone acetylation. From another perspective, the present invention provides an anti-aging agent for hepatocytes that contains an agent that brings about high histone acetylation as an active ingredient. The anti-aging agent of the present invention can be applied both in vitro (cultured cells) and in vivo (cells in the living body).

[0112] The senescence of hepatocytes can be confirmed by a decrease in cell proliferation, a decrease in the function as hepatocytes, upregulation of senescence-related genes, expression of senescence-related markers, etc. In addition, it can also be confirmed by an increase in cell volume, an increase in the inflammatory response, DNA damage, epigenetic changes, shortening of telomere length, a decrease in mitochondrial function, metabolic abnormalities, low responsiveness to growth factors, etc.

[0113] Cell proliferation can be examined by calculating the population doubling time of cells.

[0114] The function of hepatocytes and the method for measuring the same were described above in 1.

[0115] The function of senescence-related genes and the method for measuring the same were also described above in 1.

[0116] Examples of senescence-related markers can include the level of intracellular reactive oxygen species and the level of cell senescence-related β-galactosidase, and the methods for measuring these were described above in 1.

[0117] The methods for measuring increased cell volume, increased inflammatory response, DNA damage, epigenetic changes, shortening of telomere length, decreased mitochondrial function, metabolic abnormalities, and low responsiveness to growth factors were also described in 1.

[0118] Suppression of hepatocyte aging can be confirmed by measuring the above markers and events.

[0119] The present invention also provides a method for increasing the plasticity of hepatocytes with an agent that brings about histone hyperacetylation. From another perspective, the present invention provides an agent for increasing the plasticity of hepatocytes, which contains an agent that brings about histone hyperacetylation as an active ingredient. By increasing plasticity, hepatic progenitor cells can be induced from aged hepatocytes derived from pluripotent stem cells or hepatocytes derived from the elderly. Also, by increasing plasticity, the responsiveness to proliferation induction of hepatocytes can be enhanced. The agent of the present invention may be a regeneration-inducing agent that brings about improvement in the regenerative capacity of aged cells. The agent of the present invention can promote liver regeneration in the elderly. Also, the agent of the present invention can lead to the success of transplantation therapy using grafts and cells derived from elderly donors. The agent of the present invention can be applied both in vitro (cultured cells) and in vivo (cells in the living body).

[0120] The agent that brings about histone hyperacetylation may be used in combination with a member of the FGF family. Examples of members of the FGF family include FGF2, FGF1, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, or combinations thereof, and FGF2 is preferred.

[0121] Examples of agents that bring about high histone acetylation include histone deacetylase inhibitors. Examples of histone deacetylase inhibitors include Trichostatin A, Tacedinaline (CI-994), M344, ITSA-1, Sodium valproate, Sodium 4-phenylbutuyrate, Sodium Butyrate (NaB), valproic acid (VPA), Abexinostat (PCI-24781), Belinostat (PXD101), Citarinostat (ACY-241), Dacinostat (LAQ824), Depudecin, Domatinostat (4SC-202), Droxinostat, Entinostat (MS-275, SNDX-275), Fimepinostat (CUDC-907), Givinostat (ITF2357), Mocetinostat (MGCD0103), Nexturastat A, Panobinostat (LBH-589, NVP-LBH589), Pracinostat (SB939), Quisinostat (JNJ-26481585) 2HCl, Resminostat, Ricolinostat (ACY-1215), Tucidinostat (Chidamide), Vorinostat (SAHA), ACY-738, Apicidin, AR-42, BG45, BML-210, BRD73954, CAY10603, CUDC-101, Curcumin, Depudecin, H1388, HC Toxin, HPOB, LMK-235, MC1568, Oxamflatin, (-)-Parthenolide, PCI-34051, RG2833 (RGFP109), RGFP966, Romidepsin (FK228,Examples thereof include depsipeptide, Santacruzamate A (CAY10683), Scriptaid, SKLB-23bb, Splitomicin, Suberoyl bis-hydroxamic acid, Tasquinimod, TH34, Tinostamustine (EDO-S101), TMP195, TMP269, Tubacin, Tubastatin A, and combinations thereof.,

[0122] The hepatocytes that are targeted for suppressing aging or increasing plasticity by agents that bring about high histone acetylation are preferably, but not limited to, aged hepatocytes. The hepatocytes may be hepatocytes differentiated from pluripotent stem cells, cells obtained by subculturing hepatocytes differentiated from pluripotent stem cells, primary cultured hepatocytes isolated from biological tissues, cells obtained by subculturing primary cultured hepatocytes isolated from biological tissues, or any combination thereof.,

[0123] When the hepatocytes are hepatocytes differentiated from pluripotent stem cells, the "aged hepatocytes" are, for example, hepatocytes that have undergone subculture for 50 days or more in total after induction of differentiation from pluripotent stem cells (e.g., iPSCs) (22 days later).

[0124] When the hepatocytes are cells obtained by subculturing hepatocytes differentiated from pluripotent stem cells, the "aged hepatocytes" are, for example, cells obtained by subculturing hepatocytes that have undergone subculture for 25 days or more from pluripotent stem cells (e.g., iPSCs) one or more times.

[0125] When the hepatocytes are primary cultured cells isolated from liver tissue, the "aged hepatocytes" are, for example, in the case of humans, hepatocytes isolated from the tissues of humans aged 65 years or older.

[0126] When the hepatocytes are cells obtained by subculturing primary cultured hepatocytes isolated from biological tissues, the "aged hepatocytes" are, for example, in the case of humans, hepatocytes cultured for 20 days or more after isolation, etc.

[0127] The hepatocytes are preferably of human origin, but are not limited to humans and may be derived from mammals such as mice, rats, guinea pigs, hamsters, rabbits, pigs, cats, dogs, sheep, cows, horses, goats, and monkeys.

[0128] By culturing hepatocytes in a medium containing an agent that brings about high histone acetylation, aging can be suppressed and plasticity can be increased.

[0129] As the medium, it is preferable to use the medium described above in 2 (the medium used for the induction (reprogramming) of hepatocytes into hepatic progenitor cells). That is, as the basal medium, an SFD medium (described in WO2016093222), (DMEM / F12, DMEM, IMDM, RPMI1640, Williams' Medium E), etc. can be used, and it is preferable to add a member of the FGF family to the basal medium.

[0130] The concentration of the member of the FGF family in the medium may be adjusted as appropriate. For example, when using FGF2 as the member of the FGF family, the concentration of FGF2 is usually preferably 0.01 to 1000 ng / mL, more preferably 0.1 to 100 ng / mL, and even more preferably 1 to 50 ng / mL.

[0131] Regarding other components that may be added to the medium and their concentrations, they are the same as those described above in 2.

[0132] The medium may be a serum-containing medium or a serum-free medium, but in the examples described later, a serum-free medium was used.

[0133] The hepatocytes are preferably seeded on a gel and cultured. The gel to be used is not particularly limited, and GFR Matrigel (manufactured by Corning) etc. can be used.

[0134] The hepatocytes may be cultured on a container coated with a main component of the tissue basement membrane such as laminin.

[0135] The temperature during cultivation is not particularly limited, but it is preferably 30 to 40 °C, more preferably 37 °C.

[0136] The cultivation period is not particularly limited, but it is preferably 4 to 15 days, more preferably 5 to 10 days.

[0137] 4. Applications According to the present invention, it becomes possible to mass-produce hepatocytes and bile duct cells from pluripotent stem cells (for example, iPS cells, etc.).

[0138] The human hepatocytes and the like created by the method of the present invention can be applied to industrial uses such as in vitro drug metabolism tests, hepatotoxicity tests, hepatitis virus infection tests, etc.

[0139] The human hepatocytes and the like created by the method of the present invention include the following. 1. Hepatocytes produced by a method for producing hepatocytes, which includes differentiating endoderm cells into hepatocytes in the presence of a member of the FGF family, HGF, a member of the IL6 family, and dexamethasone. 2. Alpha-fetoprotein (AFP)-negative hepatic progenitor cells produced by a method for producing hepatic progenitor cells, which includes culturing hepatocytes in the presence of a member of the FGF family, and having the ability to proliferate and differentiate bidirectionally into hepatocytes and bile duct epithelial cells. 3. Cells (for example, hepatocytes, bile duct cells) differentiated from the hepatic progenitor cells of 2.

[0140] The human hepatocytes and the like created by the method of the present invention can be used in regenerative medicine as the main component of a transplantation composition. The transplantation site of the hepatocytes can be any site as long as transplantation is possible, and examples include intracranial, mesenteric, liver, spleen, kidney, subcapsular kidney, portal vein, etc. For each transplantation, the number of hepatocytes is per 1 cm of the transplantation site 2It is preferably in the range of 100,000 to 100,000,000, more preferably 1,000,000 to 50,000,000, and even more preferably 1,000,000 to 10,000,000. For transplantation, EGF, HGF, ROCK inhibitor, TGF-β receptor inhibitor, GSK-3β inhibitor, etc. may be used. The transplantation composition may contain an agent (described above) that increases the plasticity of hepatocytes.

[0141] In addition, an artificial liver can also be manufactured using human hepatocytes and the like created by the method of the present invention.

[0142] Furthermore, human hepatocytes and the like created by the method of the present invention can be transplanted into non-human animals to produce chimeric animals. The non-human animal (e.g., mouse) into which the cells are transplanted can mimic the physiological functions of the biological species (e.g., human) from which the transplanted cells are derived. Using this animal, drug metabolism tests and safety tests of drug compounds can be conducted. The non-human animal is preferably in a state of liver failure. Liver failure can be induced by administering ganciclovir. When liver progenitor cells are transplanted into non-human animals, the transplanted liver progenitor cells can proliferate and / or differentiate in the non-human animals. Transplanting the hepatocytes produced by the method of the present invention promotes liver regeneration in non-human animals. Examples of non-human animals include mice and rats. The transplantation site and the number of hepatocytes used for transplantation may be the same as in the case of transplantation into humans.

[0143] Furthermore, according to the present invention, it is possible to promote liver regeneration using an agent that brings about histone hyperacetylation. The present invention provides a liver regeneration promoter containing an agent that brings about histone hyperacetylation as an active ingredient. The present invention provides a method for promoting liver regeneration, which includes administering an agent that brings about histone hyperacetylation to a subject in a pharmaceutically effective amount. The agent that brings about histone hyperacetylation has been described above. Agents that bring about high histone acetylation can promote the regeneration of the liver that has suffered liver injury. Liver injury can be caused by viral hepatitis (caused by infection with hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus), alcoholic hepatitis, autoimmune hepatitis, primary cirrhosis, drug-induced hepatitis, fatty liver (alcoholic, non-alcoholic), liver resection by surgery, liver injury by trauma, aging, liver fibrosis, liver injury associated with obesity, etc. Agents that bring about high histone acetylation can be used as medicines for the treatment and / or prevention of these diseases and disorders. Agents that bring about high histone acetylation may be in the form of salts or solvates. A pharmaceutical preparation formulated by a conventional method using an agent that brings about high histone acetylation (for example, injection, capsule, tablet, powder, granule, etc.) can be administered to a subject (human or animal). For example, when the agent that brings about high histone acetylation is NaB, in terms of the amount of the active ingredient, it can be administered orally or parenterally (for example, intranasally, rectally, transdermally, subcutaneously, intravenously, intramuscularly, etc.) once or in several divided doses at a dose of about 10 to 100,000 mg / kg (body weight) per day, preferably about 100 to 10,000 mg / kg (body weight) per day. However, the dose and the number of administrations can be appropriately changed depending on symptoms, age, administration method, etc. When using an agent that brings about high histone acetylation other than NaB, it is advisable to use it at a dose that exhibits an effect equivalent to that of NaB at the above dose. When formulating into an injection, a carrier such as distilled water or physiological saline may be used. When formulating into capsules, tablets, powders, or granules, excipients such as starch, lactose, sucrose, calcium carbonate, binder solutions of starch, gum arabic, gelatin, sodium alginate, carboxymethyl cellulose, hydroxypropyl cellulose, lubricants such as magnesium stearate and talc, disintegrants such as starch, agar, crystalline cellulose, calcium carbonate, sodium bicarbonate, and sodium alginate may be used. The content rate of the active ingredient in the preparation can be varied between 1 and 99% by weight. For example, when taking forms such as tablets, capsules, granules, and powders, it is preferable to contain 5 to 80% by weight of the active ingredient, and in the case of an injection, it is preferable to contain 1 to 10% by weight of the active ingredient.

Example

[0144] Hereinafter, the present invention will be described in more detail by way of examples.

[0145] 〔Example 1〕 Low acetylation of histones impairs the plasticity of human hepatocytes with aging.

[0146] Our results will make liver transplantation from the elderly more useful clinically: generally, liver tissues and hepatocytes from the elderly have low regenerative capacity. We found how to easily obtain hepatocytes from human induced pluripotent stem cells. These hepatocytes reproduce liver aging in culture dishes, and we found that liver plasticity (responsiveness to proliferation induction) is impaired by histone hypoacetylation associated with aging. Promotion of histone acetylation improved the plasticity of aged hepatocytes and increased their regenerative capacity in a liver injury model.

[0147] Abstract Hepatocyte plasticity plays a crucial role in liver regeneration and declines with aging. However, the underlying mechanism of this decline is unclear. Here, to elucidate the underlying mechanism, we devised a research strategy using hepatocytes derived from human induced pluripotent stem cells (hiPSC-Hep), accumulated changes in aging-related characteristics and expression information of aging-related gene sets during long-term subculture of hiPSC-Hep. Furthermore, we identified that human hepatocyte plasticity is regulated by FGF2-MAPK-EZH2 and gradually declines with aging. Notably, the impaired plasticity of aged hepatocytes was strongly correlated with histone hypoacetylation. Selective inhibition of histone deacetylases significantly improved the plasticity of aged hiPSC-Hep and primary human hepatocytes. And this effect increased the repopulation ability of aged primary human hepatocytes in a liver injury model. Therefore, histone hypoacetylation associated with aging impairs hepatocyte plasticity, and histone acetylation could be a therapeutic target for improving liver regenerative capacity in the elderly.

[0148] Introduction Liver regeneration is a unique phenomenon that regenerates new tissues in the liver to replace the lost parts (dead cell masses, tissues). Due to this phenomenon, the liver seems to be an organ that does not age (Timchenko, 2009). However, clinical reports have shown that aging is a major risk factor for liver diseases including non-alcoholic fatty liver disease, alcoholic liver disease, and hepatitis C (Kim et al., 2015), and liver transplantation from the elderly results in worse survival rates and generally worse outcomes compared to transplantation from young donors (Durand et al., 2019; Germani et al., 2012). These facts indicate that aging can weaken the regenerative ability of the liver. In fact, the decline in liver regeneration associated with aging was discovered half a century ago when researchers noticed that the proliferative response was significantly reduced in the livers of aged rats after partial hepatectomy (Bucher et al., 1964). This phenomenon has also been confirmed in other rodent models (Fry et al., 1984; Lakova et al., 2003), and several changes associated with aging that can be linked to the deterioration of regeneration (e.g., metabolic abnormalities, epigenetic changes, low responsiveness to growth factors, and shortening of telomere length) have been found (Aikata et al., 2000; Sato et al., 2017; Sawada, 1989; Timchenko, 2009). However, how aging contributes to the deterioration of liver regeneration and how to improve this regenerative ability in the elderly, especially in humans, remains unclear.

[0149] Hepatocytes account for more than 70% of liver mass. And accumulating evidence suggests that hepatocyte plasticity plays a crucial role in maintaining liver regenerative capacity (Kopp et al.,; Li et al., 2016). Lineage tracing experiments in mice have shown that in response to liver injury, hepatocytes can convert into proliferative progenitor-like cells and replace lost stem cells and cholangiocytes (Tarlow et al., 2014; Yanger et al., 2014; Yanger et al., 2013). Such progenitor-like cells have also been detected in cirrhotic human livers (Deng et a., 2018). Therefore, studies examining the endogenous relationship between aging and hepatocyte plasticity would help to understand the role of aging in human liver regeneration. Due to the limitations of human-based tracing experiments, we wondered whether hepatocyte plasticity in humans could actually be modeled in a culture dish. Recently, several groups have reported that primary human hepatocytes (PHH) can be induced into a proliferative state with the ability to re-expand (Fu et al., 2019; Kim et al., 2019; Zhang et al., 2018), but these proliferative PHH have only limited bipotential differentiation. Furthermore, currently available PHH are only derived from cadaver or patient liver tissues with liver diseases with complex and unreliable exogenous factors. Such exogenous factors make it difficult to use the above PHH to discover the beneficial relationship between aging and hepatocyte plasticity.

[0150] To elucidate these endogenous relationships, an ideal set of experimental samples of hepatocytes needs to have the same origin. That is, they need to be collected from the same human individual at different ages. However, it is difficult to obtain such hepatocytes from the same donor. Over the past decade, there have been extensive developments in the induction of differentiation of human induced pluripotent stem cells (hiPSCs) for applications in regenerative medicine and disease model generation (Stadtfeld and Hochedlinger, 2010; Studer et al., 2015), and several labs have adopted methods that enable the use of hiPSCs to model neurodegenerative diseases associated with aging (Miller et al., 2013; Vera et al., 2016). Unfortunately, hiPSC-derived hepatocytes (hiPSC-Hep) that can effectively recapitulate the biological aging process have not been reported to date. This is, in part, due to the extremely rapid degeneration of hiPSC-Hep (Nie et al., 2018). In this paper, we established a simple and convenient method for creating hiPSC-Hep that can maintain liver function over the long term while gradually accumulating aging markers and characteristics. We noticed that the plasticity of hiPSC-Hep is significantly reduced by histone hypoacetylation associated with aging. Furthermore, treatment with histone deacetylase inhibitors (HDACi) clearly increased the plasticity of aged hiPSC-Hep and primary human hepatocytes (PHH), and this effect significantly improved the regenerative ability of aged PHH in a mouse model of liver injury.

[0151] Results Simple creation of hiPSC-Hep with improved function To generate a liver aging model using HiPSC-Hep, we developed a two-step method for HiPSC-Hep generation by relying on our experience in hiPSC-derived liver organoid formation (Nie et al., 2018) (Figures 1-1A and 1-2A). In the first step, hiPSCs were efficiently differentiated into a highly pure definitive endoderm cell population (Figures 1-1B and 1-2B). In the second step, we induced definitive endoderm cells to differentiate directly into hepatocytes using a hepatocyte differentiation medium (HDM) containing screened liver growth and differentiation factors, and found that these lineages matured into hepatocytes with ALB expression (Figures 1-2C and 1-2D).

[0152] During the differentiation of endodermal cells into hepatocytes, these lineages consistently showed maturation with changes in traits characteristic of hepatocytes and an increase in albumin (ALB) secretion (Si-Tayeb et al., 2010) (Figures 1-1 A-C and 1-2 E). Five days after the differentiation of endodermal cells, these lineages showed a rapid increase in the expression of early hepatic-fate genes and a rapid decrease in the expression of endoderm-related genes (Figure 1-2B). Immunostaining revealed that these cells were in a germ cell state with positive expression of AFP, CK19, HNF4A, and KI67 and modest secretion of ALB (Figures 1-1 C and 1-2 E). After further incubation for 10 days, these lineages matured into ALB-expressing cells (Figures 1-1 B and 1-1 C), showing the expression of E-cadherin (E-CAD), a tight junction constituent protein, and zona occludens 1 (ZO-1; Figure 1-2 F); as well as liver functions such as the uptake and excretion of ICG, glycogen storage, and the uptake of low-density lipoprotein (Figure 1-2 G). Compared with hiPSC-Hep (K-Hep and ST-Hep) obtained by conventional protocols (Kajiwara et al, 2012; Si-Tayeb et al., 2010), these newly created hiPSC-Hep (N-Hep) are characterized by enhanced expression of liver-specific genes as well as ALB secretion (Figures 1-2 H and 1-2 I). Therefore, this study provides a simple and convenient protocol for creating hiPSC-Hep with improved liver function.

[0153] Spontaneous reproduction of the liver aging process In contrast to the rapid degeneration of previously reported hiPSC-Hep (Nie et al., 2018), N-Hep maintained cell morphology and liver function over a long period (Figure 1-1 D and 1-2 J). That is, until day 72 of cell culture, we observed the formation of multinucleated cells accompanied by a decrease in ALB secretion (Figure 1-1 D and 1-2 J). On day 82 (D82), N-Hep lost its hepatocyte morphology and showed a high proportion of apoptotic cells (Figure 1-2 K). On the other hand, we noticed an increase in the cell volume of N-Hep during culture. This is a phenomenon frequently observed during cell senescence (Neurohr et al., 2019) (Figure 1-2 L). To test whether N-Hep was undergoing the process of senescence, we performed transcriptome analysis to compare D22-Hep (hepatocytes generated 22 days after hiPSC), D52-Hep, and D72-Hep, and found that senescence-related genes were abundantly expressed in D72-Hep (Figure 1-1 E). Gene ontology analysis also showed that liver changes associated with senescence (Kim et al., 2015) (including increased inflammatory response and DNA damage) were also enhanced in D72-Hep (Figure 1-2 M). Furthermore, the levels of intracellular reactive oxygen species (ROS) and senescence-associated β-galactosidase (SA-β-gal) (which are two of the cellular characteristics of senescence) gradually increased from D22 to D72 (Figure 1-1 F-H). These results indicated the spontaneous senescence of N-Hep during long-term culture. Next, we considered whether this N-Hep aging process could recapitulate the in vivo hepatocyte aging process. By comparing the transcriptional differences among D22-Hep, D72-Hep, young PHH (2 months, 2M-PHH), and old PHH (78 years, 78Y-PHH), principal component analysis showed that the gene signature of D22-Hep was similar to that of 2M-PHH, and the gene signature of D72-Hep was very close to that of 78Y-PHH (Figure 1-3 A). Furthermore, KEGG pathway analysis revealed that 35 signaling pathways whose expression increased in the in vitro aging process also increased in the in vivo aging process, and 35 signaling pathways whose expression decreased in the in vitro aging process also decreased in the in vivo aging process (Figure 1-3 B and C). These results suggest that the N-Hep aging process may represent (substitute for) the in vivo hepatocyte aging process.

[0154] Essential function of FGF2 in inducing plasticity of human hepatocytes Next, we tested whether N-Hep has the same plasticity as hepatocytes in vivo. N-Hep can be induced to proliferate while having bipotential differentiation in vivo (Figure 2-1A). To induce hepatocyte proliferation, we cultured young N-Hep (D22-Hep) in a basal medium (BM+SMs) containing a previously described small molecule cocktail (which can induce the proliferation of rodent hepatocytes) (Katsuda et al., 2017). After 6 days, the reprogrammed cells did not acquire the continuous proliferative ability seen in reprogrammed rodent hepatocytes (Katsuda et al., 2017) (Figure 2-2A), but we observed cell proliferation (Figure 2-1B and C). These data indicated that the inherent differences between rodent and human hepatocytes might have a significant impact on the results of plasticity induction. We had previously confirmed that FGF2 is a crucial factor promoting hepatocyte differentiation and maturation (Figure 2-2C and D), and is also known as an important cytokine in liver homeostasis and regeneration (Steiling et al., 2003). We found that the addition of FGF2 to the medium significantly improved the induced proliferative ability (Figure 2-1B, 2C, and 2-2B). Notably, the cells reprogrammed by FGF2 acquired the ability to continuously proliferate when FGF2 was added in the above-mentioned medium called BM+SMs. We named this FGF2-added medium the reprogramming medium (RM) (Figure 2-2A). Quantitative PCR (Q-PCR) analysis revealed that these reprogrammed cells were characterized by enhanced transcription of cell cycle genes (Figure 2-1D). Immunofluorescence analysis also showed that the proliferative cells still belonged to the hepatocyte lineage because they were positive for both KI67 and HNF4A (Figure 2-1E). Therefore, we named these reprogrammed cells (hepatic progenitor cells) proliferative hepatocytes (pHc). Time-lapse imaging suggested that, similar to other hiPSC-Hep, pHc could be obtained from a single hepatocyte (Figure 2-2C and D).Compared with D22-Hep, D22-pHC showed high transcription of liver-stem / progenitor cell-related genes (Figure 2-1 F) and down-regulation of liver function-related genes (Figure 2-1 G). Furthermore, D22-pHC showed positive staining for HNF4A, SOX9, and CK19, but not for ALB and AFP (Figure 2-1 H, 2-2 E, and 2-2 F).

[0155] To determine how FGF2 induces hepatocyte proliferation, we incubated the cells with PD0325901 and LY294002 to block the MAPK and PI3K signaling pathways, respectively. These are signaling cascades downstream of FGF2 and are involved in cell proliferation (Goetz and Mohammadi, 2013). Notably, PD0325901, but not LY294002, dose-dependently suppressed FGF2-mediated cell proliferation (Figure 2-1 I). By combining transcriptome analysis and protein-protein interaction network analysis (Jensen et al., 2009), we found that EZH2 was present at the central node of all upregulated genes, linking the gene cluster of the cell cycle and the gene cluster of epithelial-mesenchymal transition (Figure 2-2 G). Furthermore, cells treated with RM showed a distinct upregulation of EZH2 transcription suppressed by PD0325901 (Figures 2-1 J and 2-2 H). By disabling EZH2 function using the specific inhibitor 3-deazaneplanocin A hydrochloride (DZNep), we detected a significant attenuation of induced cell proliferation with only a slight change in the EZH2 transcription level (Figures 2-2 I and J). This indicates that EZH2 activated by the FGF2-MAPK axis is strongly involved in the induced proliferation of human hepatocytes. Furthermore, loss-of-function and gain-of-function experiments suggested that the FGF2-EZH2 axis is essential for maintaining the proliferative capacity of pHC (population doubling time 19.86 ± 0.70 hr) (Figures 2-2 K and L). Long-term proliferation showed that these D22-pHC were able to be passaged at least 20 times without obvious changes in their morphology, growth rate, and karyotype (Figures 2-2 M and N). Thus, we successfully induced the cell proliferation of N-Hep controlled by the FGF2-MAPK-EZH2 axis (Figure 2-1 K).

[0156] Differentiation ability of D22-pHC Next, we evaluated the bipotential differentiation of pHCs. To induce hepatocyte differentiation, we cultured D22-pHCs in freshly developed HDM. After 15 days, the differentiated cells showed limited ALB production and an atypical morphology (Figs. 3-2 A and B). To enhance the differentiation process, we modified HDM by adding retinoic acid (RA; Fig. 3-1 A), a vitamin A metabolite that mediates liver development and regeneration (Negishi et al., 2010), and ultimately obtained a significant improvement in ALB production and typical hepatocyte morphology (Figs. 3-2 A-C). Hepatocytes derived from D22-pHCs (D22-pHC-Hep) showed a clear increase in the expression of functional genes as well as ALB secretion. They were equivalent to those of the original D22-Hep (Figs. 3-1 B, C and 3-2 D). Furthermore, D22-pHC-Hep formed tight junctions among themselves and acquired ammonia excretion ability and liver functions (ICG uptake and excretion, glycogen storage, and low-density lipoprotein uptake) (Figs. 3-1 D, E and 3-2 E).

[0157] To induce differentiation into cholangiocytes, we adopted methods of cholangiogenesis and differentiation (Francis et al., 2004; Sampaziotis et al., 2015; Sampaziotis et al., 2017) and established a differentiation method into cholangiocytes shown in Fig. 3-1 F. After 2 days of incubation, 1 D22-pHC grew into a ring-shaped structure and then gradually developed into a cyst structure within 10 days (Figs. 3-1 F, 3-2 F, and G). According to Q-PCR analysis, this cyst structure was characterized by upregulated transcription of cholangiocyte characteristic genes (Figs. 3-1 G and 3-2 H). Next, the cholangiocyte fate of these cysts was confirmed by positive results of CK19, F-ACTIN, and SOX9 staining and negative results of AFP, ALB, and HNF4A staining (Fig. 3-1 H). Furthermore, transmission electron microscopy observation showed that these cysts had characteristics of mature bile ducts (including lumens with apical and basolateral cell membranes, microvilli, primary cilia on the apical membrane, tight junctions, and multivesicular bodies) (Figs. 3-1 I and 3-2 I). Also, these cholangiocytes had bile duct functions: 1) transport of rhodamine 123 into the lumen; this function was blocked by verapamil, an inhibitor of the multidrug resistance protein 1 transporter (Figs. 3-1 J and K), and 2) specific excretion of bile acids from the lumen (Fig. 3-2 J). Therefore, these data indicated that D22-Hep (young N-Hep) had sufficient plasticity to enable induction of a bipotent proliferative state.

[0158] Decrease in hepatocyte plasticity during the aging process To investigate the intrinsic relationship between aging and hepatocyte plasticity, we induced the plasticity of N-Hep during the aging process using newly developed RM (Figure 4-1A). After 6 days, we observed a sharp decline in the cell proliferation of induced pHC during the progression of aging from D22-Hep to D82-Hep (Figure 4-1B and C). Compared with the proliferation of pHC highly induced in D22-Hep, only a few proliferative hepatocyte (D52-pHC) colonies were detected in reprogrammed D52-Hep using the same procedure (Figure 4-1B and 4-2A), and almost no proliferative cells / colonies were induced in D62-, D72- and D82-Hep (Figure 4-1B and 4-2B). Next, we performed transcriptome analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to examine the changes in N-Hep during aging. Surprisingly, aging N-Hep was characterized by the attenuation of metabolic pathways of fatty acids, glucose and amino acids (Figure 4-2C and D), showing a significant downregulation of acetyl coenzyme A (CoA), a central intermediate of these metabolic pathways (Figure 4-1D and E). Acetyl-CoA is also a key node that links metabolomics to epigenetics as an acetyl source for histone acetylation (Sebastian and Mostoslavsky, 2017). Therefore, we quantitatively analyzed the changes in histone acetylation due to aging. Notably, aging N-Hep showed a tendency for a decrease in the acetylation of H3 histone at each Lys residue of H3K9, H3K18 and H3K27 (Figure 4-1F and 4-2E-G). On the other hand, these three types of histone modifications by acetylation were clearly correlated with the proliferative ability of their induced pHC (Figure 4-1G). These findings suggest that metabolic defects associated with aging are linked to histone hypoacetylation, which may impair hepatocyte plasticity (Figure 4-2H).

[0159] Upregulation of the plasticity of aged N-Hep by HDACi Next, to investigate whether histone hypoacetylation causes a decline in hepatocyte plasticity associated with aging, we stimulated D52-Hep during the induction process with sodium butyrate (NaB) or valproic acid (VPA), both of which are histone deacetylase inhibitors (HDACi), and found that each HDACi significantly promoted the proliferation of pHC induced from D52-Hep (Figs. 4-1H, I, and 4-3A). Immunostaining showed that stimulation with one HDACi significantly increased the percentage of + KI67 + cells (%) (Fig. 4-1J). On the other hand, such promotion was not detected with stimulation using other types of epigenetic regulators or inhibitors, such as tranylcypromine (Trany, a histone demethylase inhibitor), RG108 (a DNA methyltransferase inhibitor), and BIX01294 (BIX, a histone methyltransferase inhibitor) (Figs. 4-1H and I). Furthermore, while D52-pHC (pHC derived from D52-Hep) continuously proliferated and acquired the ability to differentiate into hepatocytes and cholangiocytes, it was clear that stimulation with HDACi improved the plasticity of D52-Hep (Figs. 4-3B-F).

[0160] In one of the above experiments, we found that EZH2 transcription activated by FGF2 plays a decisive role in the proliferation of proliferative hepatocytes (pHc) (Figures 2-1 I, J and 2-2 G-J). On the other hand, the transcription of activated EZH2 was much lower in reprogrammed D52-Hep (D52-pHC) than in reprogrammed D22-Hep (D22-pHC) (Figure 4-3 G). On the other hand, stimulation with HDACi enhanced EZH2 transcription and promoted the proliferation of D52-pHC induced from D52-Hep (Figures 4-1 K and 4-3 H), and D52-pHC showed greater histone acetylation compared to D52-Hep (Figure 4-3 I). These data suggested that histone hypoacetylation may be involved in the regulation of EZH2 transcription. To study the EZH2 promoter, a comparative analysis was performed using a comprehensive web tool (https: / / www.genecards.org) and the ENCODE dataset (Consortium, 2012), and we found that the two EZH2 enhancers with the highest scores (GH07J148882 and GH07J148940) are binding sites for acetylated histones (Figure 4-3 J). Furthermore, chromatin immunoprecipitation and PCR analysis (ChIP-PCR) based on anti-H3K9ac, anti-H3K18ac and anti-H3K27ac antibodies showed that the binding of acetylated histones to GH07J148882 (which is close to the transcription start site) in D52-Hep was significantly decreased (Figures 4-1 L and 4-3 J). These data implied that histone hypoacetylation leads to insufficient activation of EZH2 transcription and that this problem hinders the induction of plasticity in aged hepatocytes. Furthermore, stimulation with HDACi enhanced the proliferation of each pHc induced from D62-Hep, D72-Hep and D82-Hep (Figure 4-1 M). These results illustrated that inactivation of EZH2 transcription mediated by histone hypoacetylation impairs the plasticity of aged hepatocytes and that this plasticity can be effectively restored by HDACi (Figure 4-3 K).

[0161] Plasticity induction of PHH Next, we tested whether the mechanism of action revealed in N-Hep was consistent with that in PHH. By analyzing histone acetylation in PHH from donors aged 2 months (2M-PHH), 39 years (39Y-PHH), and 78 years (78Y-PHH), we noticed that the amounts of H3K9ac, H3K18ac, and H3K27ac also decreased with aging (Figure 5-1 A). Compared with N-Hep, the acetylation level of 2M-PHH was close to that of D52-Hep. And the acetylation levels of 39Y-PHH and 78Y-PHH were lower than that of D62-Hep (Figure 5-1 B). Indeed, the hypoacetylation associated with aging in pHH was also accompanied by a decline in plasticity induction. On the other hand, stimulation with NaB (HDACi) significantly increased the plasticity induction efficiency (Figure 5-1 C, 5D, and 5-2 A). According to the transcriptome comparison with the original PHH, in PHH-derived pHC (PHH-pHC), an increase in the expression levels of cell cycle-related and stem / progenitor cell-related characteristic genes and a decrease in the expression levels of liver function characteristic genes were shown (Figure 5-1 E, F, and 5-2 B). Immunofluorescence analysis showed that 49.8% (2M-pHC), 32.9% (39Y-pHC), and 12.2% (78Y-pHC) of PHH-pHC were positive for the expression of both HNF4A and KI67, and most of the PHH-pHC expressed HNF4A, SOX9, and CK19 (Figure 5-1 G). Furthermore, PHH-pHC showed proliferative ability on a culture dish coated with laminin 511 (Figure 5-2 C) and showed the ability to differentiate into hepatocytes and cholangiocytes (Figure 5-2 D-J). These results indicated that, similar to the aging process of N-Hep, the aging process of PHH was also related to histone hypoacetylation that impairs hepatocyte plasticity. These findings confirmed the usefulness of N-Hep as a cell source for studies on human liver aging.

[0162] In vivo regrowth of pHC after plasticity induction of aged PHH Next, we evaluated the repopulation ability of aged PHH-pHC in the liver using TK-NOG mice after ganciclovir-induced liver failure (Hasegawa et al., 2011). Four weeks after transplantation, mice transplanted with 78Y-PHH or 78Y-pHC were positive in assays for human ALB (hALB), human A1AT (hA1AT), and human ferritin (hFerritin), but almost none were positive for human AFP (hAFP; Figures 6-1 A-C and 6-2 A). In contrast to the large gap in hALB, hA1AT, and hFerritin between grafts of 2M-PHH and 78Y-PHH, there was little gap between 78Y-pHC grafts and 2M-pHC grafts, showing production much higher than the corresponding levels in 78Y-PHH and comparable to that of young primary hepatocytes (Figures 6-1 A-C). Also, detailed confirmation of gene expression levels for hALB was performed by Q-PCR (Figure 6-2 B). Furthermore, immunofluorescence analysis suggested that 78Y-pHC grafts yielded more hALB+ grafts as well as clusters (number of cells per cluster >5) than 78Y-PHH grafts (Figures 6-1 D, E and 6-2 C). Notably, more than 50% of hALB+ grafts in the 78Y-pHC graft group expressed CK19. And this percentage was much higher than that in the 78Y-PHH graft group (Figures 6-1 D and F). Furthermore, we detected several ALB+KI67+ clusters in 78Y-pHC grafts, but none in 78Y-PHH grafts (Figure 6-2 D).

[0163] Furthermore, hALB production in 78Y-pHC grafts was much higher than that in 78Y-PHH grafts (Figure 6-1G). Since a significant decrease in hepatic function gene expression was observed during plasticity induction (Figure 5-2B and 6-2E), we examined whether the transplanted PHH-pHCs matured in vivo. At 12 weeks after transplantation, according to Q-PCR using human gene-specific primers, the transplanted PHH-pHCs showed a significant recovery in the expression of hepatic function genes (especially CYP450 enzymes) and downregulation of CK19 (Figure 6-2E). In immunofluorescence analysis, PHH-pHC grafts showed positive results for the expression of hALB, CYP3A4, hNuMA, hA1AT, and hCK8 / 18, and little expression of hCK19 and hAFP (Figure 6-1H and 6-2F). The positive result of ZO-1 staining indicated that PHH-pHCs had matured into hepatic tissue featuring bile canaliculus formation and bipolar hepatocyte arrangement (Figure 6-1H). These results indicated that plasticity induction performed using aged PHH significantly enhanced the regenerative capacity and matured to exhibit sufficient hepatic function after transplantation.

[0164] HDAC inhibitors improve the induction of pHCs in the aged model. Finally, we investigated whether HDAC inhibitors could improve the induction of hepatic progenitor cells in an aged model. To induce hepatic progenitor cells in mice, young and aged mice were fed a choline-deficient, ethionine-supplemented (CDE) diet for 21 days. Detection of GOT and GPT indicated that chronic liver injury occurred in both young and aged mice, and the livers of the mouse model exhibited characteristics of non-alcoholic fatty liver disease (Figure 6-3 A-C). Furthermore, the survival rate of the aged model was only 60%, and it was revealed that the induction of Ki67-positive cells and Epcam-positive cells in the aged model was significantly lower than that in the young model, suggesting that the induction of hepatic progenitor cells in the mouse liver was also impaired with aging (Figure 6-3 D-H). Treatment with NaB significantly increased the induced Ki67-positive and Epcam-positive population in the aged model, and the survival rate of the aged model was also improved (Figure 6-3 D-H).

[0165] Discussion Liver aging is a normal physiological process in which hepatocytes gradually lose the functions and plasticity necessary for homeostasis (Timchenko, 2009). The development of strategies for liver diseases associated with aging and for improving liver regeneration in the elderly has been hampered by the lack of understanding of the mechanisms by which aging controls hepatocyte plasticity. By applying the differentiation induction technology of hiPSCs, we created young hepatocytes and aged hepatocytes with the same genetic background. This preparation helped us to clarify that histone hypoacetylation associated with aging impairs hepatocyte plasticity. On the other hand, upregulation of histone acetylation can significantly improve the plasticity of aged hepatocytes. Based on this hypothesis, we successfully obtained pHCs from aged PHHs (derived from 78-year-old donors). And in the liver injury model, this pHC showed greater repopulation ability than that of the original aged PHH.

[0166] Due to the shortage of donors for liver transplantation, the use of grafts from elderly donors is becoming increasingly necessary and helps reduce the mortality associated with the waiting list. However, the initially poor regenerative ability of aging hepatocytes can lead to transplant failure and poor outcomes (Bernal and Wendon, 2013; Durand et al., 2019; Uemura et al., 2007). Accumulating evidence indicates that hepatocytes in grafts from the elderly have a low proliferative response, and this disadvantage impairs liver regeneration (Ono et al., 2011; Schmucker and Sanchez, 2011). Compared with the transplantation of young PHH in our liver injury model, the aging PHH grafts produced a smaller amount of human liver proteins, thus reproducing the differences in clinical outcomes between young and aging grafts. Notably, the upregulation of histone acetylation significantly promoted the proliferation of aging PHH. And the aging PHH showed a substantial improvement in regenerative ability. This suggests that the transplantation of grafts from the elderly (which effectively activates or promotes the inherent proliferative ability of hepatocytes) will undoubtedly improve graft survival and clinical outcomes.

[0167] The aging process is constantly characterized by dynamic changes and epigenetic modifications in the metabolic process (Peleg et al., 2016; Ren et al., 2017), but how these changes control cell function remains a mystery in the field of liver research (Horvath et al., 2014; Sato et al., 2017). In this paper, we revealed a deep relationship among aging, hepatocyte plasticity, metabolism, and epigenetic modifications. In particular, hepatocyte aging was found to be accompanied by a decline in metabolic function, which affected the control of histone acetylation via acetyl-CoA, thereby causing damage to hepatocyte plasticity. In contrast to rodent hepatocytes (Katsuda et al., 2017), EZH2 transcription activated by FGF2 is required to induce the plasticity of human hepatocytes, and hypoacetylation of the EZH2 enhancer in aged hepatocytes was found to impair the plasticity of these cells. Several researchers noticed that in the aged mouse liver, calorie restriction can reverse the age-dependent decline in histone acetylation and activate the transcription of cell cycle genes (Sato et al., 2017), but until this study, there was no appropriate method to enhance histone acetylation by improving cell metabolism in a culture dish. Considering the reversibility of histone acetylation and deacetylation, we found that the inhibition of HDACs can significantly improve the plasticity of aged hepatocytes, further confirmed the control of the plasticity of aged hepatocytes by histone hypoacetylation, and proposed a feasible strategy to improve the plasticity of aged hepatocytes.

[0168] In addition to endangering homeostasis and controlling liver regeneration, aging also plays a crucial role in the development of liver diseases (Kim et al., 2015). A decline in mitochondrial function during aging has been reported to increase vulnerability to damage (Kim et al., 2015), and aging is considered a predictor of the unfavorable progression of alcoholic hepatitis and the progression of fibrosis in hepatitis C (Forrest et al., 2005; Poynard et al., 2001). In this study, we demonstrated that hiPSC-Hep can recapitulate the aging process of the liver. On the other hand, our bioinformatics analysis showed that aged hiPSC-Hep tend to express genes that promote angiogenesis and fibrosis (Figure 1-2M), suggesting that hiPSC-Hep may be useful for elucidating the deep connection between aging and liver fibrosis and for developing effective treatment strategies for aging-related liver diseases.

[0169] In conclusion, we reproduced the aging process of hepatocytes in a culture dish and found that hepatocyte plasticity is impaired by histone hypoacetylation associated with aging. Furthermore, the promotion of histone acetylation improves the plasticity of aged hepatocytes, enhances the repopulation ability of these cells in a liver injury model, thereby indicating a promising treatment strategy for promoting liver regeneration in the elderly.

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[0171] KEY RESOURCES TABLE

Table 1

[0172]

Table 2

[0173]

Table 3

[0174] Details of experimental models and subjects hiPSC culture The TkDA3 hiPSC clones were maintained in mTeSR1 medium on culture dishes coated with growth factor-reduced (GFR) Matrigel (Matrigel: RPMI1640 medium = 1:30). The 1383D2, 1383D6, M66, and YCU#7 hiPSC clones were maintained in StemFit AK02N medium on culture dishes coated with laminin 511 (laminin 511: PBS = 1:150). All cells were maintained at 37°C in a humidified incubator with a 5% CO2 concentration.

[0175] PHH Culture Cryopreserved PHHs were thawed according to the manufacturer's instructions. For the positive control experiment, PHHs were cultured in Williams E medium containing 5% FBS, 1 μM dexamethasone, 100 IU / mL penicillin (Thermo Fisher Scientific), 100 μg / mL streptomycin (Thermo Fisher Scientific), 4 μg / mL human recombinant insulin (Sigma), 2 mM GlutaMAX 5 cells / cm 2 at a density of, and 15 mM HEPES (Sigma) on culture dishes coated with type I collagen. After 24 hours, the supernatant of PHHs was collected for ALB analysis. For the plasticity induction experiment, PHHs were seeded at a density of 1x10 TM (Thermo Fisher Scientific) and in serum-free differentiation medium (SFD) (Nie et al., 2018a) containing 10 ng / mL FGF2, 10 ng / mL EGF, 20 ng / mL HGF, 10 μM Y-27632, 0.5 μM A83-01, 3 μM CHIR99021, and 0.25 mM NaB on culture dishes coated with laminin 511. Cell adhesion was improved using 5% FBS on day 0 of culture. After 24 hours, the medium was replaced with fresh medium and then every 2 days. 4 cells / cm 2

[0176] Mouse ​Adult 7- to 9-week-old TK-NOG mice were used in this study (Hasegawa et al., 2011). To induce liver injury, ganciclovir (50 mg / kg, Tanabe Mitsubishi Pharma) was administered twice to the TK-NOG recipients, 5 days and 7 days before transplantation. The mice were housed in a temperature- and light-controlled (12-hour light / dark cycle) specific pathogen-free animal facility and were bred according to the guidelines of Yokohama City University for the use of experimental animals. All experimental procedures were approved by the Clinical Research Review Committee of the Animal Experiment Center of Yokohama City University School of Medicine (No. 075).

[0177] Details of the method Differentiation into hiPSC-Hep Differentiation from hiPSCs into hiPSC-Hep was performed using the following two-step procedure. Step I (endoderm differentiation ) hiPSC clones were differentiated into endodermal cells for 7 days on plates coated with GFR Matrigel or laminin 511 using a medium consisting of RPMI 1640, 1% B27, 50 ng / mL WNT3A, and 100 ng / mL activin A. 10 μM Y-27632 was added on day 0, and 0.5 mM NaB was added from day 1 to day 3. The medium was changed daily. Step II (differentiation and maturation into hepatocytes ) hiPSC endodermal cells were detached from the plate using 0.05% trypsin / EDTA (Gibco). Then, on plates coated with GFR Matrigel or laminin 511, 1.5 - 2 x 10 5 cells / cm 2It was reseeded at a density of . From day 8 to day 10, the medium was replaced with HDM daily. From day 11, the medium was replaced every two days. This HDM consisted of SFD containing 10 ng / mL FGF2, 20 ng / mL HGF, 10 ng / mL OSM, 100 nM dexamethasone and 10 mM nicotinamide. Control hiPSC-Hep was differentiated according to a published protocol with minor modifications (Kajiwara et al., 2012; Si-Tayeb et al., 2010).

[0178] Induction of proliferative hepatocytes (pHc) The newly created hiPSC-Hep was harvested at a certain senescence time point using 0.05% trypsin / EDTA and seeded at a density of 5,000 cells / cm 2 onto plates coated with GFR Matrigel or laminin 511 using reprogramming medium (RM) consisting of SFD containing 10 ng / mL FGF2, 10 ng / mL EGF, 20 ng / mL HGF, 10 μM Y-27632, 0.5 μM A83-01 and 3 μM CHIR99021. The medium was replaced on day 1, day 3 and day 5. To block the PI3K and MAPK signaling pathways, 10 μM LY29002 and a certain amount of PD0325902 (0.01 μM - 1 μM) were added to the RM respectively. To block the function of EZH2, 0.1 μM DZNep was added to the RM. To control epigenetic modifications, 0.25 mM NaB, 0.5 mM VPA, 10 μM Trany, 0.5 μM RG108 and 0.5 μM BIX were added to the RM. The cell number was analyzed by using Incell analyzer 2000 (GE Healthcare) in combination with Hoechst 33342 staining (Invitrogen).

[0179] Subculture of proliferative hepatocytes (pHc) From day 6 to day 8 of induction, proliferative hepatocytes (pHc) were harvested using 0.05% trypsin / EDTA and seeded at a density of 1 x 10 4 cells / cm 2 onto plates coated with GFR-Matrigel or laminin 511 using RM. On day 1 and day 3, the medium was replaced with RM without Y-27632.

[0180] Time-lapse tracing of hepatocyte proliferation induced from a single cell D22-Hep was seeded at a density of 500 cells / cm 2 onto plates coated with GFR-Matrigel (with RM). After incubation for 4 hours, phase-contrast imaging was performed using a BZ9000 all-in-one fluorescence microscope (Keyence), and images were acquired every 24 hours.

[0181] Cell Counting Kit-8 assay According to the instructions, the cell viability at the indicated day was determined using Cell Counting Kit-8.

[0182] Drawing of cell growth curve pHc was passaged at approximately 90% confluence, and the total cell number was determined for each passage. Growth rate = (total cell number) / (seeded cell number) The growth rate of pHc was measured and the cell growth curve was calculated.

[0183] Calculation of cell population doubling time pHc was seeded at a density of 5000 cells / cm 2 onto plates coated with GFR-Matrigel. After incubation for 4 hours, the medium was replaced with fresh medium, and the number of attached cells was counted using an Incell analyzer 2000 in combination with Hoechst 33342 staining. The cell number was also counted at the following time points: 24 hours, 48 hours, and 72 hours. The cell population doubling time was calculated using GraphPad Prism according to the cell number at each time point.

[0184] Hepatocyte differentiation from pHCs Hepatocyte differentiation of pHCs was initiated from the point of approximately 90% confluence. The medium was changed to HDM containing 3 μM RA for 8 days and then to RA-free HDM for the next 7 days. The medium was changed every 2 days. The total hepatocyte differentiation period was 15 days.

[0185] Cholangiocyte differentiation from pHCs pHCs obtained by inducing plasticity at a certain point during passage or in the aging process were suspended in RM at a density of 1 x 10 6 cells / mL. A total of 5,000 pHCs mixed with 50 μL of GFR-Matrigel were seeded as a mount drop onto an untreated 24-well plate and incubated in a 5% CO2 incubator at 37 °C for 30 minutes. Next, 700 μL of cholangiocyte differentiation medium (CDM) was added to fill the well. This CDM consisted of SFD containing 10 ng / mL EGF, 20 ng / mL HGF, 50 ng / mL WNT3A, 100 ng / mL R-spondin-1, 50 ng / mL FGF10, 3 μM RA, 10 μM Y-27632, and 10 μM forskolin. The medium was changed every 3 days, and the relevant analysis was performed on the 10th day.

[0186] Assays for human ALB, A1AT, ferritin, and AFP Human albumin ELISA quantification kits, human alpha-1-antitrypsin ELISA quantification kits, human ferritin ELISA quantification kits, and human AFP ELISA quantification kits were used according to the manufacturers' instructions to measure human ALB, A1AT, ferritin, and AFP, respectively. Samples were diluted in the range of 10-fold to 5,000-fold to obtain values within the linear range of the standard curve.

[0187] Detection of intracellular senescence-associated β-galactosidase and ROS The activity of senescence-associated β-galactosidase (SA-β-Gal) was detected using a cell senescence detection and quantification kit, and CellROX was used according to the manufacturer's instructions.TM Intracellular ROS was analyzed using Deep Red Reagent. The photographs were taken using a Leica TCS SP5 confocal microscope (Leica).

[0188] Acetyl-CoA assay To determine the acetyl-CoA level in hiPSC-Hep, cells were detached from the plate using cell lysis buffer. The acetyl-CoA concentration was measured using an acetyl coenzyme A assay kit according to the manufacturer's instructions. The obtained values were normalized using the corresponding protein concentration.

[0189] Indocyanine green uptake and efflux Indocyanine green (ICG) dry powder (Daiichi Sankyo) (10 mg) was dissolved in 10 mL of hepatocyte culture medium to obtain a stock solution of 1 mg / mL. Cells were incubated in suspension or seeded with ICG for 4 hours at 37°C in a humidified incubator with 5% CO2 concentration. Next, the cells were washed three times with phosphate-buffered saline (PBS) and further incubated for 2 hours in fresh HCM (hepatocyte culture medium BulletKit TM HCM TM ) to measure ICG efflux. The images were taken using a BZ9000 all-in-one fluorescence microscope.

[0190] Periodic acid Schiff staining and low-density lipoprotein uptake Glycogen was detected using periodic acid Schiff (PAS) staining according to the manufacturer's instructions. That is, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. After washing with PBS, the cells were oxidized in a 0.5% periodic acid solution for 7 minutes, washed with PBS, and then incubated in Schiff reagent for 15 minutes. After three incubations in sulfurous acid water for 2 minutes each, the cells were washed with PBS and visualized using a BZ9000 all-in-one fluorescence microscope. For the low-density lipoprotein uptake assay, cells were incubated with 5 μg / mL Dil-Ac-LDL and 50 μL of Hoechst 33342 standard dilution solution at 37 °C for 2 hours. Next, the cells were washed with PBS. Photographs were analyzed using a BZ9000 all-in-one fluorescence microscope.

[0191] Transport assays for rhodamine 123 and cholyl-lysyl-fluorescein The rhodamine 123 transport assay and the cholyl-lysyl-fluorescein (CLF) transport assay were performed according to the previously described method (Sampaziotis et al., 2015). For the rhodamine 123 transport assay, cholangiocytes were incubated at 37 °C for 30 minutes in the presence or absence of 10 μM verapamil. Next, the cells were incubated with 100 μM rhodamine 123 at 37 °C for 5 minutes and washed three times with IMDM. Fresh CDM was added and the cells were incubated at 37 °C for an additional 40 minutes. For the CLF transport assay, cholangiocytes were loaded with 5 μM CLF or 5 μM FITC for 30 minutes at 37 °C, and then the cells were washed three times with IMDM. Fresh CDM was added and the cells were incubated at 37 °C for an additional 10 minutes. Photographs were taken using a Leica TCS SP5 confocal microscope.

[0192] Transmission electron microscopy Analysis of cholangiocyte cysts by transmission electron microscopy was performed according to the previously described method (Nie et al., 2018a). That is, pre-fixed cholangiocyte cysts were post-fixed, dehydrated, and embedded in fresh 100% resin. Next, 70 nm ultra-thin sections were cut out and stained with 2% uranyl acetate. Next, the sections were washed with distilled water and stained with Lead stain solution. Grids were observed under a JEM-1400Plus microscope (JEOL), and digital images were acquired using a VELETA camera (Olympus).

[0193] Flow cytometry The antibodies used in flow cytometry were as follows: PE-mouse anti-human CXCR4, BV421 mouse anti-human CD117, APC mouse anti-human EpCAM, rabbit anti-H3K9ac, rabbit anti-H3K14ac, rabbit anti-H3K18ac, rabbit anti-H3K27ac, rabbit anti-H3K56ac, and goat anti-rabbit IgG (H+L) Alexa Fluor 647. Cells were captured by a MoFlo Astrios system (Beckman Coulter).

[0194] Chromatin immunoprecipitation (ChIP) combined with quantitative PCR The procedures performed were according to the manufacturer's instructions. That is, 3 × 10 6 Cells were cross-linked in 1% formaldehyde for 10 minutes and then in 1x glycine for 5 minutes at room temperature, recovered by scraping, centrifuged, and resuspended in lysis buffer. DNA was digested to a length of approximately 150 - 900 bp fragments by micrococcal nuclease. The nuclear membrane was disrupted using a Bioruptor ultrasonicator (Cosmo bio) to release DNA fragments. Samples were immunoprecipitated overnight at 4°C using an antibody cocktail containing rabbit anti-human H3K9ac, rabbit anti-human H3K18ac, and rabbit anti-human H3K27ac. Immunocomplexes were captured using 30 μL of protein G magnetic beads according to the manufacturer's instructions. DNA was recovered by digestion with proteinase K at 65°C for 2 hours. The DNA was purified using a spin column. Next, ChIP and input were used for qPCR using the primers shown in the following table.

[0195] List of ChIP-PCR primers and probes for EZH2 enhancer

Table 4

[0196] The manufacturer names and product numbers of the probes are summarized below. TIFF2025094074000012.tif31149

[0197] Transplantation After two injections of ganciclovir, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using DRI-CHEM (Fujifilm) according to the manufacturer's instructions. A total of 1x10 6 individual PHHs or PHH-pHCs were transplanted into the spleens of TK-NOG recipients. Serum was collected every two weeks, and the mice were sacrificed at week 12.

[0198] RNA isolation and quantitative real-time polymerase chain reaction (Q-PCR) PureLink TM Total RNA was isolated using the PureLink RNA Mini Kit. RNA (< 2 μg) was used as a template to synthesize single-stranded cDNA using a high-performance cDNA reverse transcription kit according to the manufacturer's instructions. Q-PCR was performed using cDNA, specific primers, and probes from the Universal Probe Library. The primers and probes used in this study are listed in the table below. All data were calculated by the ΔΔCT method using β-ACTB (Thermo Fisher Scientific) as a normalization control.

[0199] List of quantitative Q-PCR primers and probes for human genes

Table 5

[0200] Sequences of the Left primer and Right primer of OCT4: SEQ ID NOs: 9 and 10 Sequences of the Left primer and Right primer of NANOG: SEQ ID NOs: 11 and 12 Sequences of the Left primer and Right primer of SOX17: SEQ ID NOs: 13 and 14 Sequences of the Left primer and Right primer of FOXA2: SEQ ID NOs: 15 and 16 Sequences of the Left primer and Right primer of TBX3: SEQ ID NOs: 17 and 18 Sequences of the Left primer and Right primer of TTR: SEQ ID NOs: 19 and 20 Sequences of the Left primer and Right primer of A1AT: SEQ ID NOs: 21 and 22 Sequences of the Left primer and Right primer of ALB: SEQ ID NOs: 23 and 24 Sequences of the Left primer and Right primer of TDO2: SEQ ID NOs: 25 and 26 Sequences of the Left primer and Right primer of G6PC: SEQ ID NOs: 27 and 28 Sequences of the Left primer and Right primer of ASGR1: SEQ ID NOs: 29 and 30 Sequences of the Left primer and Right primer of HNF4A: SEQ ID NOs: 31 and 32 Sequences of the Left primer and Right primer of TAT: SEQ ID NOs: 33 and 34 Sequences of the Left primer and Right primer of CYP2C9: SEQ ID NOs: 35 and 36 Sequences of the Left primer and Right primer of CYP2C19: SEQ ID NOs: 37 and 38 Sequences of the Left primer and Right primer of CYP3A4: SEQ ID NOs: 39 and 40 Sequences of the Left primer and Right primer of CYP7A1: SEQ ID NOs: 41 and 42 Sequences of the Left primer and Right primer of MKI67: SEQ ID NOs: 43 and 44 Sequences of the Left primer and Right primer of PCNA: SEQ ID NOs: 45 and 46 Sequences of the Left primer and Right primer of CCNB1: SEQ ID NOs: 47 and 48 Sequences of the Left primer and Right primer of CCND1: SEQ ID NOs: 49 and 50 Sequences of the Left primer and Right primer of CCNE1: SEQ ID NOs: 51 and 52 Sequences of the Left primer and Right primer of CDC20: SEQ ID NOs: 53 and 54 Sequences of the Left primer and Right primer of EZH2: SEQ ID NOs: 55 and 56 Sequences of the Left primer and Right primer of EpCAM: SEQ ID NOs: 57 and 58 Sequences of the Left primer and Right primer of C-MET: SEQ ID NOs: 59 and 60 Sequences of the Left primer and Right primer of LGR5: SEQ ID NOs: 61 and 62 Sequences of the Left primer and Right primer of RBP4: SEQ ID NOs: 63 and 64 Sequences of the Left primer and Right primer of SOX9: SEQ ID NOs: 65 and 66 Sequences of the Left primer and Right primer of HNF6: SEQ ID NOs: 67 and 68 Sequences of the Left primer and Right primer of GGT: SEQ ID NOs: 69 and 70 Sequences of the Left primer and Right primer of CFTR: SEQ ID NOs: 71 and 72 Sequences of the Left primer and Right primer of AQP1: SEQ ID NOs: 73 and 74 Sequences of the Left primer and Right primer of SSTR2: SEQ ID NOs: 75 and 76 Sequences of the left and right primers of ACLY: SEQ ID NOs: 77 and 78 Sequences of the left and right primers of PDHB: SEQ ID NOs: 79 and 80 Sequences of the left and right primers of ACSS2: SEQ ID NOs: 81 and 82 Sequences of the left and right primers of CPT1A: SEQ ID NOs: 83 and 84

[0201] The manufacturer names and product numbers of the probes are summarized below. JPEG2025094074000014.jpg249123

[0202] Histological examination and immunofluorescence staining Liver tissue samples were embedded in O.C.T. (optimal cutting temperature) compound (Sakura Finetek Japan), 5-μm sections were prepared, and mounted on MAS-GP type A coated slides (Matsunami Glass). For immunofluorescence staining, sections or cultured cells were fixed in 4% paraformaldehyde solution dissolved in PBS for 10 minutes, washed three times with PBS, blocked for 30 minutes using 10% ECL prime blocking agent dissolved in PBS containing 0.3% Triton X-100, and further washed three times with PBS. Next, sections or cells were incubated overnight at 4 °C with primary antibodies in blocking buffer. Sections or cells were washed three times with PBS and further incubated for 60 minutes at room temperature with fluorescently labeled secondary antibodies. Finally, sections or cells were washed three times with PBS and covered with mounting solution containing DAPI. Fluorescence was detected using a Zeiss Axio Imager M1 microscope (Carl Zeiss AG, Oberkochen, Germany). The antibodies used for immunofluorescence staining were as follows: rabbit anti-human AFP (1:100), goat anti-human ALB (1:100), mouse anti-human CK19 (1:50), rabbit anti-human A1AT (1:100), chicken anti-human A1AT (1:100), mouse anti-human KI67 (1:50), goat anti-human HNF4A (1:100), mouse anti-ZO1 (1:100), mouse anti-human E-cadherin (1:100), mouse anti-human SOX9 (1:100), F-ACTIN (1:100), mouse anti-human CK8 / 18 (1:100), mouse anti-human NuMA (1:100), donkey anti-mouse IgG (H+L) Alexa Fluor 488 (1:500), donkey anti-rabbit IgG(H+L) Alexa Fluor 488 (1:500), donkey anti-goat IgG (H+L) Alexa Fluor 555 (1:500), goat anti-guinea pig IgG (H+L) Alexa Fluor 555 (1:500) and goat anti-chicken IgY (H+L) Alexa Fluor 488 (1:500).

[0203] Microarray and Data Analysis Total RNA was prepared from hiPSC-derived hepatocytes (D22-Hep, D52-Hep, D72-Hep and D22-pHC), PHH (2M-PHH and 78Y-PHH) and PHH-pHC (2M-pHC and 78Y-pHC) using the PureLink TM RNA Mini Kit. RNA for gene expression profiling was hybridized using SurePrint G3 Human Gene Expression 8x60K (Agilent Technologies) according to the manufacturer's instructions. The data were normalized using GeneSpring. Gene ontology enrichment analysis and KEGG pathway analysis were performed using DAVID Bioinformatics Resources 6.8 (https: / / david.ncifcrf.gov / home.jsp) (Huang da et al., 2009). The STRING interaction network was analyzed using STRING 9.05 (http: / / string905.embl.de / ) (Franceschini et al., 2013).

[0204] Statistics Values were shown as mean ± standard deviation (SD). Statistical significance was evaluated using the Mann–Whitney U test when comparing two groups and one-way ANOVA and Bonferroni's multiple comparison test when comparing three or more groups. p < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism.

[0205] Availability of Data and Software Data Resources The accession number for the microarray data reported in this paper is GEO: GSE131806.

[0206] References Franceschini, A., Szklarczyk, D., Frankild, S., Kuhn, M., Simonovic, M., Roth, A., Lin, J., Minguez, P., Bork, P., von Mering, C., et al. (2013). STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic acids research 41, D808-815. Hasegawa, M., Kawai, K., Mitsui, T., Taniguchi, K., Monnai, M., Wakui, M., Ito, M., Suematsu, M., Peltz, G., Nakamura, M., et al. (2011). The reconstituted 'humanized liver' in TK-NOG mice is mature and functional. Biochemical and biophysical research communications 405, 405-410. Huang da, W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature protocols 4, 44-57. Kajiwara, M., Aoi, T., Okita, K., Takahashi, R., Inoue, H., Takayama, N., Endo, H., Eto, K., Toguchida, J., Uemoto, S., et al. (2012). Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America 109, 12538-12543. Nie, Y.Z., Zheng, Y.W., Miyakawa, K., Murata, S., Zhang, R.R., Sekine, K., Ueno, Y., Takebe, T., Wakita, T., Ryo, A., et al. (2018a). Recapitulation of hepatitis B virus-host interactions in liver organoids from human induced pluripotent stem cells. EBioMedicine 35, 114-123. Nie, Y.Z., Zheng, Y.W., Ogawa, M., Miyagi, E., and Taniguchi, H. (2018b). Human liver organoids generated with single donor-derived multiple cells rescue mice from acute liver failure. Stem cell research & therapy 9, 5. Sampaziotis, F., de Brito, M.C., Madrigal, P., Bertero, A., Saeb-Parsy, K., Soares, F.A.C., Schrumpf, E., Melum, E., Karlsen, T.H., Bradley, J.A., et al. (2015). Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation. Nature biotechnology 33, 845-852. Si-Tayeb, K., Noto, F.K., Nagaoka, M., Li, J., Battle, M.A., Duris, C., North, P.E., Dalton, S., and Duncan, S.A. (2010). Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51, 297-305. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Industrial Applicability

[0207] The present invention can be used for mass production of hepatocytes and cholangiocytes. Human hepatocytes and the like created by the present invention can be used in in vitro drug metabolism tests, hepatotoxicity tests, hepatitis virus infection tests, or in regenerative medicine, bioartificial livers, and the like. A chimeric animal in which human hepatocytes are replaced with the human hepatocytes and the like created by the present invention can be produced, and by using this chimeric animal, drug metabolism tests and safety tests of drug discovery compounds can be performed. Further, according to the present invention, liver regeneration can be promoted by using a drug that brings about high histone acetylation.

Claims

1. A method for producing hepatoblasts, comprising the steps of culturing endoderm cells derived from iPS cells in a serum-free medium containing FGF2, HGF, oncostatin M and dexamethasone, and differentiating the cells into hepatoblasts.

2. The method of claim 1 , further comprising comprising the step of: culturing the culture medium comprising:

3. The method of claim 1, wherein the serum-free medium is a serum-free differentiation medium (SFD).

4. The method of claim 1 , wherein the iPS cells are of human origin.

5. The method of claim 1 , further comprising comprising the step of: culturing the culture medium comprising:

6. The method according to claim 1, wherein the culture period of the endoderm cells derived from iPS cells is from 5 days to less than 10 days.

7. The method according to claim 1, wherein the endoderm cells derived from iPS cells are cultured on a plate coated with Matrigel or laminin.

8. The method according to claim 1, wherein the endoderm cells derived from iPS cells are cells differentiated into endoderm cells by culturing iPS cells in a medium containing B27, WNT3A and activin A.

9. The method according to claim 8, wherein Y-27632 is added to a medium containing B27, WNT3A and activin A on day 0, and NaB is added from day 1 to day 3, and the iPS cells are cultured for 7 days.

10. The method of claim 1, wherein the hepatoblast is a cell expressing TTR.

11. The method of claim 10, wherein the hepatoblast cells further express at least one marker selected from the group consisting of AFP, CK19, HNF4A and KI67.

12. Hepatoblasts expressing TTR, AFP, CK19, HNF4A and KI67.

13. A method for producing hepatocytes, comprising the step of further culturing the hepatoblasts obtained by the method of claim 1 in a medium containing a member of the FGF family, HGF, a member of the IL6 family and dexamethasone to differentiate them into hepatocytes.

14. The method according to claim 13, wherein the hepatocytes are cells that highly express TAT.

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