Method for inducing hepatocyte plasticity

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 technology]

[0002] Approximately 70% of liver tissue is composed 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)). Specifically, it has been shown that when liver injury occurs in mice, mature hepatocytes transform into proliferative hepatic progenitor cells, repairing lost 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)). Similar hepatic progenitor cells have also been detected in human liver cirrhosis (Cell Stem Cell. 23(1):114-22(2018) (Non-Patent Document 7)). Furthermore, liver regeneration capacity is associated with the aging state of hepatocytes, and it has been shown that liver grafts derived from elderly donors have significantly poorer survival rates (J Hepatol. 70(4):745-58(2019) (Non-Patent Document 8); J Hepatol. 57(2):288-96(2012) (Non-Patent Document 9)).

[0003] T. Ochiya et al. demonstrated that by treating mouse and rat hepatocytes in vitro with small molecule compounds (YAC, Y-27632, A83-01, CHIR99021), they were able to induce proliferative hepatic progenitor cells from mature hepatocytes and culture them for more than 20 passages (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, due to significant differences in the properties of human hepatocytes and rodent-derived hepatocytes, attempts to induce human hepatic progenitor cells failed to culture for more than three passages (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)). Furthermore, not only did they not maintain proliferation ability, but they also did not maintain the pluripotency to differentiate into human hepatocytes and bile duct cells, and induction of hepatic progenitor cells based on plasticity control of human hepatocytes had not been achieved. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nat Cell Biol.18(3):238-45(2016) [Non-patent document 2] Hepatology.64(6):2244-6 (2016) [Non-patent document 3] Cell Stem Cell.15(3):340-9(2014) [Non-patent document 4] Cell Stem Cell.15(5):605-18(2014) [Non-Patent Document 5] Cell. 157,1324-38(2014) [Non-patent document 6] Genes Dev.27(7):719-24(2013) [Non-Patent Document 7] Cell Stem Cell.23(1):114-22(2018) [Non-patent document 8] J Hepatol.70(4):745–58 (2019) [Non-Patent Document 9] J Hepatol.57(2):288–96 (2012) [Non-Patent Document 10] Cell Stem Cell. 20(1):41-55 (2017) [Non-Patent Document 11] Cell Stem Cell. 23(6):806-819,2018 [Non-Patent Document 12] Cell Research.29,8-22, 2019 [Non-Patent Document 13] J Hepatol.70(1):97-107,2019 Summary of the Invention [Problem 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 conventional methods lack high proliferation and pluripotency. 3. There is no technology to control the plasticity or aging of human hepatocytes.

[0006] The present invention aims to overcome these drawbacks. [Means for solving the problem]

[0007] The present inventors have discovered a novel method for inducing iPS cell-derived hepatocytes and have succeeded in generating hepatocytes that can maintain liver function for a long period of time while gradually accumulating aging markers and characteristics.

[0008] Furthermore, we have newly discovered that, unlike rodent-derived hepatocytes, the plasticity of human hepatocytes is regulated by the FGF2-MAPK-EZH2 axis. Specifically, we found that by increasing the transcriptional activity of EZH2 through the addition of FGF2, human hepatocytes can be converted into hepatic progenitor cells with proliferation and multipotency (bipotentiality to differentiate into hepatocytes and bile duct cells), whereas this plasticity is impaired in aged hepatocytes.

[0009] Furthermore, we demonstrated that decreased histone acetylation inhibits the acquisition of plasticity in senescent cells. We also found that selective inhibition of histone deacetylase (HDAC) can improve the plasticity of senescent human hepatocytes derived from human iPS cells and primary human hepatocytes derived from elderly individuals (78 years old). Artificially induced hepatic progenitor cells from these senescent cells can be cultured for more than 20 passages in an in vitro culture system, making them suitable for the mass production of hepatocytes.

[0010] The gist of the present invention is as follows. (1) A method for producing hepatocytes, which comprises 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) The method described in (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 endoderm 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 prepared by the method according to any one of (1) to (5), which are viable and maintain the ability to secrete albumin in ex vivo monolayer culture for 12 days or more. (7) The hepatocytes according to (6), which can survive for 12 days or more and have the functions of hepatocytes other than the ability to secrete albumin. (8) The hepatocytes described in (7), wherein the hepatocyte functions other than the ability to secrete albumin are at least one selected from the group consisting of drug metabolism, indocyanine green uptake and excretion, glycogen storage, low-density lipoprotein uptake, and gene expression. (9) Hepatocytes according to any one of (6) to (8), which exhibit characteristics of aging. (10) The hepatocytes described in (9), wherein the characteristics of aging are at least one selected from the group consisting of increased cell volume, expression of aging-related genes, increased inflammatory response, DNA damage, increased levels of intracellular reactive oxygen species, increased levels of cellular senescence-associated beta-galactosidase, epigenetic changes, shortening of telomere length, and decreased 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), which comprises culturing hepatocytes in the presence of a member of the FGF family and an agent that induces 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 drug that induces histone hyperacetylation is a histone deacetylase inhibitor. (15) 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, and 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, The method according to any one of (12) to (14), wherein the compound is at least one selected from the group consisting of Depsipeptide, Santacruzamate A (CAY10683), Scriptaid, SKLB-23bb, Splitomicin, Suberoyl bis-hydroxamic acid, Tasquinimod, TH34, Tinostamustine (EDO-S101), TMP195, TMP269, Tubacin, and Tubastatin A. (16) The method according to any one of (11) to (15), wherein the hepatocytes are cells differentiated from pluripotent stem cells, cells subcultured from cells differentiated from pluripotent stem cells, primary cultured cells isolated from biological tissue, cells subcultured from primary cultured cells isolated from 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) Alpha-fetoprotein (AFP)-negative hepatic progenitor cells prepared by any of the methods described in (11) to (17), which have the ability to proliferate and the ability to differentiate into hepatocytes and bile duct epithelial cells. (19) The hepatic progenitor cells according to (18), which are capable of differentiating into hepatocytes and / or bile duct cells. (20) A method for producing hepatocytes, comprising inducing differentiation of the hepatic progenitor cells according to (18) or (19) into hepatocytes. (21) A method for producing bile duct cells, comprising inducing differentiation of the hepatic progenitor cells according to (18) or (19) into bile duct cells. (22) A method for suppressing hepatocyte aging using a drug that induces histone hyperacetylation. (23) The method according to (22), wherein a drug that induces histone hyperacetylation is used in combination with a member of the FGF family. (24) A hepatocyte aging inhibitor containing, as an active ingredient, a drug that induces histone hyperacetylation. (25) The agent according to (24), wherein the drug that induces histone hyperacetylation is a histone deacetylase inhibitor. (26) 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, and 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, The agent according to (25), which is at least one selected from the group consisting of Depsipeptide, Santacruzamate A (CAY10683), Scriptaid, SKLB-23bb, Splitomicin, Suberoyl bis-hydroxamic acid, Tasquinimod, TH34, Tinostamustine (EDO-S101), TMP195, TMP269, Tubacin, and Tubastatin A. (27) The agent according to any one of (24) to (26), wherein the agent that induces histone hyperacetylation is used in combination with a member of the FGF family. (28) A method for increasing the plasticity of hepatocytes using drugs that induce histone hyperacetylation. (29) The method described in (28), wherein a drug that induces histone hyperacetylation is used in combination with a member of the FGF family. (30) An agent for increasing the plasticity of liver cells, comprising as an active ingredient a drug that induces histone hyperacetylation. (31) The agent according to (30), wherein the drug that induces histone hyperacetylation is a histone deacetylase inhibitor. (32) 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, and 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, The agent according to (31), which is at least one selected from the group consisting of Depsipeptide, Santacruzamate A (CAY10683), Scriptaid, SKLB-23bb, Splitomicin, Suberoyl bis-hydroxamic acid, Tasquinimod, TH34, Tinostamustine (EDO-S101), TMP195, TMP269, Tubacin, and Tubastatin A. (33) The agent according to any one of (30) to (32), wherein the agent that induces histone hyperacetylation 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 described in (6), the hepatic progenitor cells described in (18), and cells induced to differentiate from the hepatic progenitor cells described in (18) into a non-human animal. (35) A method for producing a chimeric animal described in (34), which comprises transplanting at least one cell selected from the group consisting of the hepatocytes described in (6), the hepatic progenitor cells described in (18), and cells induced to differentiate from the hepatic progenitor cells described in (18) into a non-human animal with liver failure. (36) The method for producing a chimeric animal according to (34) or (35), wherein the transplanted hepatic progenitor cells proliferate and / or differentiate. (37) The method for producing a chimeric animal according to any one of (34) to (36), wherein liver regeneration is promoted. (38) A transplant composition comprising at least one cell selected from the group consisting of the hepatocytes described in (6), the hepatic progenitor cells described in (18), and cells induced to differentiate from the hepatic progenitor cells described in (18). (39) A kit for a culture medium for differentiation induction, comprising: an agent containing FGF10, retinoic acid, and forskolin for use in a culture medium used to induce the differentiation of hepatic progenitor cells according to (18) or (19) into bile duct cells; and instructions for using the agent in the culture medium used for 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, as an active ingredient, a drug that induces histone hyperacetylation. (42) A method for promoting liver regeneration, comprising administering to a subject a pharmaceutically effective amount of an agent that induces histone hyperacetylation. [Effects of the Invention]

[0011] According to the present invention, hepatocytes that can be used as an aging model have been obtained.

[0012] The present invention makes it possible to induce plasticity in hepatocytes. Hepatocytes with induced plasticity (hepatic progenitor cells) can be differentiated into hepatocytes or bile duct cells.

[0013] This invention makes it possible to induce plasticity in aged hepatocytes. This specification includes the contents disclosed in the specification and / or drawings of Japanese Patent Application No. 2019-177843, which is a priority document of this application. [Brief explanation of the drawings]

[0014] [Figure 1-1]Recapitulation of the aging process by means of human induced pluripotent stem cell-derived hepatocytes (hiPSC-Hep). (A) Schematic representation of hepatocyte differentiation, maturation, and aging using a novel hiPSC-Hep-based strategy. (B) Q-PCR analysis of lineage-related gene expression during hiPSC-to-hepatocyte differentiation: OCT4 and NANOG (pluripotent cells), SOX17 and FOXA2 (endoderm), TBX3 and TTR (hepatoblasts), and A1AT and ALB (hepatocytes). (C) Immunofluorescence staining of ALB, AFP, CK19, A1AT, HNF4A, and KI67. (D) Analysis of ALB secretion from N-Hep (hepatocytes derived from human induced pluripotent stem cells generated by the inventors using the method described in the Examples) from day 22 (D22) to day 72 (D72) by enzyme-linked immunosorbent assay (ELISA). (E) Transcriptome analysis shows upregulation of several genes in D72-Hep (hepatocytes differentiated from hiPSCs and subcultured after 22 days of differentiation, corresponding to "Young Hepatocytes" in Figure 1-1A) compared with D22-Hep (hepatocytes differentiated from hiPSCs and subcultured after 22 days of differentiation, corresponding to "Old Hepatocytes" in Figure 1-1A) and D52-Hep (hepatocytes differentiated from hiPSCs and subcultured after 52 days of differentiation, corresponding to "Old Hepatocytes" in Figure 1-1A). Gene names are indicated by large dots: senescence-related genes. (F) N-Hep from D32 to D72 Images of intracellular ROS (top images, red) and SA-β-gal staining (bottom images, green) in N-Hep from D22 to D72. (G) Quantification of the percentage of intracellular ROS-positive cells in N-Hep from D22 to D72. (H) Quantification of SA-β-gal-positive cells in N-Hep from D22 to D72. See also Figure 1-2 and Figure 1-3. [Figure 1-2]Development of a two-step protocol for generating hiPSC-Hep, its functional characteristics, and changes associated with aging (related to Figure 1). (A) Schematic diagram of the two-step hepatocyte differentiation from hiPSCs. (B) Representative flow cytometry profiles showing the expression of CXCR4, CD117, and EpCAM in hiPSC-derived definitive endoderm. (C) Q-PCR analysis of ALB expression in lineages differentiated with the indicated media in step II: "Δ" means "without"; SFD: serum-free defined medium; 6FM: SFD containing six selected factors; OSM: oncostatin M; DEX: dexamethasone. (D) ELISA-based assay of ALB secretion in lineages cultured with the indicated media in step II. (E) ELISA-based dynamic assay of ALB secretion during hepatocyte differentiation from multiple donor-derived hiPSC clones. (F) Immunostaining of N-Hep with ALB (red), E-cadherin (E-CAD) (green), and zona occludens 1 (ZO-1) (green). (G) Analysis of indocyanine green (ICG) uptake and efflux in N-Hep (green), detection of glycogen stores by periodic acid-Schiff staining (purple), and examination of low-density lipoprotein uptake using Dil-labeled acetyl-LDL (Dil-Ac-LDL) (red). (H) Q-PCR analysis of liver-related gene expression in N-Hep, K-Hep, and ST-Hep (hiPSC-Hep obtained by the conventional protocol shown below, denoted K-Hep and ST-Hep using the initials of their names; Kajiwara et al., 2012; Si-Tayeb et al., 2010). (I) ELISA-based assay of ALB secretion from N-Hep, K-Hep, ST-Hep, and PHH. (J) Cell morphology of D52-Hep, D62-Hep, and D72-Hep; square boxes: multinucleated cells. (K) Cell morphology (left) and LIVE / DEAD staining (right) of D82-Hep; white arrowheads: multinucleated cells. (L) Quantification of average cell size of N-Hep from D22 to D72. (M) Gene ontology analysis of enriched pathways and molecular functions in D72-Hep. [Figure 1-3] Comparison of the hepatocyte aging process in vitro and in vivo. (A) Principal component analysis provides a three-dimensional graphical representation of gene expression clustering among D22-Hep, D72-Hep, young PHH (2 months old, 2M-PHH), and elderly PHH (78 years old, 78Y-PHH). (B) KEGG pathway analysis was used to compare genes whose expression increases with aging between D72-Hep, 78Y-PHH, and D22-Hep and 2M-PHH, respectively. (C) KEGG pathway analysis was used to compare genes whose expression decreases with aging between D72-Hep, 78Y-PHH, and D22-Hep and 2M-PHH, respectively. [Figure 2-1]FGF2 enhances proliferation induced in D22-Hep. (A) Schematic overview of the characterization of plasticity in D22-Hep. (B) Phase-contrast images (D1 and D6) of D22-Hep cultured in BM, BM + SMs, BM + FGF2 (F), or BM + SMs + F (the latter is reprogramming medium; RM). BM is SFD medium (described in WO2016093222) containing 10 ng / mL EGF and 20 ng / mL HGF; BM + SMs is BM medium containing 10 μM Y-27632, 0.5 μM A83-01, and 3 μM CHIR99021. (C) Quantification of cell number 6 days after plasticity induction; values ​​were normalized to the cell number at D1. (D) Q-PCR measurement of cell cycle-related gene expression in cells before culture (D0) and after 6 days of culture in RM. These time points are indicated as RM-D0 and RM-D6 in the figure, respectively. (E) Immunofluorescence staining of KI67 and HNF4A on cells in the RM-D0 and RM-D6 groups. (F) Q-PCR measurement of the expression of hepatic progenitor / stem cell-related genes HNF4A, EpCAM, c-MET, TBX3, and LGR5 in D22-Hep and D22-pHC (hepatic progenitor cells induced by the inventors using the method described in the Examples). (G) Q-PCR analysis of the expression of liver function genes ALB, A1AT, TTR, and RBP4 in D22-Hep and D22-pHC. (H) Immunostaining for HNF4A, SOX9, CK19, and ALB in D22-pHC. (I) Analysis of cell morphology after 6 days of treatment with RM plus DMSO, PD0325901 (PD, 1 μM), or LY2940002 (LY, 10 μM); and quantification of cell number after 6 days of incubation in RM plus DMSO, PD0325901 (0.01, 0.03, 0.1, or 1 μM), or LY290002 (10 μM). Values ​​were normalized to cell number at D1. (J) Transcription of EZH2 was measured by Q-PCR under the indicated conditions. (K) Diagram illustrating how the FGF2-MAPK-EZH2 axis promotes hepatocyte proliferation. See also Figure 2-2. [Figure 2-2] The MAPK-EZH2 axis activated by FGF2 promotes hepatocyte plasticity induction. (Related to Figure 2-1) (A) Cell Counting Kit-8-based analysis of the proliferative potential of reprogrammed cells subcultured in BM+SMs and BM+SMs+F, and phase-contrast images of cells subcultured in BM+SMs on day 4. (B) Cell counts after 6 days of treatment of BM+SMs with FGF2 (0, 0.1, 1, or 10 ng / ml); values ​​were normalized to cell counts on day 1. (C) Time-lapse imaging analysis of induced stem cell proliferation at the single-cell level. (D) Induction of hepatocyte proliferation using RM from D22-Hep derived from different hiPSC clones. (E) Immunofluorescence analysis of AFP on hiPSC-hepatoblasts (HB) and proliferating hepatocytes (D22-pHC). (F) ELISA-based analysis of AFP secretion from HB and D22-pHC. (G) STRING interaction network of enriched genes in D22-pHC compared to D22-Hep. (H) Q-PCR analysis of EZH2 expression in D22-Hep, cells reprogrammed in BM + SMs, and cells reprogrammed in RM. (I) Quantification of cell number at day 6 in RM containing DMSO or 3-deazaneplanocin A hydrochloride (DZNep) (0.1 μM); numbers were normalized to cell number at day 1. (J) Q-PCR analysis of EZH2 expression at day 6 in cells cultured in RM containing DMSO or DZNep (0.1 μM). (K) Phase-contrast images of D22-pHC reseeded in RM, RMΔFGF2 (RM without FGF2), and RM + DNZep (0.1 μM) on day 4. (L) Cell counts of D22-pHC cultured in RM, RMΔFGF2, and RM + DNZep (0.1 μM) on day 4; values ​​were normalized to cell counts on day 1. (M) Extended proliferation curves of D22-pHC (from P0 to P20). These results were obtained from pHCs derived from five different hiPSCs (TKDA). Phase-contrast images of pHCs at passage 20. (N) Representative karyotype images of D22-pHC (TKDA) at passage 18. [Figure 3-1] D22-pHC, which possesses bipotential differentiation potential, differentiates into hepatocytes and cholangiocytes. (A) Top: Timeline of hepatic differentiation of D22-pHC. Bottom: Phase-contrast images of D22-pHC (left) and D22-pHC-derived hepatocytes (D22-pHC-Hep, right). (B) Q-PCR analysis of the expression of ALB, A1AT, CYP2C9, and CYP2C19 in D22-Hep, D22-pHC, and D22-pHC-Hep. (C) ELISA-based analysis of ALB secretion from D22-Hep, D22-pHC, D22-pHC-Hep(P1) (passage 1 D22-pHC-derived hepatocytes), D22-pHC-Hep(P10) (passage 10 D22-pHC-derived hepatocytes), and D22-pHC-Hep(P20) (passage 20 pHC-derived hepatocytes). (D) Immunofluorescence analysis of ALB, E-CAD, and ZO-1 in D22-pHC-Hep. (E) Ammonia excretion in D22-pHC-Hep(P1), D22-pHC-Hep(P20), and D22-Hep; the no-cell (NC) group was used as a negative control. (F) Top: Timeline of D22-pHC differentiation into cholangiocytes. Bottom: Macroscopic image of D22-pHC-derived cholangiocytes (D22-pHC-Cho) (left) and a single cholangiocyte cyst (right). (G) Q-PCR measurement of SOX9, HNF6, GGT, and CFTR expression in D22-pHC and D22-pHC-Cho. (H) Immunofluorescent staining of CK19, ALB, AFP, F-ACTIN, HNF4A, and SOX9 in D22-pHC-Cho. (I) Transmission electron microscopy image of a cholangiocyte cyst: integrated structure of a D22-pHC-Cho cyst with lumen, apical (red arrowhead) and basolateral (blue arrowhead) plasma membranes, and microvilli (black arrowheads); partial longitudinal section (red arrow) and axial section (blue arrow) of a primary cilium. (J) Representative images of rhodamine 123 accumulation in the lumen of D22-pHC-Cho cysts in the absence and presence of verapamil. (K) Calculation of the fluorescence intensity along the white line in the image (J) above. See also Figure 3-2. [Figure 3-2]Characterization of D22-pHC-derived hepatocytes and cholangiocytes (related to Figure 3-1). (A) Phase-contrast images of D22-pHC cultured for 15 days in HDM and HDM+RA, respectively. (B) ELISA-based dynamic assay of ALB secreted into HDM and HDM+RA during hepatocyte differentiation. HDM consisted of SFD containing 10 ng / mL FGF2, 20 ng / mL HGF, 10 ng / mL OSM, 100 nM dexamethasone, and 10 mM nicotinamide. (C) Images of hepatocytes (D22-pHC-Hep) differentiated from the multi-donor hiPSC clone-derived D22-pHC. (D) Q-PCR analysis of ALB, A1AT, CYP2C9, and CYP2C19 expression in hepatocytes differentiated from D22-pHC at different passages (P1, P10, and P20) and from clonal D22-pHC at P5 (C1). (E) Analysis of ICG uptake and efflux in D22-pHC-Hep (green), detection of glycogen stores by periodic acid-Schiff staining (purple), and examination of low-density lipoprotein uptake using DiI-ac-LDL (red). (F) Time-lapse imaging analysis of cholangiocyte cyst formation. Arrowheads indicate a single D22-pHC cell forming a ring-shaped structure. (G) Macroscopic image of cholangiocytes (D22-pHC-Cho) derived from multi-donor hiPSC clone-derived D22-pHC. (H) Q-PCR analysis of SOX9, GGT, and CFTR expression in cholangiocytes differentiated from D22-pHC at different passages (P1, P10, and P20) and from clonal D22-pHC at P5 (C1). (I) Transmission electron microscopy images of tight junctions (blue arrowheads) (top) and multivesicular bodies (red arrowheads) (bottom) in cholangiocytes. (J) Representative images of the active export of the fluorescent bile acid CLF from the cyst lumen and FITC (fluorescein isothiocyanate) loading as a control. [Figure 4-1]Senescence-associated histone hypoacetylation impairs induced stem cell proliferation. (A) Schematic analysis of senescence-associated plasticity in N-Hep. (B) Hoechst staining-based image of proliferative cells obtained from N-Hep after 6 days of culture in RM. (C) Quantification of cells obtained from N-Hep after 6 days of culture in RM; data were normalized to cell number at D1. (D) Expression of acetyl-CoA synthesis-related genes (ACLY, PDHB, ACSS2, and CPT1A) in D22-Hep, D52-Hep, and D72-Hep was assessed by Q-PCR. (E) Analysis of intracellular acetyl-CoA levels in D22-Hep and D52-Hep. (F) Fluorescence-activated cell sorting-based analysis of H3K9ac, H3K18ac, and H3K27ac levels in D22-Hep, D32-Hep, D52-Hep, and D62-Hep. (G) Correlation of H3K9ac (left), H3K18ac (middle), and H3K27ac (right) levels with induced proliferation rate during N-Hep aging. (H) Hoechst staining-based image analysis of reprogrammed cells derived from D52-Hep cultured in RM with the addition of sodium butyrate (NaB), valproic acid (VPA), tranylcypromine (Trany, a histone demethylase inhibitor), RG108 (a DNA methyltransferase inhibitor), or BIX01294 (BIX, a histone methyltransferase inhibitor) at D8. Controls cultured in RM alone served as controls. (I) Quantification of fold changes in proliferation rate after the addition of NaB, VPA, Trany, RG108, or BIX. (J) Immunofluorescence assay of HNF4A (red) and KI67 (green) in D52-Hep-derived reprogrammed cells cultured in RM (Con) or RM + NaB (NaB added on D8); the percentage of HNF4A+KI67+ cells is shown in the graph on the right. (K) Q-PCR analysis of EZH2 expression in D52-Hep-derived reprogrammed cells cultured in RM (Con), RM + NaB (NaB), or RM + VPA (VPA).(L) Chromatin immunoprecipitation and PCR analysis (ChIP-PCR) detection of acetylated histone binding within the EZH2 enhancer in D22-Hep and D52-Hep using four primer pairs (Pr). (M) Quantification of NaB (added on D8) promotion of hepatocyte plasticity induction in D62-Hep, D72-Hep, and D82-Hep: All data were normalized to the corresponding RM-only group. Immunofluorescence staining of HNF4A (red) and KI67 (green) in reprogrammed cells derived from D82-Hep under RM+NaB conditions. See also Figures 4-2 and 4-3. [Figure 4-2] Histone hypoacetylation impairs plasticity induction in senescent hepatocytes. (Related to Figure 4-1) (A) Immunofluorescence analysis of HNF4A (red) and KI67 (green) in reprogrammed D52-Hep at day 6. (B) Immunofluorescence analysis of HNF4A (red) and KI67 (green) in reprogrammed D82-Hep at day 6. (C) Venn diagram showing the total and common number of down-regulated genes (<0.5-fold) in D52-Hep and D72-Hep compared to D22-Hep. (D) KEGG pathway analysis of commonly down-regulated genes (<0.5-fold) in D52-Hep and D72-Hep compared to D22-Hep. (E) FACS-based analysis of H3K9ac, H3K18ac, H3K27ac, H3K14ac, and H3K56ac in D22-Hep. (F) Comparison of H3K9ac, H3K18ac, and H3K27ac levels in D22-Hep and D52-Hep. (G) Quantification of median fluorescence intensity (MFI) of H3K9ac, H3K18ac, and H3K27ac in senescent N-Hep. (H) Schematic illustration of the deterioration of hepatocyte plasticity induction mediated by histone hypoacetylation in senescent hiPSC-Hep. [Figure 4-3]HDAC inhibitors improve the plasticity of D52-Hep. (Related to Figure 4-1) (A) Left: Image analysis based on Hoechst staining of cells reprogrammed from D52-Hep in RM containing NaB (0, 0.125, 0.25, or 0.5 mM) on day 8. Right: Quantification of improved cell proliferation of D52-pHC in RM containing NaB (0, 0.125, 0.25, or 0.5 mM). All values ​​were normalized to those of the NaB=0 group. (B) Prolonged proliferation period curve of D52-pHC (from P0 to P10). (C) Phase contrast images of D52-pHC (left) and D52-pHC-derived hepatocytes (D52-pHC-Hep, right). (D) Macroscopic image of D52-pHC-derived cholangiocytes (D52-pHC-Cho, left) and a single cholangiocyte cyst (right). (E) Q-PCR analysis of ALB, A1AT, CYP2C9, and CYP2C19 expression in D52-pHC and D52-pHC-Hep. (F) Q-PCR analysis of SOX9, HNF6, CFTR, and GGT expression in D52-pHC and D52-pHC-Cho. (G) Analysis of EZH2 transcription in D22-Hep and D52-Hep before (RM-D0) and after (RM-D6) RM treatment. (H) Images of cells reprogrammed from D52-Hep in RM+NaB (NaB) and RM+VPA (VPA) with or without DNZep: Cell numbers were counted on day 8 and normalized to day 1. (I) FACS-based analysis of H3K9ac, H3K18ac, and H3K27ac levels in D52-Hep and D52-pHC. (J) Analysis of histone acetylation binding sites in the EZH2 promoter from the ENCODE dataset; GH07J148882 and GH07J148940 are the two highest-scoring EZH2 enhancers. (K) Schematic diagram of HDAC inhibitors (HDACi) improving hepatocyte plasticity in senescent hepatocytes. [Figure 5-1]Induction of pHC formation from PHHs. Fluorescence-activated cell sorting-based analysis of H3K9ac, H3K18ac, and H3K27ac in primary human hepatocytes (PHHs from donors aged 2 months (2M-PHH), 39 years (39Y-PHH), and 78 years (78Y-PHH)). (B) Quantification of the median fluorescence intensity of H3K9ac, H3K18ac, and H3K27ac in PHHs, N-Heps, and pHCs. (C) Phase-contrast images at D1 and D6 of PHHs cultured in RM + NaB. (D) Quantification of proliferation induced by PHHs incubated in medium consisting of RM or RM + NaB: data were normalized to the respective cell numbers at D1. (E) Microarray analysis of mRNA expression of cell cycle-related genes in PHHs and PHH-pHCs. (F) Microarray analysis of mRNA expression of hepatic progenitor cell markers in PHH and PHH-pHC. (G) Immunofluorescence staining of KI67, HNF4A, SOX9, ALB, and CK19 in PHH-pHC. See also Figure 5-2. [Figure 5-2]Characteristics of PHH plasticity (related to Figure 5-1). (A) Quantification of the improvement in PHH-pHC proliferation by NaB and FGF2 in inducing plasticity of 2M-PHH and 39Y-PHH on laminin-511-coated plates. Values ​​were normalized by the cell counts in each RM+NaB group. (B) Comparison of liver functional characteristics between PHH and PHH-pHC. (C) Images of 2M-PHH-pHC passage on plates coated with different matrices. (D) Phase-contrast images of PHH-pHC (left) and PHH-pHC-derived hepatocytes. (E) Q-PCR analysis of ALB, A1AT, CYP2C9, and CYP2C19 expression in PHH-pHC and PHH-pHC-Hep. (F) Immunostaining of ALB in PHH-pHC-Hep. (G) ELISA-based assay of ALB secretion during hepatocyte differentiation from PHH-pHC to PHH-pHC-Hep. (H) Macroscopic image of PHH-pHC-derived cholangiocytes (PHH-pHC-Cho, top) and a single cholangiocyte cyst (bottom). (I) Q-PCR analysis of CK19, SOX9, HNF6, CFTR, AQP1, and SSTR2 expression in PHH-pHC and PHH-pHC-Cho. Values ​​were normalized to gene expression in PHH-pHC. (J) Representative images of active CLF export from the cyst lumen of PHH-pHC-Cho and FITC loading as a control. Fluorescence intensity was calculated along the white arrow in the left image. [Figure 6-1]Engraftment of PHH-pHC in a mouse model of liver injury. (A) ELISA-based analysis of hALB secretion in PHH-Tx and PHH-pHC-Tx mice 4 weeks after transplantation. (B) ELISA-based analysis of hA1AT secretion in PHH-Tx and PHH-pHC-Tx mice 4 weeks after transplantation. (C) ELISA-based analysis of hFerritin secretion in PHH-Tx and PHH-pHC-Tx mice 4 weeks after transplantation. (D) Immunofluorescence assay of hALB, hNUMA, and hCK19 in the livers of PHH-Tx and PHH-pHC-Tx mice 4 weeks after transplantation. (E) Quantification of engraftment size in the livers of PHH-Tx and PHH-pHC-Tx mice 4 weeks after transplantation. (The numbers 1, 2-4, 5-9, and 10 in the captions indicate the number of cells in each cluster.) (F) Quantification of hALB+hCK19+ and hALB+hCK19- engraftment in the livers of PHH-Tx and PHH-pHC-Tx mice 4 weeks after transplantation. (G) ELISA-based kinetic analysis of human ALB secretion in 78Y-PHH-Tx and 78Y-pHC-Tx mice after transplantation (n=3 per group). (H) Immunofluorescence assay of human ALB, CYP3A4, and ZO-1 in the livers of 78Y-pHC-Tx mice 12 weeks after transplantation. [Figure 6-2]PHH-pHC engraftment in a mouse model of liver injury (related to Figure 6-1). (A) ELISA-based assay of AFP secretion in PHH Tx and PHH-pHC Tx mice at 4 weeks post-transplant. (B) Amplification curves of human ALB in PHH Tx and PHH-pHC Tx mice, determined by PCR based on human-specific markers. Non-Tx: mice transplanted with PBS only; NTC: no template control. (C) Comparison of ALB+ engraftment in 78Y-PHH Tx and 78Y-pHC Tx mice at 4 weeks post-transplant. (D) Immunofluorescence analysis of ALB and KI67 in PHH Tx and PHH-pHC Tx mice at 4 weeks post-transplant. (E) Q-PCR analysis of PHH-pHC maturation at 12 weeks post-transplant based on human-specific markers. (F) Immunofluorescence analysis of hALB, hNuMA, hA1AT, hCK8 / 18, hKI67, and hAFP in PHH-pHC Tx mice 12 weeks after transplantation. [Figure 6-3] HDAC inhibitors improve the induction of pHCs in aging models. (A) Detection of GOT and GPT in young mice (n = 6), aged mice (Aged, n = 4), and NaB-treated aged mice (Aged + NaB, n = 6) after 21 days of feeding a choline-deficient, ethionine-supplemented (CDE) diet. (B) Macroscopic images of mouse livers after 21 days of feeding a CDE diet. (C) Hematoxylin and eosin staining of mouse livers after 21 days of feeding a CDE diet. (D) Kaplan-Meier survival curves of mice fed a CDE diet. (E, F) Immunofluorescent staining (E) and quantification (F) of Ki67 in liver sections from CDE-fed mice. (G, H) Immunofluorescent staining (G) and quantification (H) of Epcam (epithelial cell adhesion molecule) in liver sections from CDE-fed mice. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below. 1. A novel method for hepatocyte induction and the creation of a hepatocyte aging model The present invention provides a method for producing hepatocytes, which comprises differentiating endoderm cells into hepatocytes in the presence of an FGF family member, HGF, an IL6 family member, and dexamethasone. The method of the present invention is a simple method for producing hepatocytes, which can differentiate endoderm cells into hepatocytes in a single step.

[0016] Fibroblast growth factors (FGFs) are a type of growth factor 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, with FGF2 being preferred.

[0017] Hepatocyte growth factor (HGF) was discovered in the blood of partially hepatectomized rats as a hepatocyte proliferation promoter. It was purified as a cytokine that strongly promoted the proliferation of primary cultured hepatocytes, making it a promising candidate for a liver regeneration factor that supports the liver's robust regenerative capacity. HGF is a large growth factor, and HGF mRNA is a single-chain precursor of 728 amino acid residues that is translated as biologically inactive pro-HGF (94 kD). After removal of the N-terminal signal peptide and secretion, HGF undergoes specific cleavage (processing) at the Arg-Val site by proteases such as HGF activator (HGFA), coagulation factor (XIa), urokinase (u-PA), and matriptase, resulting in the production of mature (active) HGF, a heterodimer consisting of a disulfide-bonded α-chain (62 kD) and a β-chain (34 kD). HGF promotes the proliferation of primary cultured rat hepatocytes and not only hepatocytes but also various epithelial cells, endothelial cells, and some mesenchymal cells. It also possesses versatile physiological activities, such as promoting cell proliferation, cell scattering and cell motility, anti-apoptosis (cell death), morphogenesis (tubule formation, etc.), angiogenesis, anti-fibrotic effects, and immune response regulation, contributing to the regeneration and protection of tissues and organs. The HGF receptor is a product of the proto-oncogene c-met and has tyrosine kinase activity, and its diverse biological effects are mediated by c-Met. During liver regeneration, increased HGF gene expression in the liver, spleen, and lungs, as well as elevated HGF levels in the blood and liver, precede the induction of hepatic DNA synthesis. Liver regeneration is inhibited by the administration of anti-HGF antibodies. HGF is produced and secreted primarily by mesenchymal cells, such as fibroblasts, adipocytes, neutrophils, macrophages, and vascular endothelial cells, and acts paracrinely on normal epithelial cells and cancer cells. On the other hand, cancer cells also produce HGF and have been reported to activate their own c-MET through an autocrine mechanism. Cells known to produce HGF in the liver are stellate cells and sinusoidal endothelial cells of the liver sinusoids. Taking liver regeneration as an example, various acute injuries, such as partial hepatectomy, hepatitis, and hepatic ischemia, increase HGF expression not only in the injured liver but also in distant intact organs such as the lungs and kidneys, resulting in elevated blood HGF levels.In fact, administration of antibodies neutralizing HGF activity to rats and mice with liver injury significantly exacerbates liver injury and leads to liver regeneration failure. Similar results have been observed in other organ injuries, demonstrating that HGF is an endogenous factor involved in the regeneration and protection of various tissues and organs, including the liver, kidneys, lungs, cardiovascular system, and nervous system. HGF production in cultured cells is induced by PKC activators, PKA activators, cAMP elevators, various growth factors, and inflammatory cytokines (e.g., IL-1 and TNF-α), and is suppressed by TGF-β, glucocorticoids, active vitamin D, and retinoic acid. Taking advantage of its potent proliferation-promoting effect on various cells, numerous studies have demonstrated the potential for HGF as a therapeutic agent for various intractable organ diseases, including liver cirrhosis, chronic renal failure, pulmonary fibrosis, myocardial infarction, and arteriosclerosis obliterans. Furthermore, serum and plasma HGF quantification ELISA kits are used to predict the progression 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, with oncostatin being preferred.

[0019] Dexamethasone is a synthetic corticosteroid that exerts anti-inflammatory effects through the same mechanism as natural glucocorticoids and is used to treat acute inflammation, chronic inflammation, autoimmune diseases, allergic diseases, and other conditions.

[0020] Endodermal cells express endodermal markers and can differentiate into various endodermal cells (such as lung, liver, pancreas, stomach, small intestine, and large intestine cells).

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

[0022] Endodermal cells can be induced to differentiate from pluripotent stem cells. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). The method for differentiating iPS cells into endoderm cells is described in the Examples below. Specifically, hiPSC clones were differentiated into endoderm cells by culturing them for 7 days on plates coated with GFR Matrigel or laminin 511 in 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 days 1 to 3. This method may be modified as appropriate.

[0023] The endoderm cells may also be collected from a living organism.

[0024] The endoderm cells may be derived from humans, 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.

[0025] Endodermal cells can be differentiated into hepatocytes by culturing them in a medium containing a member of the FGF family, HGF, a member of the IL6 family, and dexamethasone.

[0026] Examples of basal media that can be used include SFD medium (described in WO2016093222), DMEM / F12, DMEM, IMDM, RPMI1640, and Williams' Medium E. 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 the FGF family member, HGF, IL6 family member, and dexamethasone in the medium may be adjusted appropriately. For example, when FGF2 is used as the FGF family member, the FGF2 concentration is usually 0.01 to 1000 ng / mL, preferably 0.1 to 100 ng / mL, and more preferably 1 to 50 ng / mL. The HGF concentration is usually 1 to 1000 ng / mL, preferably 5 to 100 ng / mL, and more preferably 5 to 50 ng / mL. When oncostatin (OSM) is used as the IL6 family member, the OSM concentration is usually 1 to 1000 ng / mL, preferably 5 to 100 ng / mL, and more preferably 10 to 50 ng / mL. The concentration of dexamethasone is usually 1 to 1,000,000 nM, preferably 10 to 10,000 nM, and more preferably 50 to 500 nM.

[0028] Nicotinamide may be added to the medium. The concentration of nicotinamide in the medium is usually 0.01 to 1000 mM, preferably 0.1 to 100 mM, and more preferably 1 to 10 mM. Nicotinamide is preferably added from the 8th day of culture onwards.

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

[0030] Endodermal cells are preferably cultured by seeding them on a gel. The gel to be used is not particularly limited, but examples thereof include GFR Matrigel (manufactured by Corning).

[0031] Endodermal cells may be cultured on vessels coated with a major component of tissue basement membranes, such as laminin.

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

[0033] The culture period is not particularly limited, but is preferably 1 to 150 days, and 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 hepatocyte-like cell morphology can be observed. Examples of hepatocyte-specific genes include TBX3, TTR, DLK, CK19, and EPCAM (hepatoblasts), as well as A1AT, ALB, G6PC, ASGR1, TAT, TDO2, CYP2C9, CYP2C19, CYP3A4, and CYP7A1 (hepatocytes). In the examples described below, during the differentiation process from endoderm cells to hepatocytes, the cells were in a hepatoblast state at an early stage of differentiation (e.g., day 5 of differentiation), and with further culture, they began to express liver function. Liver function will be discussed later.

[0035] According to the present invention, pluripotent stem cells can be induced to become hepatocytes in a simple manner.

[0036] Hepatocytes produced by the method of the present invention can survive and maintain their hepatocyte functions for a long period of time, making it possible to achieve long-term culture of hepatocytes in vitro and mimic the aging process.

[0037] Hepatocytes produced by the methods of the present invention can survive in vitro in monolayer culture while retaining hepatocyte function for periods 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. There is no particular upper limit to the period during which hepatocytes produced by the methods of the present invention can survive while retaining hepatocyte function. The present inventors have observed that hepatocytes produced by the methods of the present invention can survive for 82 days while retaining the ability to secrete albumin.

[0038] Hepatocyte functions maintained by hepatocytes prepared by the method of the present invention include, for example, albumin secretion ability, drug metabolism ability, indocyanine green (ICG) uptake and excretion ability, glycogen storage ability, low-density lipoprotein uptake ability, gene expression ability, etc. In one embodiment of the present invention, hepatocytes prepared by the method of the present invention can survive for 12 days or more while maintaining the ability to secrete albumin, and can also survive for 12 days or more while maintaining hepatocyte functions other than the ability to secrete albumin.

[0039] Albumin secretion ability can be measured using a commercially available albumin ELISA assay set.

[0040] Drug-metabolizing capacity can be measured using commercially available cytochrome P450-related assay kits.

[0041] The uptake and excretion of ICG can be examined by seeding cells in a medium containing ICG, incubating for an appropriate period of time, and then observing the cells under a microscope.

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

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

[0044] Gene expression testing can be performed as follows: PureLink TM Isolate total RNA using the RNA Mini Kit. Use RNA (<2 μg) as a template to synthesize first-stranded cDNA using the High-Performance cDNA Reverse Transcription Kit according to the manufacturer's instructions. Perform Q-PCR using the cDNA and specific primers and probes from the Universal Probe Library.

[0045] Hepatocytes produced by the methods of the present invention may exhibit characteristics of senescence, such as increased cell volume, expression of senescence-associated genes, increased inflammatory responses, DNA damage, increased levels of intracellular reactive oxygen species, increased levels of senescence-associated β-galactosidase, epigenetic changes, shortened telomeres, decreased mitochondrial function, metabolic abnormalities, and poor responsiveness to growth factors.

[0046] The cell volume can be determined by incubating the cells with a standard dilution of Hoechst 33342, washing the cells with PBS, and analyzing the photographs using a fluorescent microscope.

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

[0048] An example of an inflammatory response is an increase in intracellular inflammation-related factors, 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] Intracellular reactive oxygen species levels were measured using CellROX TM Measurement can be performed by adding a reagent such as Deep Red Reagent (Thermo Fisher Scientific) to cells, observing them under a microscope, or counting the number of cells that emit fluorescence.

[0051] Senescence-associated β-galactosidase levels 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] Telomere length can be measured using a commercially available telomere length qPCR kit (ScienCell Research Laboratories).

[0054] An example of mitochondrial function is the production of ATP through oxidative phosphorylation (phosphorylation of ADP) via the electron transport chain. A decline in mitochondrial function can be confirmed by measuring a 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 those related to 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 metabolic-related gene expression using the commercially available SurePrint G3 Human Gene Expression 8x60K (Agilent Technologies).

[0056] Poor response to growth factors can be assessed by adding growth factors, culturing for several days, incubating the cells with a standard dilution of Hoechst 33342, washing the cells with PBS, and analyzing photographs taken under a fluorescence microscope.

[0057] Hepatocytes generated by the method of the present invention are different from hepatocytes derived from living organisms. Furthermore, compared with hepatocytes induced by conventional methods, hepatocytes generated by the method of the present invention exhibit significantly upregulated expression of liver-specific genes (ALB, G6PC, ASGR1, TAT, TDO2, CYP2C9, CYP2C19, CYP3A4, and CYP7A1), ALB secretion, and CYP3A4 activity. While hepatocytes induced by conventional methods cannot be cultured in vitro for more than 30 days, hepatocytes generated by the method of the present invention can be cultured in vitro for more than 50 days, reproducing the hepatocyte aging process.

[0058] The following are examples of the upregulated values ​​of "liver-specific gene expression, ALB secretion, and CYP3A4 activity" in hepatocytes produced by the method of the present invention (N-Hep on day 22 of culture in the Examples described below) compared to hepatocytes induced by conventional methods. TIFF0007824587000001.tif57135

[0059] TIFF0007824587000002.tif21135

[0060] TIFF0007824587000003.tif21141

[0061] Conventional method A: Si-Tayeb, K., Noto, FK, Nagaoka, M., Li, J., Battle, MA, Duris, C., North, PE, Dalton, S., and Duncan, SA (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. The cells in the table have not been passaged. Measurement method for liver-specific genes: Q-PCR (see Examples below) ALB secretion measurement method: Measured by ELISA (see Examples below) Measurement of CYP3A4 activity: Commercially available P450-Glo TM Measurement was performed using the CYP3A4 Assay kit (Promega, V8801).

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

[0063] Furthermore, the secretion of ALB from hepatocytes produced by the method of the present invention can be increased by at least two times, preferably at least three times, and more preferably at least four times, compared to hepatocytes induced by conventional method A.

[0064] Furthermore, hepatocytes produced by the method of the present invention have CYP3A4 activity, whereas hepatocytes induced by conventional method A do not have detectable CYP3A4 activity.

[0065] Compared to hepatocytes induced by conventional method B, hepatocytes produced by the method of the present invention can exhibit increased expression of ALB by at least 2-fold, preferably at least 4-fold, and more preferably at least 5-fold; G6PC by at least 20-fold, preferably at least 25-fold, and more preferably at least 29-fold; ASGR1 by approximately the same amount; TAT by at least 30-fold, preferably at least 40-fold, and more preferably at least 50-fold; TDO2 by at least 2-fold, preferably at least 4-fold, and more preferably at least 5-fold; CYP2C9 by approximately the same amount; CYP2C19 by at least 1.2-fold, preferably at least 1.5-fold, and more preferably at least 2-fold; CYP3A4 by at least 3-fold, preferably at least 5-fold, and more preferably at least 8-fold; and CYP7A1 by at least 2-fold, preferably at least 4-fold, and more preferably at least 5-fold.

[0066] Furthermore, the secretion of ALB from hepatocytes produced by the method of the present invention can be increased by 1.2 times or more, preferably 1.5 times or more, and more preferably 2 times or more, compared to hepatocytes induced by conventional method B.

[0067] Furthermore, the CYP3A4 activity of hepatocytes produced by the method of the present invention can be increased by 30 times or more, preferably 40 times or more, and more preferably 47 times or more, compared to hepatocytes induced by conventional method B.

[0068] Examples of measured values ​​for liver-specific gene expression, ALB secretion, and CYP3A4 activity for hepatocytes prepared by the method of the present invention (N-Hep on day 22 of culture in the Examples described below), hepatocytes induced by conventional method A, and hepatocytes induced by conventional method B are described below. JPEG0007824587000004.jpg250111

[0069] TIFF0007824587000005.tif62132

[0070] TIFF0007824587000006.tif62149

[0071] N-Hep: Hepatocytes produced by the method of the present invention (N-Hep on day 22 of culture in the Examples described below) Human hepatocytes: Bioreclamation IVT IVT-F00995-P-AKB (donor: female, age 39) The cells in the table have not been passaged. Measurement method for liver-specific genes: Q-PCR (see Examples below) ALB secretion measurement method: Measured by ELISA (see Examples below) Measurement of CYP3A4 activity: Commercially available P450-Glo TM Measured using a CYP3A4 Assay kit (Promega, V8801).

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

[0073] The hepatocytes produced by the method of the present invention may have an ALB secretion level, as quantified by ELISA, of 2 μg / ml / 24 h / 1 million or more, preferably 4 μg / ml / 24 h / 1 million or more, and more preferably 5 μg / ml / 24 h / 1 million or more.

[0074] The hepatocytes produced by the method of the present invention were obtained by the commercially available P450-Glo TM CYP3A4 activity measured using a CYP3A4 Assay kit (Promega, V8801) may be 50,000 RLU / 4h / ml / million cells or more, preferably 100,000 RLU / 4h / ml / million cells or more, and more preferably 200,000 RLU / 4h / ml / million cells or more.

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

[0076] 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, and FGF23, with FGF2 being preferred.

[0077] The hepatocytes used for inducing hepatic progenitor cells or pHC or plasticity may be any cells that express liver-specific genes and exhibit liver function, and the hepatocytes may be hepatocytes differentiated from pluripotent stem cells, cells obtained by subculture of hepatocytes differentiated from pluripotent stem cells, primary cultured hepatocytes isolated from biological tissue, cells obtained by subculture of primary cultured hepatocytes isolated from biological tissue, or a 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 onto plates coated with GFR Matrigel or laminin 511 using nicotinamide-free hepatic differentiation medium (HDM). The medium was replaced with HDM daily from days 8 to 10, and from day 11 onward, the medium was replaced every two days to induce differentiation into 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 may be modified as appropriate. To subculture these hepatocytes, they were seeded onto plates coated with GFR Matrigel or laminin 511 in HDM containing nicotinamide, and the medium was changed every two days. 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 needed.

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

[0081] Hepatocytes may be primary cultured hepatocytes isolated from biological tissue or subcultured cells. In the examples described below, primary human hepatocytes from donors aged 2 months, 39 years, and 78 years were used. To culture or subculture primary human hepatocytes, they were seeded on plates coated with GFR Matrigel or laminin 511 using human hepatocyte culture medium, and the medium was changed every two days. Two types of human hepatocyte culture medium were used. 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 may be modified as needed.

[0082] When hepatic progenitor cells are derived from primary cultured cells isolated from human biological tissue, cells subcultured therefrom, or a combination thereof, the age of the human is typically 0 to 120 years, preferably 0 to 100 years, and more preferably 0 to 80 years. When primary cultured cells isolated from biological tissue of an animal other than a human, cells subcultured therefrom, or a combination thereof are used, the appropriate age in weeks, months, and years can be estimated from the human age. The number of passages of the cultured cells is typically 1 to 5, preferably 1 to 4, and more preferably 1 to 3, and the number of days the cells are cultured is typically 1 to 30 days, preferably 1 to 20 days, and more preferably 1 to 10 days.

[0083] The hepatocytes may be hepatocytes differentiated from pluripotent stem cells, cells obtained by subculture of hepatocytes differentiated from pluripotent stem cells, primary cultured hepatocytes isolated from biological tissue, or a combination of cells obtained by subculture of primary cultured hepatocytes isolated from biological tissue.

[0084] The hepatocytes may be derived from humans, 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.

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

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

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

[0088] The medium may be supplemented with EGF, HGF, a ROCK inhibitor, a selective inhibitor of ALK4, ALK5, or ALK7 (TGF-β receptor inhibitor), or a GSK-3β inhibitor.

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

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

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

[0092] ALK4, ALK5, and ALK7 are TGF-β1 activin receptor-like kinases (ALKs), and 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, and Vactosertib. When A83-01 is used as a selective inhibitor of ALK4, ALK5, and ALK7 (TGF-β inhibitor), the concentration of A83-01 is generally 0.01 to 100 μM, preferably 0.1 to 10 μM, and more preferably 0.1 to 5 μM.

[0093] GSK-3β is a serine-threonine protein kinase, and 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, and TWS119. When using CHIR99021 as a GSK-3β inhibitor, the concentration of CHIR99021 is usually 0.01 to 100 μM, preferably 0.01 to 20 μM, and more preferably 0.1 to 20 μM.

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

[0095] Hepatocytes are preferably cultured by seeding them on a gel. The gel to be used is not particularly limited, but examples thereof include GFR Matrigel (manufactured by Corning).

[0096] Hepatocytes may be cultured on vessels coated with a major component of tissue basement membranes, such as laminin.

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

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

[0099] The hepatic progenitor cells induced from hepatocytes by the method of the present invention are proliferative and have the ability to differentiate, for example, alpha-fetoprotein (AFP)-negative hepatic progenitor cells that have the ability to proliferate and the ability to differentiate in two directions into hepatocytes and bile duct epithelial cells.

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

[0101] Hepatic progenitor cells can be subcultured by seeding them on a matrigel or laminin coating using a medium containing a member of the FGF family, and then replacing the medium with a ROCK inhibitor-free medium at an appropriate time.

[0102] The proliferation potential of hepatic progenitor cells can be examined by calculating the cell population doubling time, as described in the Examples below. Hepatic progenitor cells can have high proliferation potential, with a cell population doubling time of 14 to 36 hours.

[0103] Hepatic progenitor cells induced from hepatocytes by the method of the present invention can differentiate into hepatocytes, bile duct cells, and the like.

[0104] To differentiate hepatic progenitor cells into hepatocytes, culture in a hepatic differentiation medium can be performed. HDM (described above in 1) can be used as the hepatic differentiation medium. Vitamin A metabolites (e.g., retinoic acid (RA)) can be added to the hepatic differentiation medium at an appropriate time and for an appropriate period. In the examples described below, the medium was replaced with HDM containing RA for 8 days, followed by HDM without RA for the next 7 days. The medium was replaced every two days. The total hepatocyte differentiation period was 15 days. This method can be modified as needed. Hepatocytes differentiated from hepatic progenitor cells can express functional genes (e.g., AAT, CTP2C9, CYP2C19, etc.) and exhibit increased ALB secretion. Furthermore, hepatocytes differentiated from hepatic progenitor cells can form tight junctions between their own cells and acquire ammonia excretion and liver functions (uptake and excretion of ICG, glycogen storage, and low-density lipoprotein uptake).

[0105] To differentiate hepatic progenitor cells into cholangiocytes, hepatic progenitor cells can 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 mount drops into untreated 24-well plates and incubated at 37°C in a 5% CO2 incubator for 30 minutes. The wells were then filled with cholangiocyte differentiation medium (CDM). The 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 may be modified as needed. Cholangiocytes differentiated from hepatic progenitor cells can develop into ring-shaped structures and then into cyst structures. Q-PCR analysis showed that these cyst structures were characterized by upregulated transcription of cholangiocyte-characteristic genes (Figures 3-1G and 3-2H). Cholangiocytes in these cysts stained positive for CK19, F-ACTIN, and SOX9, and negative for AFP, ALB, and HNF4A. Furthermore, these cysts may have characteristics of mature bile ducts (including a lumen with apical and basolateral plasma membranes, microvilli, primary cilia on the apical membrane, tight junctions, and multivesicular bodies). These cholangiocytes may have biliary functions: 1) transport of rhodamine 123 into the lumen and 2) specific export of bile acids from the lumen.

[0106] Thus, the present invention provides a method for producing hepatocytes, comprising inducing the differentiation of hepatic progenitor cells obtained by a method for producing hepatic progenitor cells, including culturing hepatocytes in the presence of an FGF family member, into hepatocytes. The present invention also provides a method for producing cholangiocytes, comprising inducing the differentiation of hepatic progenitor cells obtained by a method for producing hepatic progenitor cells, including culturing hepatocytes in the presence of an FGF family member. The present invention also provides a culture medium containing FGF10, retinoic acid, and forskolin for inducing the differentiation of the hepatic progenitor cells into cholangiocytes. The culture medium of the present invention may contain other components contained in the above-mentioned cholangiocyte differentiation medium (CDM). Furthermore, the present invention also provides a culture medium kit for inducing the differentiation of the hepatic progenitor cells into cholangiocytes, comprising an agent containing FGF10, retinoic acid, and forskolin for use in the culture medium used to induce the differentiation of the hepatic progenitor cells into cholangiocytes; and instructions for using the agent in the culture medium used for the differentiation induction. The agent containing FGF10, retinoic acid, and forskolin may contain other components contained in the above-mentioned cholangiocyte differentiation medium (CDM).

[0107] Hepatic progenitor cells induced from hepatocytes by the method of the present invention can proliferate over a long period of time, for example, at least 20 passages, thereby enabling the mass production of hepatocytes and bile duct cells.

[0108] 3. Improved plasticity of senescent hepatocytes In the above-mentioned 2, by further adding a drug that induces histone hyperacetylation to the culture medium containing a member of the FGF family, the plasticity of aged hepatocytes can be improved and hepatic progenitor cells can be induced.

[0109] The present invention provides a method for producing hepatic progenitor cells, which comprises culturing hepatocytes in the presence of a member of the FGF family and an agent that induces histone hyperacetylation.

[0110] Hepatocyte aging can be suppressed and plasticity can be induced by drugs that induce histone hyperacetylation. Herein, hepatocyte plasticity refers to the ability of hepatocytes to transform into proliferative hepatic progenitor cells that have the ability to differentiate into both hepatocytes and bile duct cells. By improving hepatocyte plasticity, hepatocytes become more responsive to induction of proliferation.

[0111] Therefore, the present invention provides a method for suppressing hepatocyte aging using an agent that induces histone hyperacetylation. From another perspective, the present invention provides an agent for suppressing hepatocyte aging (anti-aging drug) that contains an agent that induces histone hyperacetylation as an active ingredient. The agent for suppressing hepatocyte aging of the present invention can be applied both in vitro (cultured cells) and in vivo (cells in a living organism).

[0112] Hepatocyte senescence can be confirmed by decreased cell proliferation, decreased hepatocyte function, upregulation of senescence-related genes, and expression of senescence-related markers, as well as by increased cell volume, increased inflammatory response, DNA damage, epigenetic changes, shortened telomere length, decreased mitochondrial function, metabolic abnormalities, and poor responsiveness to growth factors.

[0113] Cell proliferation can be determined by calculating the population doubling time.

[0114] The functions of hepatocytes and their measurement methods were described above in 1.

[0115] The functions of aging-related genes and their measurement methods were also described above in 1.

[0116] Examples of senescence-related markers include intracellular reactive oxygen species levels and cellular senescence-related β-galactosidase levels, and methods for measuring these are described above in 1.

[0117] Measurements of increased cell volume, increased inflammatory response, DNA damage, epigenetic changes, shortened telomere length, decreased mitochondrial function, metabolic abnormalities, and poor responsiveness to growth factors are also discussed above in 1.

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

[0119] The present invention also provides a method for increasing hepatocyte plasticity using an agent that induces histone hyperacetylation. From another perspective, the present invention provides an agent for increasing hepatocyte plasticity, comprising as an active ingredient an agent that induces histone hyperacetylation. By increasing plasticity, hepatic progenitor cells can be induced even from aged hepatocytes derived from pluripotent stem cells or from hepatocytes derived from elderly people. Furthermore, by increasing plasticity, the responsiveness of hepatocytes to proliferation induction can be enhanced. The agent of the present invention can be a regeneration inducer that improves the regenerative capacity of senescent cells. The agent of the present invention can promote liver regeneration in elderly people. Furthermore, the agent of the present invention enables successful transplantation therapy using grafts or cells derived from elderly donors. The agent of the present invention can be used both in vitro (cultured cells) and in vivo (cells in the living body).

[0120] The agent that induces histone hyperacetylation may be used in combination with a member of the FGF family, such as FGF2, FGF1, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, or a combination thereof, with FGF2 being preferred.

[0121] Drugs that induce histone hyperacetylation include histone deacetylase inhibitors, such as 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, and 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 of such antihistamines include cyclosporine, ...

[0122] The hepatocytes targeted for suppressing aging or increasing plasticity by a drug that induces histone hyperacetylation are preferably, but not limited to, senescent hepatocytes. The hepatocytes may be hepatocytes differentiated from pluripotent stem cells, cells subcultured from hepatocytes differentiated from pluripotent stem cells, primary cultured hepatocytes isolated from biological tissue, cells subcultured from primary cultured hepatocytes isolated from biological tissue, or a combination thereof.

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

[0124] When the hepatocytes are cells obtained by subculture of hepatocytes differentiated from pluripotent stem cells, "senescent hepatocytes" are, for example, cells obtained by subculture of hepatocytes differentiated from pluripotent stem cells (e.g., iPSCs) for 25 days or more and then subcultured once or more.

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

[0126] When hepatocytes are cells obtained by subculture of primary cultured hepatocytes isolated from biological tissue, "senescent hepatocytes" refer to, for example, human hepatocytes that have been cultured for 20 days or more after isolation.

[0127] The hepatocytes may be derived from humans, 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] Cultivating hepatocytes in a medium containing agents that induce histone hyperacetylation can suppress aging and increase plasticity.

[0129] The medium used may be the medium described in 2 (the medium used for inducing (reprogramming) hepatocytes into hepatic progenitor cells). That is, the basal medium may be SFD medium (described in WO2016093222), (DMEM / F12, DMEM, IMDM, RPMI1640, Williams' Medium E), or the like, and may be supplemented with a member of the FGF family.

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

[0131] Other components that may be added to the medium and their concentrations are also as described above in 2.

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

[0133] Hepatocytes are preferably cultured by seeding them on a gel. The gel to be used is not particularly limited, but examples thereof include GFR Matrigel (manufactured by Corning).

[0134] Hepatocytes may be cultured on vessels coated with a major component of tissue basement membranes, such as laminin.

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

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

[0137] 4. Application The present invention enables the mass production of hepatocytes and bile duct cells from pluripotent stem cells (for example, iPS cells).

[0138] The human hepatocytes and other cells created by the method of the present invention can be used for industrial purposes such as in vitro drug metabolism tests, liver toxicity tests, and hepatitis virus infection tests.

[0139] Human hepatocytes etc. created by the method of the present invention include the following. 1. Hepatocytes produced by a method for producing hepatocytes, which comprises 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 are produced by a method for producing hepatic progenitor cells, which includes culturing hepatocytes in the presence of a member of the FGF family. These cells have the ability to proliferate and differentiate into both hepatocytes and bile duct epithelial cells. 3.2 Cells induced to differentiate from the hepatic progenitor cells (e.g., hepatocytes, bile duct cells).

[0140] 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 transplant composition. The transplant site for hepatocytes may be any site that allows transplantation, including intracranial, mesenteric, liver, spleen, kidney, subrenal capsule, and portal vein. The number of hepatocytes per transplant is determined based on the number of cells per 1 cm of the transplant site. 2The number of cells is preferably 100,000 to 100,000,000 per cell, more preferably 1,000,000 to 50,000,000 cells, and even more preferably 1,000,000 to 10,000,000 cells per cell. For transplantation, EGF, HGF, a ROCK inhibitor, a TGF-β receptor inhibitor, a GSK-3β inhibitor, or the like may be used. The transplant composition may also contain an agent (described above) that increases the plasticity of hepatocytes.

[0141] Furthermore, an artificial liver can be produced using human hepatocytes and the like created by the method of the present invention.

[0142] Furthermore, human hepatocytes, etc., created by the method of the present invention can be transplanted into non-human animals to produce chimeric animals. A non-human animal (e.g., a mouse) into which the cells are transplanted can mimic the physiological functions of the species (e.g., a human) from which the transplanted cells originate. Using this animal, drug metabolism tests and safety tests of drug discovery compounds can be conducted. The non-human animal preferably has liver failure. Liver failure can be induced by administering ganciclovir. When hepatic progenitor cells are transplanted into a non-human animal, the transplanted hepatic progenitor cells can proliferate and / or differentiate within the non-human animal. Liver regeneration in non-human animals is promoted by transplanting hepatocytes created by the method of the present invention. 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 those for transplantation into humans.

[0143] Furthermore, the present invention makes it possible to promote liver regeneration using an agent that induces histone hyperacetylation. The present invention provides a liver regeneration promoter comprising, as an active ingredient, an agent that induces histone hyperacetylation. The present invention also provides a method for promoting liver regeneration, comprising administering to a subject a pharmaceutically effective amount of an agent that induces histone hyperacetylation. The agent that induces histone hyperacetylation has been described above. Drugs that induce histone hyperacetylation can promote liver regeneration after liver damage. Liver damage can be caused by viral hepatitis (caused by infection with hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, or hepatitis E virus), alcoholic hepatitis, autoimmune hepatitis, primary cirrhosis, drug-induced hepatitis, fatty liver (alcoholic or non-alcoholic), surgical liver resection, traumatic liver damage, aging, liver fibrosis, obesity-related liver damage, and the like. Drugs that induce histone hyperacetylation can be used as pharmaceuticals to treat and / or prevent these diseases and disorders. The agent that induces histone hyperacetylation may be in the form of a salt or a solvate. Drugs that induce histone hyperacetylation can be formulated by standard methods into pharmaceutical preparations (e.g., injections, capsules, tablets, powders, granules, etc.) and administered to subjects (humans or animals). For example, when the drug that induces histone hyperacetylation is NaB, it can be administered orally or parenterally (e.g., nasally, rectally, transdermally, subcutaneously, intravenously, intramuscularly, etc.) at a dose of approximately 10 to 100,000 mg / kg (body weight) per day, preferably approximately 100 to 10,000 mg / kg (body weight) per day, in terms of the amount of active ingredient, in a single dose or in divided doses. The dose and frequency of administration can be adjusted depending on the patient's symptoms, age, and administration method. When using drugs that induce histone hyperacetylation other than NaB, it is recommended to use a dose that produces an effect equivalent to that of NaB at the above-mentioned dose. When formulating into injections, carriers such as distilled water and physiological saline are preferably used. When formulating into capsules, tablets, powders, or granules, excipients such as starch, lactose, sucrose, and calcium carbonate are preferably used; binders such as starch paste, gum arabic, gelatin, sodium alginate, carboxymethylcellulose, and hydroxypropylcellulose are preferably used; lubricants such as magnesium stearate and talc are preferably used; and disintegrants such as starch, agar, crystalline cellulose, calcium carbonate, sodium bicarbonate, and sodium alginate are preferably used. The content of the active ingredient in the formulation can vary between 1 and 99% by weight. For example, when taking the form of tablets, capsules, granules, or powders, the active ingredient is preferably contained in an amount of 5 to 80% by weight, while when taking into account injections, the active ingredient is preferably contained in an amount of 1 to 10% by weight. [Example]

[0144] The present invention will be described in more detail below with reference to examples.

[0145] Example 1 Histone hypoacetylation impairs the plasticity of human hepatocytes during aging.

[0146] Our results may make liver transplants from elderly individuals more useful in clinical practice: liver tissue and hepatocytes from elderly individuals typically have poor regenerative capacity. We discovered how to easily derive hepatocytes from human induced pluripotent stem cells. These hepatocytes mimicked liver aging in a culture dish, and we found that liver plasticity (responsiveness to proliferation induction) was impaired by histone hypoacetylation associated with aging. Promoting histone acetylation improved the plasticity of aged hepatocytes and increased their regenerative capacity in a liver injury model.

[0147] summary Hepatocyte plasticity plays a crucial role in liver regeneration and declines with aging. However, the underlying mechanisms underlying this decline remain unclear. Here, we devised a research strategy using human induced pluripotent stem cell-derived hepatocytes (hiPSC-Hep) to elucidate the underlying mechanisms. We then accumulated information on changes in senescence-related characteristics and expression of senescence-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 senescent hepatocytes was strongly correlated with histone hypoacetylation. Selective inhibition of histone deacetylase significantly improved the plasticity of senescent hiPSC-Hep and primary human hepatocytes. This effect also enhanced the repopulation capacity of senescent primary human hepatocytes in a liver injury model. Thus, aging-associated histone hypoacetylation impairs hepatocyte plasticity, and histone acetylation may be a therapeutic target for improving liver regenerative capacity in elderly individuals.

[0148] Introduction Liver regeneration is a unique phenomenon in which the liver regenerates new tissue to replace lost tissue (dead cell mass, tissue). Because of this phenomenon, the liver appears 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 nonalcoholic fatty liver disease, alcoholic liver disease, and hepatitis C (Kim et al., 2015), and liver transplants from elderly donors have poorer survival rates and overall outcomes compared with transplants from younger donors (Durand et al., 2019; Germani et al., 2012). These findings suggest that aging may weaken the liver's regenerative capacity. In fact, the decline in liver regeneration associated with aging was discovered half a century ago when researchers noticed a significantly reduced proliferative response in aged rat livers 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 aging-associated changes (e.g., metabolic abnormalities, epigenetic changes, reduced responsiveness to growth factors, and shortened telomere length) have been found that may be linked to impaired regeneration (Aikata et al., 2000; Sato et al., 2017; Sawada, 1989; Timchenko, 2009). However, how aging contributes to impaired liver regeneration and how to improve this regenerative capacity in elderly individuals remains unclear, especially in humans.

[0149] Hepatocytes account for over 70% of liver mass, and accumulating evidence suggests that hepatocyte plasticity plays a crucial role in maintaining liver regenerative capacity (Kopp et al., 2016; Li et al., 2016). Lineage tracing experiments in mice have shown that in response to liver injury, hepatocytes can transform into proliferative progenitor-like cells to replace lost stem cells and bile duct cells (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 al., 2018). Therefore, studies investigating the intrinsic link between aging and hepatocyte plasticity will help us understand the role of aging in human liver regeneration. Due to the limitations of human tracing experiments, we wondered whether human hepatocyte plasticity could be modeled in a culture dish. Recently, several groups have reported that primary human hepatocytes (PHHs) can be induced to a proliferative state with repopulation potential (Fu et al., 2019; Kim et al., 2019; Zhang et al., 2018). However, these proliferative PHHs only have limited bipotential differentiation. Furthermore, currently available PHHs are derived only from liver tissue of cadavers or patients with liver disease, which have complex and unreliable extrinsic factors. These factors make it difficult to use these PHHs to discover useful links between aging and hepatocyte plasticity.

[0150] To elucidate these endogenous relationships, an ideal experimental set of hepatocytes would need to be of identical origin—i.e., harvested from the same human individual at different ages. However, obtaining such hepatocytes from the same donor is challenging. Over the past decade, extensive progress has been made in directing the differentiation of human induced pluripotent stem cells (hiPSCs) for applications in regenerative medicine and disease modeling (Stadtfeld and Hochedlinger, 2010; Studer et al., 2015), and several laboratories have adopted methods that enable the use of hiPSCs to model aging-associated neurodegenerative diseases (Miller et al., 2013; Vera et al., 2016). Unfortunately, to date, no hiPSC-derived hepatocytes (hiPSC-Hep) capable of effectively recapitulating the biological aging process have been reported. 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 generating hiPSC-Hep cells that can gradually accumulate senescence markers and characteristics while maintaining long-term liver function. We found that the plasticity of hiPSC-Hep cells was significantly reduced by histone hypoacetylation associated with senescence. Furthermore, treatment with histone deacetylase inhibitors (HDACi) significantly increased the plasticity of senescent hiPSC-Hep and PHH cells, and this effect significantly improved the regenerative capacity of senescent PHH cells in a mouse model of liver injury.

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

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

[0153] Spontaneous reproduction of the liver aging process In contrast to the previously reported rapid degeneration of hiPSC-Hep (Nie et al., 2018), N-Hep maintained cell morphology and hepatic function for a long time (Figures 1-1 D and 1-2 J). Specifically, by day 72 of cell culture, we observed the formation of multinucleated cells accompanied by a decrease in ALB secretion (Figures 1-1 D and 1-2 J). At day 82 (D82), N-Hep lost its hepatocyte morphology and exhibited a high proportion of apoptotic cells (Figure 1-2 K). Meanwhile, we noticed that the cell volume of N-Hep increased during culture, a phenomenon frequently observed during cellular senescence (Neurohr et al., 2019) (Figure 1-2 L). To test whether N-Hep cells were undergoing a senescence process, we performed transcriptome analysis comparing D22-Hep (hepatocytes generated from hiPSCs 22 days later), D52-Hep, and D72-Hep cells and found that senescence-related genes were abundantly expressed in D72-Hep (Figure 1E). Gene ontology analysis indicated that senescence-related hepatic changes (Kim et al., 2015), including increased inflammatory responses and DNA damage, were also elevated in D72-Hep (Figure 1M). Furthermore, levels of intracellular reactive oxygen species (ROS) and senescence-associated β-galactosidase (SA-β-gal), two cellular hallmarks of senescence, gradually increased from D22 to D72 (Figure 1FH). These results indicated spontaneous senescence in N-Hep cells during long-term culture. Next, we investigated whether the aging process of N-Hep could mimic the in vivo hepatocyte aging process. By comparing transcriptional differences between D22-Hep, D72-Hep, young PHH (2 months, 2M-PHH), and elderly PHH (78 years, 78Y-PHH), principal component analysis (PCA) demonstrated that the gene signature of D22-Hep was similar to that of 2M-PHH, and that of D72-Hep was very similar to that of 78Y-PHH (Figure 1-3 A). Furthermore, KEGG pathway analysis revealed that 35 signaling pathways whose expression increased during in vitro aging were also upregulated during in vivo aging, and that 35 signaling pathways whose expression decreased during in vitro aging were also downregulated during in vivo aging (Figure 1-3 B and C). These results suggest that the aging process of N-Hep may represent (or substitute for) the aging process of hepatocytes in vivo.

[0154] Essential role of FGF2 in inducing plasticity in human hepatocytes Next, we tested whether N-Hep cells possess the same plasticity as hepatocytes in vivo. N-Hep cells can be induced to proliferate while undergoing bipotential differentiation in vivo (Figure 2-1A). To induce hepatocyte proliferation, we cultured young N-Hep cells (D22-Hep) in a basal medium containing a previously described cocktail of small molecules (BM+SMs) (which can induce proliferation of rodent hepatocytes) (Katsuda et al., 2017). After 6 days, the reprogrammed cells did not acquire the sustained proliferation potential seen in reprogrammed rodent hepatocytes (Katsuda et al., 2017) (Figure 2-2A), but we did observe cell proliferation (Figure 2-1B and C). These data indicated that inherent differences between rodent and human hepatocytes may have a significant impact on the outcome of plasticity induction. We previously confirmed that FGF2 is a crucial factor promoting hepatocyte differentiation and maturation (Figures 2-2 C 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 culture medium significantly enhanced the induced proliferation potential (Figures 2-1 B, 2C, and 2-2 B). Notably, FGF2-reprogrammed cells acquired the ability to proliferate continuously when FGF2 was added to the above-mentioned medium, called BM+SMs. We termed this FGF2-supplemented medium (RM) (Figure 2-2 A). Quantitative PCR (Q-PCR) analysis revealed that these reprogrammed cells were characterized by enhanced transcription of cell cycle genes (Figure 2-1 D). Furthermore, immunofluorescence analysis indicated that the proliferative cells still belonged to the hepatocyte lineage, because they were positive for both KI67 and HNF4A (Figure 2-1 E). Therefore, we named these reprogrammed cells (hepatic progenitor cells) proliferative hepatocytes (pHCs). Time-lapse imaging suggested that pHCs could be derived from single hepatocytes, similar to other hiPSC-Hep cells (Figure 2C and D).Compared with D22-Hep, D22-pHC showed elevated transcription of hepatic stem / progenitor cell-related genes (Figure 2-1F) and downregulation of liver function-related genes (Figure 2-1G). Furthermore, D22-pHC showed positive staining for HNF4A, SOX9, and CK19, but not for ALB and AFP (Figures 2-1H, 2-2E, and 2-2F).

[0155] To determine how FGF2 induces hepatocyte proliferation, we incubated 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 2-1I). Using a combination of transcriptome analysis and protein-protein interaction network analysis (Jensen et al., 2009), we found that EZH2 was present at the central node of all up-regulated genes, linking the cell cycle gene cluster and the epithelial-mesenchymal transition gene cluster (Figure 2-2G). Furthermore, cells treated with RM showed a clear upregulation of EZH2 transcription, which was suppressed by PD0325901 (Figures 2-1J and 2-2H). By abolishing 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 EZH2 transcription (Figures 2-2I and J). This indicates that EZH2, activated by the FGF2-MAPK axis, is strongly involved in induced proliferation of human hepatocytes. Furthermore, loss- and gain-of-function experiments suggested that the FGF2-EZH2 axis is essential for maintaining the proliferative potential of pHCs (population doubling time: 19.86 ± 0.70 hr) (Figures 2-2K and L). Long-term growth indicated that these D22-pHCs could be passaged at least 20 times without obvious changes in their morphology, proliferation rate, or karyotype (Figures 2-2 M and N). Thus, we successfully induced N-Hep cell proliferation regulated by the FGF2-MAPK-EZH2 axis (Figure 2-1 K).

[0156] Bipotent differentiation of D22-pHC Next, we evaluated the bipotential differentiation of pHC. To induce hepatocyte differentiation, we cultured D22-pHC in a newly developed HDM. After 15 days, differentiated cells exhibited limited ALB production and atypical morphology (Figure 3-2 A and B). To enhance the differentiation process, we modified HDM by adding retinoic acid (RA; Figure 3-1 A), a vitamin A metabolite that mediates liver development and regeneration (Negishi et al., 2010). Finally, we obtained significant improvements in ALB production and typical hepatocyte morphology (Figure 3-2 A and C). D22-pHC-derived hepatocytes (D22-pHC-Hep) showed a clear increase in functional gene expression and ALB secretion, which were equivalent to those of the original D22-Hep (Figure 3-1 B and C and 3-2 D). Furthermore, D22-pHC-Hep cells formed tight junctions between their own cells and acquired ammonia excretion and liver functions (uptake and excretion of ICG, glycogen storage, and low-density lipoprotein uptake) (Figures 3-1 D, E and 3-2 E).

[0157] To induce cholangiocyte differentiation, we adapted the bile duct development and differentiation method (Francis et al., 2004; Sampaziotis et al., 2015; Sampaziotis et al., 2017) and established the cholangiocyte differentiation method shown in Figure 3-1F. After 2 days of incubation, a single D22-pHC developed into a ring-shaped structure, which then gradually developed into a cyst within 10 days (Figures 3-1F, 3-2F and G). Q-PCR analysis revealed that the cyst structures featured upregulated transcription of cholangiocyte-characteristic genes (Figures 3-1G and 3-2H). The cholangiocyte fate of these cysts was then confirmed by positive staining for CK19, F-ACTIN, and SOX9, and negative staining for AFP, ALB, and HNF4A (Figure 3-1H). Furthermore, transmission electron microscopy revealed that these cysts possessed characteristics of mature bile ducts, including a lumen with apical and basolateral membranes, microvilli, primary cilia on the apical membrane, tight junctions, and multivesicular bodies (Figures 3-1 I and 3-2 I). These cholangiocytes also possessed bile ductular functions: 1) transport of rhodamine 123 into the lumen; this function was blocked by verapamil, an inhibitor of the multidrug resistance protein 1 transporter (Figures 3-1 J and K), and 2) specific export of bile acids from the lumen (Figure 3-2 J). These data therefore demonstrated that D22-Hep (young N-Hep) possesses sufficient plasticity to induce a bipotential proliferative state.

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

[0159] HDACi-mediated upregulation of plasticity in aged N-Hep Next, to investigate whether histone hypoacetylation causes the decline in hepatocyte plasticity associated with aging, we stimulated D52-Hep with sodium butyrate (NaB) or valproic acid (VPA), both of which are histone deacetylase inhibitors (HDACi), during the induction process, and found that each HDACi significantly promoted the proliferation of pHCs induced from D52-Hep (Fig. 4-1 H, I and 4-3 A). Immunostaining revealed that stimulation with one HDACi significantly increased the proliferation of HNF4. + KI67 + The results showed that stimulation with HDACi significantly enhanced the percentage of D52-Hep cells (Figure 4-1 J). However, this enhancement was not observed with other epigenetic regulators or inhibitors, such as tranylcypromine (Trany, a histone demethylase inhibitor), RG108 (a DNA methyltransferase inhibitor), and BIX01294 (BIX, a histone methyltransferase inhibitor) (Figure 4-1 H and I). Furthermore, D52-pHC (D52-Hep-derived pHC) continuously proliferated and acquired the ability to differentiate into hepatocytes and cholangiocytes, whereas stimulation with HDACi enhanced the plasticity of D52-Hep cells (Figure 4-3 B-F).

[0160] In one of the above experiments, we found that FGF2-activated EZH2 transcription plays a crucial role in the proliferation of proliferative hepatocytes (pHCs) (Figures 2-1 I, J, and 2-2 GJ). On the other hand, activated EZH2 transcription 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 increased EZH2 transcription and promoted the proliferation of D52-pHC derived from D52-Hep (Figures 4-1 K and 4-3 H), and D52-pHC showed greater histone acetylation than 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, we performed a comparative analysis using a comprehensive web tool (https: / / www.genecards.org) and the ENCODE dataset (Consortium, 2012). We found that the two highest-scoring EZH2 enhancers (GH07J148882 and GH07J148940) were acetylated histone binding sites (Figure 4-3J). Furthermore, chromatin immunoprecipitation and PCR analysis (ChIP-PCR) using anti-H3K9ac, anti-H3K18ac, and anti-H3K27ac antibodies demonstrated significantly reduced acetylated histone binding to GH07J148882, which is close to the transcription start site (Figures 4-1L and 4-3J). These data suggest 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 proliferation of pHCs derived from D62-Hep, D72-Hep, and D82-Hep (Fig. 4-1M). These results demonstrated that histone hypoacetylation-mediated inactivation of EZH2 transcription impairs the plasticity of senescent hepatocytes, and that this plasticity can be effectively restored by HDACi (Fig. 4-3K).

[0161] Induction of plasticity in 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 derived from donors aged 2 months (2M-PHH), 39 years (39Y-PHH), and 78 years (78Y-PHH), we found that the amounts of H3K9ac, H3K18ac, and H3K27ac also decreased with aging (Figure 5-1A). Compared with N-Hep, the acetylation level in 2M-PHH was closer to that in D52-Hep. Furthermore, the acetylation levels in 39Y-PHH and 78Y-PHH were lower than those in D62-Hep (Figure 5-1B). Indeed, the aging-associated hypoacetylation in PHH was also accompanied by a weakening of plasticity induction. On the other hand, stimulation with NaB (an HDACi) significantly increased the efficiency of plasticity induction (Figures 5-1C, 5D, and 5-2A). Transcriptome comparison with the original PHHs revealed elevated expression levels of cell cycle- and stem / progenitor cell-related genes and decreased expression levels of liver function-related genes in PHH-derived pHCs (PHH-pHCs) (Figures 5-1E, 5F, and 5-2B). Immunofluorescence analysis showed that 49.8% (2M-pHC), 32.9% (39Y-pHC), and 12.2% (78Y-pHC) of PHH-pHCs were positive for both HNF4A and KI67, and most PHH-pHCs expressed HNF4A, SOX9, and CK19 (Figure 5-1G). Furthermore, PHH-pHC cells demonstrated proliferation in laminin-511-coated culture dishes (Figure 5-2C) and bipotent differentiation into hepatocytes and cholangiocytes (Figure 5-2DJ). These results indicated that, like the aging process in N-Hep cells, the aging process in PHH cells is also associated with histone hypoacetylation, which impairs hepatocyte plasticity. These findings confirmed the usefulness of N-Hep cells as a cell source for research on human liver aging.

[0162] In vivo repopulation of pHCs after induction of plasticity in aging PHHs Next, we evaluated the repopulation potential 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 tested positive for human ALB (hALB), human A1AT (hA1AT), and human ferritin (hFerritin), but few tested positive for human AFP (hAFP; Figures 6-1 AC and 6-2 A). In contrast to the large gaps in hALB, hA1AT, and hFerritin levels between 2M-PHH and 78Y-PHH grafts, almost no gap was observed between 78Y-pHC and 2M-pHC grafts, with production levels much higher than those in 78Y-PHH and comparable to those of young primary hepatocytes (Figure 6-1A-C). Further, detailed confirmation of hALB gene expression levels was performed by Q-PCR (Figure 6-2B). Furthermore, immunofluorescence analysis suggested that 78Y-pHC grafts yielded more hALB+ grafts and clusters (>5 cells per cluster) than 78Y-PHH grafts (Figures 6-1D, E, and 6-2C). 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 6-1D and F). Furthermore, we detected some 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). Because a significant decline in liver function gene expression was observed during plasticity induction (Figures 5-2B and 6-2E), we investigated whether the transplanted PHH-pHCs matured in vivo. At 12 weeks posttransplantation, Q-PCR analysis using human gene-specific primers revealed a significant recovery in the expression of liver function genes (especially CYP450 enzymes) and a downregulation of CK19 (Figure 6-2E). Immunofluorescence analysis showed that PHH-pHC grafts were positive for the expression of hALB, CYP3A4, hNuMA, hA1AT, and hCK8 / 18, but barely expressed hCK19 and hAFP (Figures 6-1H and 6-2F). Positive ZO-1 staining indicated that PHH-pHCs had matured into hepatic tissue characterized by bile canaliculi formation and bipolar hepatocyte arrangement (Figure 6H). These results suggest that plasticity induction using aged PHHs significantly enhanced their repopulation potential and enabled them to mature and demonstrate full hepatic function after transplantation.

[0164] HDAC inhibitors improve the induction of pHCs in aged models. Finally, we investigated whether HDAC inhibitors could improve hepatic progenitor cell induction in aged mice. 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 models exhibited characteristics of nonalcoholic fatty liver disease (Figure 6-3 AC). Furthermore, the survival rate of the aged model was only 60%, and the induction of Ki67- and Epcam-positive cells in the aged model was significantly lower than that in the young model, suggesting that hepatic progenitor cell induction in the mouse liver is also impaired with aging (Figure 6-3 DH). NaB treatment significantly increased the induced Ki67- and Epcam-positive populations in the aged model, and the survival rate of the aged model was also improved (Figure 6-3 DH).

[0165] Consideration Liver aging is a normal physiological process in which hepatocytes gradually lose the functions and plasticity required for homeostasis (Timchenko, 2009). The development of strategies for aging-associated liver diseases and for improving liver regeneration in elderly individuals has been hindered by a lack of understanding of the mechanisms by which aging regulates hepatocyte plasticity. By applying hiPSC differentiation techniques, we generated young and aged hepatocytes with identical genetic backgrounds. This preparation helped us reveal that aging-associated histone hypoacetylation 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 derived pHCs from aged PHHs (derived from a 78-year-old donor). In a liver injury model, these pHCs exhibited greater repopulation potential than the original aged PHHs.

[0166] Due to the shortage of donors for liver transplantation, the use of grafts derived from older donors is becoming increasingly necessary, helping to reduce the mortality rate associated with waiting lists. However, the initially poor regenerative capacity of senescent 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 derived from elderly individuals have a reduced proliferative response, and this disadvantage impairs liver regeneration (Ono et al., 2011; Schmucker and Sanchez, 2011). Compared with transplants of young PHHs in our liver injury model, senescent PHH grafts produced lower amounts of human liver proteins, thus recapitulating the discrepancy in clinical outcomes between young and senescent grafts. Notably, upregulation of histone acetylation significantly promoted the proliferation of senescent PHHs. Aged PHHs also showed significantly improved regenerative capacity, suggesting that transplantation of aged-derived grafts (in which the intrinsic proliferative potential of hepatocytes has been effectively activated or promoted) should improve graft survival and clinical outcomes.

[0167] The aging process is always characterized by dynamic changes in metabolic processes and epigenetic modifications (Peleg et al., 2016; Ren et al., 2017). However, how these changes control cellular function remains a mystery in the field of liver research (Horvath et al., 2014; Sato et al., 2017). In this paper, we uncovered the profound relationship between aging, hepatocyte plasticity, metabolism, and epigenetic modifications. In particular, we found that hepatocyte aging is accompanied by a decline in metabolic function, which affects the regulation of acetyl-CoA-mediated histone acetylation, thereby impairing hepatocyte plasticity. In contrast to rodent hepatocytes (Katsuda et al., 2017), we found that FGF2-activated EZH2 transcription is required to induce plasticity in human hepatocytes, and hypoacetylation of the EZH2 enhancer in senescent hepatocytes impairs their plasticity. Although several researchers have found that calorie restriction can reverse the aging-dependent decline in histone acetylation and activate the transcription of cell cycle genes in aging mouse livers (Sato et al., 2017), until this study, there was no suitable method to enhance histone acetylation by improving cellular metabolism in a culture dish. Considering the reversibility of histone acetylation and deacetylation, we found that suppression of HDACs can significantly improve the plasticity of aging hepatocytes, further confirming the control of aging hepatocyte plasticity by histone hypoacetylation and proposing a viable strategy for improving the plasticity of aging hepatocytes.

[0168] In addition to compromising hepatic homeostasis and regulating liver regeneration, aging also plays a crucial role in the development of liver disease (Kim et al., 2015). It has been reported that declines in mitochondrial function during aging increase vulnerability to damage (Kim et al., 2015), and aging is thought to be a predictor of the unfavorable outcome of alcoholic liver disease and the progression of fibrosis in hepatitis C (Forrest et al., 2005; Poynard et al., 2001). In this study, we demonstrated that hiPSC-Hep cells can recapitulate the aging process in the liver. Furthermore, our bioinformatics analysis showed that aged hiPSC-Hep cells tend to express genes that promote angiogenesis and fibrogenesis (Figures 1-2M), suggesting that hiPSC-Hep cells may be useful for clarifying the deep connection between aging and liver fibrosis and for developing effective therapeutic strategies for aging-associated liver diseases.

[0169] In conclusion, we recapitulated the aging process of hepatocytes in a culture dish and found that hepatocyte plasticity was impaired by aging-associated histone hypoacetylation. Furthermore, promoting histone acetylation improved the plasticity of aged hepatocytes and enhanced their repopulation capacity in liver injury models, thereby representing a promising therapeutic strategy for promoting liver regeneration in elderly patients.

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[0171] KEY RESOURCES TABLE [Table 1]

[0172] [Table 2]

[0173] [Table 3]

[0174] Experimental model and subject details hiPSC culture The TkDA3 hiPSC clone was 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 5% CO2.

[0175] PHH culture Cryopreserved PHHs were thawed according to the manufacturer's instructions. For positive control experiments, PHHs were cultured at 1x10 on type I collagen-coated culture dishes. 5 cells / cm 2 at a density of 1000 μg / mL in 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 TM PHHs were cultured in Williams E medium 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 laminin-511-coated culture dishes. 4 cells / cm 2 Cells were seeded at a density of 1000 x g. 5% FBS was added on day 0 of culture to improve cell attachment. The medium was replaced with fresh medium after 24 hours and then every 2 days.

[0176] mouse Adult 7-9 week-old TK-NOG mice were used in this study (Hasegawa et al., 2011). To induce liver injury, TK-NOG recipients were intraperitoneally administered ganciclovir (50 mg / kg, Mitsubishi Tanabe Pharma) twice, 5 and 7 days before transplantation. Mice were housed in a temperature- and light-controlled (12-hour light / dark cycle), specific pathogen-free animal facility and maintained in accordance with the Yokohama City University Guidelines for the Use of Laboratory Animals. All experimental procedures were approved by the Clinical Institutional Review Board of the Animal Experimentation Center, Yokohama City University School of Medicine (No. 075).

[0177] Learn more about the method Differentiation into hiPSC-Hep The following two steps were used to differentiate hiPSCs into hiPSC-Hep. Step I (endoderm differentiation ) hiPSC clones were differentiated into endoderm cells for 7 days on plates coated with GFR Matrigel or laminin 511 in 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 days 1 to 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) and plated onto GFR Matrigel or laminin 511-coated plates at 1.5-2 x 10 cells per well in nicotinamide-free hepatic differentiation medium (HDM). 5 cells / cm 2The cells were replated at a density of 1000 μg / mL. From day 8 to day 10, the medium was replaced with HDM every day. From day 11 onward, the medium was replaced every 2 days. 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. Control hiPSC-Hep cells were differentiated according to published protocols with minor modifications (Kajiwara et al., 2012; Si-Tayeb et al., 2010).

[0178] Induction of proliferative hepatocytes (pHC) Freshly generated hiPSC-Hep cells were harvested at certain senescence time points using 0.05% trypsin / EDTA and plated onto GFR Matrigel or laminin 511-coated plates at 5,000 cells / cm. 2 Cells were seeded at a density of 1000 x g in 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 changed on days 1, 3, and 5. To block PI3K and MAPK signaling pathways, 10 μM LY29002 and a fixed amount of PD0325902 (0.01 μM–1 μM), respectively, were added to the RM. To block EZH2 function, 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 using an Incell analyzer 2000 (GE Healthcare) in conjunction with Hoechst 33342 staining (Invitrogen).

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

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

[0181] Cell Counting Kit-8 Assay Cell viability was determined on the indicated days using the Cell Counting Kit-8 according to the manufacturer's instructions.

[0182] Cell proliferation curve pHCs were passaged at approximately 90% confluence, and the total cell number was determined at each passage. Proliferation rate = (total cell number) / (seeded cell number). The proliferation rate of pHC was measured and the cell proliferation curve was calculated.

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

[0184] Hepatocyte differentiation from pHC Hepatocyte differentiation of pHCs was initiated at approximately 90% confluence. The medium was changed to HDM containing 3 μM RA for 8 days, followed by HDM without RA for the next 7 days. The medium was changed every 2 days. The total hepatocyte differentiation period was 15 days.

[0185] Bile duct cell differentiation from pHCs 1 x 10 pHCs were obtained by inducing plasticity at a certain point during passage or aging. 6 pHCs were suspended in RM at a density of 1000 cells / mL. A total of 5,000 pHCs mixed with 50 μL of GFR-Matrigel were seeded as mount drops into untreated 24-well plates and incubated at 37°C in a 5% CO2 incubator for 30 minutes. Next, 700 μL of cholangiocyte differentiation medium (CDM) was added to each 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 relevant analyses were performed on day 10.

[0186] Assays for human ALB, A1AT, ferritin, and AFP Human ALB, A1AT, ferritin, and AFP were measured using the Human Albumin ELISA Kit, Human Alpha-1-Antitrypsin ELISA Kit, Human Ferritin ELISA Kit, and Human AFP ELISA Kit according to the manufacturer's instructions. Samples were diluted 10- to 5000-fold to obtain values ​​within the linear range of the standard curve.

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

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

[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 1 mg / mL stock solution. Cells were incubated with ICG in suspension or seeded form for 4 hours at 37°C in a humidified incubator with 5% CO2. Cells were then washed three times with phosphate-buffered saline (PBS) and resuspended in fresh HCM (Hepatocyte Culture Medium BulletKit). TM HCM TM The cells were then incubated for an additional 2 hours in PBS (1000 mL) to measure ICG excretion. 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. Cells were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. After washing with PBS, cells were oxidized in 0.5% periodic acid solution for 7 minutes, washed with PBS, and then incubated in Schiff's reagent for 15 minutes. After three 2-minute incubations in sulfite water, cells were washed with PBS and visualized using a BZ9000 all-in-one fluorescence microscope. For low-density lipoprotein uptake analysis, 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. Cells were then washed with PBS. Photographs were analyzed using a BZ9000 all-in-one fluorescence microscope.

[0191] Rhodamine 123 and cholyl-lysyl-fluorescein transport assays Rhodamine 123 transport assays and cholyl-lysyl-fluorescein (CLF) transport assays were performed as previously described (Sampaziotis et al., 2015). For rhodamine 123 transport assays, cholangiocytes were incubated with or without 10 μM verapamil at 37°C for 30 min. Next, cells were incubated with 100 μM rhodamine 123 at 37°C for 5 min and washed three times with IMDM. Fresh CDM was added and incubated for an additional 40 min at 37°C. For CLF transport assays, cholangiocytes were loaded with 5 μM CLF or 5 μM FITC for 30 min at 37°C, and then cells were washed three times with IMDM. Fresh CDM was added and incubated for an additional 10 min at 37°C. Photographs were taken using a Leica TCS SP5 confocal microscope.

[0192] Transmission electron microscopy Transmission electron microscopy of cholangiocyte cysts was performed according to a previously described method (Nie et al., 2018a). Prefixed cholangiocyte cysts were postfixed, dehydrated, and embedded in fresh 100% resin. Ultrathin sections of 70 nm were then cut and stained with 2% uranyl acetate. The sections were then washed with distilled water and stained with lead stain solution. Grids were observed under a JEM-1400Plus microscope (JEOL), and digital images were captured using a VELETA camera (Olympus).

[0193] Flow cytometry Antibodies used for 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 procedure followed was in accordance with the manufacturer's instructions. 6 Cells were crosslinked in 1% formaldehyde for 10 minutes and 1x glycine for 5 minutes at room temperature, harvested by scraping, centrifuged, and resuspended in lysis buffer. DNA was digested with micrococcal nuclease to fragments of approximately 150-900 bp in length. Nuclear membranes were 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. Immune complexes 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. ChIP and input samples were then used for qPCR using the primers listed in the table below.

[0195] List of ChIP-PCR primers and probes for the EZH2 enhancer [Table 4] Sequences of the left and right primers of Pr1: SEQ ID NOs: 1 and 2 Sequences of the left and right primers of Pr2: SEQ ID NOs: 3 and 4 Sequences of the left and right primers of Pr3: SEQ ID NOs: 5 and 6 Sequences of the left and right primers of Pr4: SEQ ID NOs: 7 and 8

[0196] The probe manufacturers and product numbers are listed below. TIFF0007824587000011.tif31149

[0197] transplant After two ganciclovir injections, 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 PHHs or PHH-pHCs were transplanted intraspleenically into TK-NOG recipients. Serum was collected every 2 weeks, and mice were sacrificed at 12 weeks.

[0198] RNA isolation and quantitative real-time polymerase chain reaction (Q-PCR) PureLink TM Total RNA was isolated using the RNA Mini Kit. RNA (<2 μg) was used as a template to synthesize first-stranded cDNA using the High-Performance cDNA Reverse Transcription Kit according to the manufacturer's instructions. Q-PCR was performed using the cDNA and 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 using 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 and right primers of OCT4: SEQ ID NOs: 9 and 10 Sequences of the left and right primers of NANOG: SEQ ID NOs: 11 and 12 Sequences of the left and right primers of SOX17: SEQ ID NOs: 13 and 14 Sequences of the left and right primers of FOXA2: SEQ ID NOs: 15 and 16 Sequences of the left and right primers of TBX3: SEQ ID NOs: 17 and 18 Sequences of the left and right primers for TTR: SEQ ID NOs: 19 and 20 Sequences of the left and right primers of A1AT: SEQ ID NOs: 21 and 22 Sequences of the left and right primers of ALB: SEQ ID NOs: 23 and 24 Sequences of the left and right primers of TDO2: SEQ ID NOs: 25 and 26 Sequences of the left and right primers of G6PC: SEQ ID NOs: 27 and 28 Sequences of the left and right primers of ASGR1: SEQ ID NOs: 29 and 30 Sequences of the left and right primers of HNF4A: SEQ ID NOs: 31 and 32 Sequences of the left and right primers of TAT: SEQ ID NOs: 33 and 34 Sequences of the left and right primers of CYP2C9: SEQ ID NOs: 35 and 36 Sequences of the left and right primers for CYP2C19: SEQ ID NOs: 37 and 38 Sequences of the left and right primers of CYP3A4: SEQ ID NOs: 39 and 40 Sequences of the left and right primers for CYP7A1: SEQ ID NOs: 41 and 42 Sequences of the left and right primers of MKI67: SEQ ID NOs: 43 and 44 Sequences of the left and right primers of PCNA: SEQ ID NOs: 45 and 46 Sequences of the left and right primers of CCNB1: SEQ ID NOs: 47 and 48 Sequences of the left and right primers of CCND1: SEQ ID NOs: 49 and 50 Sequences of the left and right primers of CCNE1: SEQ ID NOs: 51 and 52 Sequences of the left and right primers of CDC20: SEQ ID NOs: 53 and 54 Sequences of the left and right primers of EZH2: SEQ ID NOs: 55 and 56 Sequences of the left and right primers of EpCAM: SEQ ID NOs: 57 and 58 Sequences of the left and right primers of C-MET: SEQ ID NOs: 59 and 60 Sequences of the left and right primers of LGR5: SEQ ID NOs: 61 and 62 Sequences of the left and right primers of RBP4: SEQ ID NOs: 63 and 64 Sequences of the left and right primers of SOX9: SEQ ID NOs: 65 and 66 Sequences of the left and right primers of HNF6: SEQ ID NOs: 67 and 68 Sequences of the left and right primers of GGT: SEQ ID NOs: 69 and 70 Sequences of the left and right primers for CFTR: SEQ ID NOs: 71 and 72 Sequences of the left and right primers of AQP1: SEQ ID NOs: 73 and 74 Sequences of the left and right primers 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 probe manufacturers and product numbers are listed below. JPEG0007824587000013.jpg249123

[0202] Histological examination and immunofluorescence staining Liver tissue samples were embedded in optimal cutting temperature (OCT) compound (Sakura Finetech Japan), and 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 in PBS for 10 minutes, washed three times with PBS, and blocked with 10% ECL prime blocking agent in PBS containing 0.3% Triton X-100 for 30 minutes, followed by three further washes with PBS. Next, sections or cells were incubated with primary antibodies in blocking buffer overnight at 4°C. Sections or cells were washed three times with PBS and then incubated with fluorescently labeled secondary antibodies for an additional 60 minutes at room temperature. 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), and 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 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) were purified using PureLink. TM Total RNA was prepared using the RNA Mini Kit. RNA for gene expression profiling was hybridized using the SurePrint G3 Human Gene Expression 8x60K (Agilent Technologies) according to the manufacturer's instructions. Data were normalized using GeneSpring. Gene ontology enrichment and KEGG pathway analyses were performed using DAVID Bioinformatics Resources 6.8 (https: / / david.ncifcrf.gov / home.jsp) (Huang da et al., 2009). STRING interaction networks were analyzed using STRING 9.05 (http: / / string905.embl.de / ) (Franceschini et al., 2013).

[0204] statistics Values ​​are presented as mean ± standard deviation (SD). Statistical significance was assessed 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, MC, Madrigal, P., Bertero, A., Saeb-Parsy, K., Soares, FAC, Schrumpf, E., Melum, E., Karlsen, TH, Bradley, JA, 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, FK, Nagaoka, M., Li, J., Battle, MA, Duris, C., North, PE, Dalton, S., and Duncan, SA (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 the mass production of hepatocytes and bile duct cells. Human hepatocytes, etc., created by the present invention can be used for in vitro drug metabolism tests, hepatotoxicity tests, hepatitis virus infection tests, or for regenerative medicine and bioartificial livers. Chimeric animals in which human hepatocytes have been replaced with human hepatocytes can be produced using the human hepatocytes, etc., created by the present invention, and these chimeric animals can be used to perform drug metabolism tests and safety tests on drug discovery compounds. Furthermore, the present invention can promote liver regeneration using drugs that induce histone hyperacetylation.

Claims

1. A method for producing hepatoblasts, comprising the steps of inducing differentiation of iPS cells into endoderm cells in a serum-free medium that does not contain nicotinamide, and then culturing the endoderm cells in a serum-free medium that contains FGF2, HGF, oncostatin M, dexamethasone, and nicotinamide to differentiate them into hepatoblasts.

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

3. The method of claim 1, wherein the iPS cells are derived from a human.

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

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

6. The method described in claim 1, wherein the nicotinamide-free serum-free medium contains B27, WNT3A and activin A.

7. The method according to claim 6, wherein iPS cells are cultured for 7 days in a serum-free medium containing B27, WNT3A and activin A, to which Y-27632 is added on day 0, and NaB is added from day 1 to day 3.

8. The method of claim 1, wherein the hepatoblasts are cells expressing TTR.

9. The method of claim 8, wherein the hepatoblasts are cells that further express at least one marker selected from the group consisting of AFP, CK19, HNF4A, and KI67.

10. A method for producing hepatocytes, comprising the step of further culturing 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.

11. The method according to claim 10, wherein the hepatocytes are cells that highly express TAT.

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