Compounds and methods for culturing and / or expanding hepatocytes

Abstract

The invention relates to the field of biological medicines, in particular to a compound and a method for culturing and / or amplifying hepatocytes, and application of the cultured and / or amplified hepatocytes.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to compounds and methods for culturing and / or expanding hepatocytes, and uses of the cultured and / or expanded hepatocytes. Background of the Invention

[0003] In vitro cultured hepatocytes are widely used as a research platform for liver pathophysiology and as an alternative to animal models in drug screening and treatment. 1 . Nevertheless, freshly isolated primary hepatocytes rapidly lose many of their hepatocyte functions when cultured in vitro, as the conditions of in vitro culture are very different from those of the native liver. In 2019, Xiang et al. successfully achieved long-term in vitro maintenance of primary human hepatocytes (PHH) by using a combination of five small molecule compounds (X5C) to regulate the complex signaling network required for hepatocyte maintenance. 2 Infection of these cells with hepatitis B virus (HBV) effectively reproduced the entire infection process within one month and produced viral particles forming HBV covalently closed circular DNA. 2 However, the ability of these cells to be maintained and expanded in culture is rather limited.

[0004] Although it remains a challenge to directly expand functional hepatocytes in large quantities in vitro, it is relatively easy to expand hepatocytes after they have dedifferentiated into hepatic progenitor cells. In mice, genetic tracing experiments have shown that during chronic liver injury, mature hepatocytes spontaneously revert to the immature state of hepatic progenitors and can subsequently redifferentiate into hepatocytes during recovery, thereby regenerating liver tissue. 3-5 Although in vitro culture can also induce hepatocyte dedifferentiation, it is difficult to induce cell proliferation. Even the supplementation of growth factors (such as HGF) can only induce limited expansion of in vitro cultured cells. 6 In 2017, Takahiro et al. used Y-27632, A-83-01, and CHIR99021 (YAC) to reprogram mature rat and mouse hepatocytes into bipotential hepatic progenitor cells in vitro. The reprogrammed cells showed the ability to differentiate into hepatocytes or bile duct epithelial cells. 7 . A similar approach has also been applied to the in vitro expansion of human hepatocytes. However, since YAC does not support the process of promoting the transformation of human hepatocyte precursor cells, the researchers further screened and found that the HAC combination containing hepatocyte growth factor (HGF), A-83-01 and CHIR99021, which are important for liver regeneration, has the highest efficiency in converting primary isolated human hepatocytes into precursor cells. 8 .

[0005] Two other studies reported a human hepatocyte culture medium containing Wnt3a as a key factor and another transformation culture medium that required activation of the SIRT1 signaling pathway to promote the efficient transformation of primary human hepatocytes into bipotential precursor cells or precursor-like cells, allowing human hepatocytes to expand in vitro. 9-10 The proliferated cells can be redifferentiated back into metabolically competent hepatocytes, undergo HBV infection and reactivation, or construct organoids under three-dimensional culture conditions and transplanted into the body to reconstruct mouse livers, etc. 9-10 In addition, treating primary human hepatocytes with oncostatin M (OSM) and modulating the expression of its receptor gp130 can also induce hepatocyte proliferation; after removing OSM, polarized hepatocytes can be formed, which can be used to simulate drug-induced cholestasis (i.e., inhibiting the production of bile acids). 11 However, the induced hepatocytes obtained by this "dedifferentiation and redifferentiation" method do not exhibit all the functions of primary human hepatocytes.

[0006] Notably, functional hepatocytes are able to self-renew in vivo and rapidly proliferate to regenerate the entire organ in response to partial hepatectomy (PHx) injury. Therefore, it is unclear whether and how human hepatocytes cultured in long-term two-dimensional culture in vitro can maintain their in vivo proliferative capacity and metabolic functions.

[0007] Current methods of culturing human hepatocytes are unable to maintain the function and self-renewal capacity of hepatocytes, hindering the in vitro modeling of metabolic diseases of the liver, drug toxicity, and liver regeneration. For example, the only short-term (24-hour) induction system reported for the metabolic dysfunction-associated fatty liver (MAFLD) model in a 2D monolayer culture system of primary human hepatocytes is a short-term (24-hour) induction system. Although treatment of HepG2 hepatoma cells with palmitic acid (PA) and oleic acid (OA) can simulate a process of excessive fat accumulation similar to that in MAFLD, there are huge differences between the gene expression and metabolism of these cancerous cells and normal cells, which may affect the model's ability to accurately reproduce the response of normal hepatocytes. 13 In addition, MAFLD and some toxic substances have a long development and treatment process, so accurate in vitro modeling of this disease and hepatotoxicity requires long-term culture and expansion of human hepatocytes with intact liver function.

[0008] There is still a need in the art for methods of culturing human hepatocytes in vitro, such as long-term culture, where the cultured hepatocytes can maintain the self-renewal ability and metabolic function of primary human hepatocytes, can be used to establish metabolic disease models and evaluate drug toxicity or therapeutic effects in vitro, and can also regenerate liver tissue in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1.Effects of seven compounds on the morphology and functional or proliferation marker gene expression of cultured PHHs. a, Top: Representative bright field images of PHH cultures exposed to 7C or X5C chemical cocktails for 9 days. Bottom: Glycogen storage in cultured PHHs confirmed by cytoplasmic periodic acid Schiff (PAS) staining. b, Immunofluorescence detection of ALB, Ki67, CYP3A4, and MRP2 in PHHs after 18 days of culture, with each compound subtracted from 7C. c, Percentage of cells expressing ALB, CYP3A4, MRP2, and Ki67 on day 18 of culture, proportionally increased relative to PHH abundance in 7C cultures. d, Immunofluorescence detection of ALB in EdU-labeled PHHs. Arrows indicate EdU+ proliferating hepatocytes on day 18 of 7C culture. e, Cell number during long-term culture assessed by ATP content. Significance assessed by one-way ANOVA. ***p<0.001; **p<0.01; *p<0.05; ns=not significant.

[0011] Figure 2 Long-term maintenance of metabolic function in isolated PHHs. a, Drug metabolism in long-term cultured PHHs. CYP enzyme activity was measured by LC-MS / MS via conversion of phenacetin to acetaminophen, diclofenac to 4-hydroxydiclofenac, dextromethorphan to dextrorphan, and midazolam to 1-hydroxymidazolam. FHHs were used as positive controls. bc, Measurement of human ALB and urea secretion in PHHs cultured for 90 days. d, PAS staining and ICG uptake assays of metabolic capacity of human hepatocytes treated with different culture systems at 90 days of culture. e, Functional gene expression analysis of fresh control, 48-hour WE (William's E medium) control, WE, X5C-treated, 2C-treated, and 7C-treated for 30 or 90 days. Significance was assessed by one-way ANOVA. ***p<0.001; **p<0.01; *p<0.05; ns=not significant.

[0012] Figure 3.Establishment of acute liver failure, MAFLD, and cholestasis models in PHH. a, Schematic diagram of establishment of APAP-induced liver failure PHH model and rescue by NAC / 4MP after 60 days of 2C culture. b, AST levels in the culture medium of PHH exposed to APAP and treated or not with different chemical combinations. c, Anti-γH2AX and propidium iodide (PI) staining of 2C-cultured PHH exposed to 10 mM APAP and treated or not with different chemical mixtures. d, JC-1 staining to evaluate mitochondrial membrane potential in PHH exposed to 10 mM APAP and treated or not with different chemical combinations. ef, Lipid droplets were detected using BODIPY 493 / 503 in PHHs induced with or without MAFLD induction with different concentrations of palmitic acid (P) (10 μM or 50 μM) and oleic acid (O) (5 μM or 25 μM) for 72 h and treated with or without 30 μM ETC-1002 for 16 h in control (e) 2C cultures (f) at day 30. g, Oil red staining to assess lipid accumulation in 2C cultured PHHs exposed to PO (50 μM: 25 μM) and treated with or without 30 μM ETC at day 30. h, Lipid abundance was proportionally reduced in ETC treatment compared with control treatment. i, Relative expression analysis of liver function-related markers based on QPCR at day 30 compared with control. Left: 2C cultures; Right: Ctr cultures. j, CMFDA staining to detect functional bile canaliculi on the apical surface of polar PHHs treated or untreated with different chemical combinations. k, Schematic diagram of the establishment of the PHH model of cholestatic toxicity. lm, To evaluate the effects of cyclosporine A or troglitazone transport inhibitor on the accumulation of CMF (green) in bile canaliculi of PHH cultured in 2C under the same treatment of negative control melatonin. Significance was assessed using unpaired Student's test or one-way ANOVA. ***p<0.001; **p<0.01; *p<0.05; ns=not significant.

[0013] Figure 4Modeling chronic liver injury caused by hazardous substances in 2C-cultured PHHs. a, Schematic representation of the chronic hepatotoxicity modeled by long-term treatment of self-renewing functional hepatocytes with specific substances. b, Experimental design involved short-term and long-term treatment with NTPs or NFT drugs, with exposure times ranging from days 3 to 4, days 3 to 18, days 13 to 18, or days 17 to 18. c, EC50 / Cmax values ​​of all 12 DILI / non-DILI compounds in 2C-cultured PHHs at 24 h (D3-4 or D17-18), 72 h (D15-18), and 15 days (D3-18). d, Cell viability of 2C-cultured PHHs was measured after exposure to NTPs for different lengths of time. AST levels in the culture medium of 2C-cultured PHHs exposed to NTPs. e, Cell viability and AST levels of PHHs exposed to NFTs. fg, Co-staining of CMFDA and Hoechst (f) and its statistical results (g) for PHH treated with NFT at different concentrations and time periods.

[0014] Figure 5 .scRNA-seq analysis of PHHs cultured with different chemical combinations. a, Representative brightfield views of PHHs cultured with different combinations before scRNA-seq. b, Uniform manifold approximation and projection (UMAP) plots of sequencing data from different culture conditions, colored by cell type and further subdivided into PHH subpopulations. c, Proportion of different cell types in culture. de, Distribution of PHH subpopulations in different cultures and their proportions. f, Hepatocyte Ki67 and functional gene expression of PHH subpopulations. g, Schematic diagram of dedifferentiation and chemical maintenance of human hepatocytes cultured in vitro. h, Cluster analysis of transcriptional profiles of seven hepatocyte subpopulations (hepatocytes 1-7) and cholangiocyte-like cells from cultured PHHs using Bray-Curtis differences. These maps were compared with maps of fetal hepatocytes at human embryonic development stages 5, 12, and 16, adult hepatocytes, iPS-differentiated hepatocytes and cholangiocytes, and the liver cancer cell line HepG2. ij, Pseudo-time distribution of hepatocyte subsets and their marker genes, different colors represent different cell types. k, RNA rate analysis under different culture conditions, showing the differentiation direction of each cell, arrows indicate the differentiation direction.

[0015] Figure 6.Effects of seven compounds on the morphology and function or expression of proliferation marker genes in cultured PHHs. a, Representative bright field images and magnifications of PHH cultures exposed to 7C or X5C compound combinations or WE control for 18 days at different initial cell densities. Insets show magnified views. b, Immunofluorescence detection of ALB and Ki67 expression in PHHs. Arrows indicate Ki67+ hepatocytes at day 18 of culture. c, Proportional increase in PHH abundance relative to control cultures (left) at day 18 of culture, percentage of ALB (middle) and Ki67 expressing (right) cells. de, Same as bc, except for different initial cell densities. f, Co-staining of CYP3A4, MRP2 expression and EdU in 7C-treated PHHs at day 18. Significance was assessed by one-way ANOVA.

[0016] Figure 7 Minimal compound combinations to maintain PHH metabolic function in vitro for long periods of time. ac, Immunofluorescence detection of ALB, KRT7, Ki67, CYP3A4, MRP2, and BSEP expression and EdU incorporation in PHH after 18 days of culture, with each compound subtracted from 4C. d, Proportional increase in PHH abundance and percentage of cells expressing KRT7, Ki67, CYP3A4, MRP2, BSEP, and EdU relative to 4C cultures at day 18 of culture. e, Drug metabolism in long-term cultured PHH measured by CYP enzyme activity using LC-MS / MS. Fresh PHH (FHH) thawed for 6 hours were used as positive controls. f, Bright field images of PHH cultures treated with 7C or 4C for 4 weeks and 1 week after passaging before and after passaging. Significance was assessed by one-way ANOVA. ***p<0.001; **p<0.01; *p<0.05; ns=not significant.

[0017] Figure 8 .Direct comparison of PHHs maintained with 2C and 4C. a, Immunofluorescence detection of ALB, MRP2, and Ki67 in PHHs treated with 4C or 2C on day 18 of culture. b, Relative cell abundance, percentage of ALB+, Ki67+, and MRP2+ cells on day 18. c, EdU incorporation in PHHs on day 18 of culture with 4C or 2C, where the EdU incubation time was 12 h. d, Calculation of results for group c. Significance was assessed using an unpaired t-test.

[0018] Fig. 9 Replacement of GSK or CID in the 2C compound set with molecules with the same target or similar structure. a, Chemical structure of the modified compound similar to CID. bc, Relative cell abundance, percentage of ALB+, Ki67+, and MRP2+ cells on day 18 when CID (b) or GSK (c) were replaced by molecules with the same target or similar structure, respectively.

[0019] Fig.10 .2C-maintained PHHs respond to liver regeneration signals. ab, Immunofluorescence detection of ALB, Ki67, and MRP2 (a) or CYP3A4 and KRT7 (b) in PHHs treated with 2C and different mitogens at day 18 of culture. cg, Relative cell abundance, percentage of ALB+, Ki67+, MRP2+, CYP3A4+, and KRT7+ cells at day 18. Significance was assessed using one-way ANOVA test. ***p<0.001; **p<0.01; ns=not significant.

[0020] Fig.11 APAP-induced acute liver failure was modeled in PHH. a, Anti-γH2AX staining of PHH treated or untreated with different compound combinations and exposed to 10 mM APAP or 20 mM APAP after 60 days of culture with 2C. b, Percentage of γH2AX+ cells in panel a. c, Anti-γH2AX and PI staining of 7C-cultured PHH exposed to 10 mM APAP with or without NAC, 4MP, or 2C. d, JC-1 staining to assess mitochondrial membrane potential in 7C-cultured PHH exposed to 10 mM APAP with or without NAC, 4MP, or 2C. Significance was assessed using an unpaired t-test.

[0021] Fig.12Fatty acid-induced changes in inflammatory and metabolic genes in ex vivo cultured PHHs mimic the development of MAFLD. ad, qPCR-based relative expression analysis of inflammation- and metabolism-related genes in PHHs cultured in 2C (ab) or WE (cd) at day 30, treated or not with PO, and then rescued with ETC or 2C, compared with controls. e, Oil red staining to assess lipid accumulation in 7C cultured PHHs exposed to PO (50 μM: 25 μM) at day 30 and treated or not with 30 μM ETC. f, Proportional decrease in lipid abundance in ETC-treated PHHs compared with control-treated PHHs. gh, Lipid droplets were detected using BODIPY 493 / 503 in PHHs cultured with or without MAFLD induction in different concentrations of palmitic acid (P) (10 μM, 50 μM) and oleic acid (O) (5 μM, 25 μM) for 72 h and treated or not with ETC for 16 h on day 30 of culture in 7C (g) or X5C (h). i, CMFDA staining was used to detect functional bile canaliculi on the apical surface of polar PHHs cultured in WE or 7C, 2C for 112 days. no, Effects of cholestasis-related compounds CsA or troglitazone and control melatonin on CMF (green) accumulation in bile canaliculi of PHHs cultured in 7C. Significance was assessed using unpaired t-test or one-way ANOVA. ***p<0.001; **p<0.01; *p<0.05; ns=not significant.

[0022] Fig.13 .2C Drug-induced liver injury (DILI) testing of standard compounds in culture. a, Cell viability assessed by ATP content assay of twelve compounds, including eight standard DILI compounds and four standard non-DILI compounds. b, Peak concentrations (Cmax) of the twelve standard compounds. cf, Cell viability and AST levels of human hepatoma cell lines (Huh7 and HepG2) were measured after exposure to NTPs or NFTs for different time periods.

[0023] Fig.14. scRNA-seq analysis of cell types under different culture conditions. a, UMAP plots of sequencing data from all five culture samples, colored by cell clusters. bc, Differential expression of marker genes that differ by cell type. d, UMAP plots of cholangiocyte marker genes KRT7 (keratin 7), KRT19 (keratin 19), MMP7 (matrix metallopeptidase 7), EPCAM (epithelial cell adhesion molecule), and CFTR (cystic fibrosis transmembrane conductance regulator) under all five culture conditions. e, UMAP plots of sequencing data from all five culture samples, colored by cell sample. f, UMAP plots of endothelial cell (EC) marker genes CLDN5 (Claudin-5), VWF (von Willebrand factor) and PECAM1 (platelet endothelial cell adhesion molecule 1); mononuclear phagocyte (MP) marker genes LYZ (lysozyme), FCER1G (Fc fragment of IgE receptor Ig), C1QB (complement C1q subcomponent subunit B) and AIF1 (allogeneic graft inflammatory factor 1) in all 5 culture samples; T cell marker genes TRBC2 (T cell receptor β constant region 2), TRAC (T cell receptor α constant region), CD3D (CD3d molecule) and CD2 (CD2 molecule).

[0024] Fig.15 .scRNA-seq analysis of cell differentiation between hepatocyte subsets. ac, Monocle 2 analysis of hepatocyte subset trajectories. d, Differential expression of marker genes that differ in cell type among hepatocyte subsets. e, Pseudo-temporal distribution of the most differentially expressed genes in different clusters of hepatocyte subsets. f, Network diagram proposed by Partition-Based Graph Abstraction (PAGA) for temporal analysis, with the corresponding UMAP shown on the left and the undirected network diagram on the right; the thickness of the line indicates the strength of the differential relationship. g, The differentiation direction of each cell is shown with an arrow representing the direction of differentiation. DETAILED DESCRIPTION OF THE INVENTION

[0026] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are the terms and routine procedures widely used in the corresponding fields.

[0027] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0028] As used herein, "in vitro" and "ex vivo" have similar meanings, are well known in the art, and are often used interchangeably.

[0029] In one aspect, the present invention provides a small molecule compound combination for in vitro culture and / or expansion of hepatocytes, the combination comprising a selective inhibitor of Rho-associated kinase (ROCK) such as GSK429286A (C 21 H 16 F4N4O2) or its functional analogs, protein kinase D (PKD) inhibitors such as CID755673 (C 12 H 11 NO3) or its functional analogs, Notch inhibitors such as DAPT (C 23 H 26 F2N2O4) or its functional analogs, BMP inhibitors such as LDN193189 (C 25 H 22 N6) or its functional analogs, ATP-citrate lyase inhibitors such as ETC1002 (C 19 H 36 O5) or its functional analogs, Wnt inhibitors such as IWP2 (C 22 H 18 N4O2S3) or its functional analogs, and prolyl endopeptidase inhibitors such as Salidroside (C 14 H 20 (07) or one or more compounds thereof.

[0030] The small molecule compound combination of the present invention may be a mixture of all the small molecule compounds. However, the small molecule compounds in the combination may also exist independently but eventually act together, for example, they are added to the culture medium together.

[0031] As used herein, a "functional analogue" of a given compound refers to a compound that has the same or similar function as the given compound, such as activating or inhibiting the same target, activating or inhibiting the same signaling pathway, etc.

[0032] In some embodiments, "functional analogs" include structural analogs. It is worth noting that each specific small molecule compound mentioned herein includes its pharmaceutically acceptable salt. For example, the functional analogs of GSK429286A described in the present invention can be other Rho-associated kinase (ROCK) selective inhibitors. Exemplary functional analogs of GSK429286A include: Y27632 (C 14 H 21 N3O)、Y39983(C 16 H 16N4O), Chroman 1(C 24 H 28 N4O4)、AT13148(C 17 H 16 ClN3O) or AR13324(C 28 H 27 N3O3).

[0033] The functional analogs of CID755673 described in the present invention may be other protein kinase D (PKD) inhibitors. Exemplary functional analogs of CID755673 include: 3-IN-PP1 (C 17 H 18 N6)、CID 2011756(C 22 H 21 ClN2O3)、CRT5(C 28 H 30 N4O2), 01-56, 01-41, 01-6, 01-27, 01-38, 01-45 or 01-71.

[0034] In some embodiments, the combination described herein comprises one or more compounds selected from the group consisting of GSK429286A, CID755673, DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0035] In some embodiments, the combination of the present invention comprises a Rho-associated kinase (ROCK) selective inhibitor such as GSK429286A or a functional analog thereof, a protein kinase D (PKD) inhibitor such as CID755673 or a functional analog thereof, and optionally, one or more compounds selected from Notch inhibitors such as DAPT or a functional analog thereof, BMP inhibitors such as LDN193189 or a functional analog thereof, ATP-citrate lyase inhibitors such as ETC1002 or a functional analog thereof, Wnt inhibitors such as IWP2 or a functional analog thereof, and prolyl endopeptidase inhibitors such as Salidroside or a functional analog thereof.

[0036] In some embodiments, the combination of the present invention does not comprise one or more compounds selected from DAPT or a functional analog thereof, LDN193189 or a functional analog thereof, ETC1002 or a functional analog thereof, IWP2 or a functional analog thereof, and Salidroside or a functional analog thereof.

[0037] In some embodiments, the combination of the present invention does not comprise DAPT or a functional analogue thereof, LDN193189 or a functional analogue thereof, and IWP2 or a functional analogue thereof.

[0038] In some embodiments, the combination of the present invention does not comprise ETC1002 or a functional analogue thereof and Salidroside or a functional analogue thereof.

[0039] In some embodiments, the combination of the present invention does not comprise DAPT or a functional analogue thereof, LDN193189 or a functional analogue thereof, ETC1002 or a functional analogue thereof, IWP2 or a functional analogue thereof, and Salidroside or a functional analogue thereof.

[0040] In some embodiments, the combination described herein comprises GSK429286A, CID755673, and, optionally, one or more compounds selected from DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0041] In some embodiments, the combination described herein does not comprise one or more compounds selected from DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0042] In some embodiments, the combinations described herein do not include DAPT, LDN193189, and IWP2.

[0043] In some embodiments, the combinations described herein do not comprise ETC1002 and Salidroside.

[0044] In some embodiments, the combinations described herein do not comprise DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0045] In some embodiments, the combination of the present invention comprises a Rho-associated kinase (ROCK) selective inhibitor such as GSK429286A or a functional analog thereof, and a protein kinase D (PKD) inhibitor such as CID755673 or a functional analog thereof.

[0046] In some embodiments, the combination of the present invention comprises a compound selected from GSK429286A, Y27632, Y39983, Chroman 1, AT13148 and AR13324, and a compound selected from CID755673, 3-IN-PP1, CID2011, CRT5, 01-56, 01-41, 01-6, 01-27, 01-38, 01-45 and 01-71.

[0047] In some embodiments, the combination of the present invention comprises GSK429286A and CID755673. In some embodiments, the combination comprises GSK429286A and a compound selected from 3-IN-PP1, CID2011, CRT5, 01-56, 01-41, 01-6, 01-27, 01-38, 01-45 and 01-71.

[0048] In some embodiments, the combination described herein comprises CID755673, and a compound selected from the group consisting of Y27632, Y39983, Chroman 1, AT13148, and AR13324.

[0049] In some embodiments, the combination of the present invention comprises a Rho-associated kinase (ROCK) selective inhibitor such as GSK429286A or a functional analog thereof, a protein kinase D (PKD) inhibitor such as CID755673 or a functional analog thereof, a Notch inhibitor such as DAPT or a functional analog thereof, a BMP inhibitor such as LDN193189 or a functional analog thereof, and optionally, one or more compounds selected from ATP-citrate lyase inhibitors such as ETC1002 or a functional analog thereof, a Wnt inhibitor such as IWP2 or a functional analog thereof, and a prolyl endopeptidase inhibitor such as Salidroside or a functional analog thereof.

[0050] In some embodiments, the combination of the present invention comprises a compound selected from GSK429286A, Y27632, Y39983, Chroman 1, AT13148 and AR13324, a compound selected from CID755673, 3-IN-PP1, CID2011, CRT5, 01-56, 01-41, 01-6, 01-27, 01-38, 01-45 and 01-71, DAPT and LDN193189, and optionally one or more compounds selected from ETC1002, IWP2, and Salidroside.

[0051] In some embodiments, the combination of the invention comprises GSK429286A, CID755673, DAPT, LDN193189, and optionally, one or more compounds selected from ETC1002, IWP2, and Salidroside.

[0052] In some embodiments, the combination of the present invention comprises a Rho-associated kinase (ROCK) selective inhibitor such as GSK429286A or a functional analog thereof, a protein kinase D (PKD) inhibitor such as CID755673 or a functional analog thereof, a Notch inhibitor such as DAPT or a functional analog thereof, and a BMP inhibitor such as LDN193189 or a functional analog thereof.

[0053] In some embodiments, the combination described herein comprises GSK429286A, CID755673, DAPT, and LDN193189.

[0054] In some embodiments, the combination of the present invention comprises a Rho-associated kinase (ROCK) selective inhibitor such as GSK429286A or a functional analog thereof, a protein kinase D (PKD) inhibitor such as CID755673 or a functional analog thereof, a Notch inhibitor such as DAPT or a functional analog thereof, a BMP inhibitor such as LDN193189 or a functional analog thereof, an ATP-citrate lyase inhibitor such as ETC1002 or a functional analog thereof, a Wnt inhibitor such as IWP2 or a functional analog thereof, and a prolyl endopeptidase inhibitor such as Salidroside or a functional analog thereof.

[0055] In some embodiments, the combination of the present invention comprises a compound selected from GSK429286A, Y27632, Y39983, Chroman 1, AT13148 and AR13324, a compound selected from CID755673, 3-IN-PP1, CID2011, CRT5, 01-56, 01-41, 01-6, 01-27, 01-38, 01-45 and 01-71, DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0056] In some embodiments, the combination described herein comprises GSK429286A, CID755673, DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0057] In some embodiments, the small molecule compound combination of the present invention is used as a cell (such as hepatocyte) culture medium supplement. In some embodiments, the culture medium supplement is used to culture and / or expand hepatocytes in vitro.

[0058] In some embodiments, the present invention provides a cell (such as hepatocyte) culture medium supplement, which comprises or consists of the small molecule compound combination of the present invention. In some embodiments, the culture medium supplement is used for in vitro culture and / or expansion of hepatocytes.

[0059] In one aspect, the present invention provides a culture medium comprising the small molecule compound combination of the present invention or the cell (eg, hepatocyte) culture medium supplement of the present invention. In some embodiments, the culture medium is used for in vitro culture and / or expansion of hepatocytes.

[0060] On the other hand, the present invention provides a culture medium, the culture medium comprises a selective inhibitor of Rho-associated kinase (ROCK) such as GSK429286A or a functional analog thereof, a protein kinase D (PKD) inhibitor such as CID755673 or a functional analog thereof, a Notch inhibitor such as DAPT or a functional analog thereof, a BMP inhibitor such as LDN193189 or a functional analog thereof, an ATP-citrate lyase inhibitor such as ETC1002 or a functional analog thereof, a Wnt inhibitor such as IWP2 or a functional analog thereof, and a prolyl endopeptidase inhibitor such as Salidroside or a functional analog thereof. In some embodiments, the culture medium is used for in vitro culture and / or expansion of hepatocytes.

[0061] In some embodiments, the culture medium of the present invention comprises one or more compounds selected from the group consisting of GSK429286A, CID755673, DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0062] In some embodiments, the culture medium of the present invention comprises a Rho-associated kinase (ROCK) selective inhibitor such as GSK429286A or a functional analog thereof, a protein kinase D (PKD) inhibitor such as CID755673 or a functional analog thereof, and optionally, one or more compounds selected from Notch inhibitors such as DAPT or a functional analog thereof, BMP inhibitors such as LDN193189 or a functional analog thereof, ATP-citrate lyase inhibitors such as ETC1002 or a functional analog thereof, Wnt inhibitors such as IWP2 or a functional analog thereof, and prolyl endopeptidase inhibitors such as Salidroside or a functional analog thereof.

[0063] In some embodiments, the culture medium of the present invention does not contain one or more compounds selected from DAPT or a functional analog thereof, LDN193189 or a functional analog thereof, ETC1002 or a functional analog thereof, IWP2 or a functional analog thereof, and Salidroside or a functional analog thereof.

[0064] In some embodiments, the culture medium of the present invention does not contain DAPT or a functional analog thereof, LDN193189 or a functional analog thereof, and IWP2 or a functional analog thereof.

[0065] In some embodiments, the culture medium of the present invention does not contain ETC1002 or a functional analog thereof and Salidroside or a functional analog thereof.

[0066] In some embodiments, the culture medium of the present invention does not contain DAPT or a functional analog thereof, LDN193189 or a functional analog thereof, ETC1002 or a functional analog thereof, IWP2 or a functional analog thereof, and Salidroside or a functional analog thereof.

[0067] In some embodiments, the culture medium of the present invention comprises GSK429286A, CID755673, and optionally, one or more compounds selected from DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0068] In some embodiments, the culture medium of the present invention does not contain one or more compounds selected from DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0069] In some embodiments, the culture medium described herein does not contain DAPT, LDN193189, and IWP2.

[0070] In some embodiments, the culture medium of the present invention does not contain ETC1002 and Salidroside.

[0071] In some embodiments, the culture medium described herein does not contain DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0072] In some embodiments, the culture medium of the present invention comprises a Rho-associated kinase (ROCK) selective inhibitor such as GSK429286A or a functional analog thereof, and a protein kinase D (PKD) inhibitor such as CID755673 or a functional analog thereof.

[0073] In some embodiments, the culture medium of the present invention comprises a compound selected from GSK429286A, Y27632, Y39983, Chroman 1, AT13148 and AR13324, and a compound selected from CID755673, 3-IN-PP1, CID2011, CRT5, 01-56, 01-41, 01-6, 01-27, 01-38, 01-45 and 01-71.

[0074] In some embodiments, the culture medium of the present invention comprises GSK429286A and CID755673. In some embodiments, the culture medium comprises GSK429286A and a compound selected from PP1, CID2011, CRT5, 01-56, 01-41, 01-6, 01-27, 01-38, 01-45 and 01-71.

[0075] In some embodiments, the culture medium of the present invention comprises CID755673, and a compound selected from the group consisting of Y27632, Y39983, Chroman 1, AT13148, and AR13324.

[0076] In some embodiments, the culture medium of the present invention comprises a Rho-associated kinase (ROCK) selective inhibitor such as GSK429286A or a functional analog thereof, a protein kinase D (PKD) inhibitor such as CID755673 or a functional analog thereof, a Notch inhibitor such as DAPT or a functional analog thereof, a BMP inhibitor such as LDN193189 or a functional analog thereof, and optionally, one or more compounds selected from ATP-citrate lyase inhibitors such as ETC1002 or a functional analog thereof, a Wnt inhibitor such as IWP2 or a functional analog thereof, and a prolyl endopeptidase inhibitor such as Salidroside or a functional analog thereof.

[0077] In some embodiments, the culture medium of the present invention comprises a compound selected from GSK429286A, Y27632, Y39983, Chroman 1, AT13148 and AR13324, a compound selected from CID755673, 3-IN-PP1, CID2011, CRT5, 01-56, 01-41, 01-6, 01-27, 01-38, 01-45 and 01-71, DAPT and LDN193189, and optionally one or more compounds selected from ETC1002, IWP2, and Salidroside.

[0078] In some embodiments, the culture medium of the present invention comprises GSK429286A, CID755673, DAPT, LDN193189, and optionally, one or more compounds selected from ETC1002, IWP2, and Salidroside.

[0079] In some embodiments, the culture medium of the present invention comprises a Rho-associated kinase (ROCK) selective inhibitor such as GSK429286A or a functional analog thereof, a protein kinase D (PKD) inhibitor such as CID755673 or a functional analog thereof, a Notch inhibitor such as DAPT or a functional analog thereof, and a BMP inhibitor such as LDN193189 or a functional analog thereof.

[0080] In some embodiments, the culture medium described herein comprises GSK429286A, CID755673, DAPT, and LDN193189.

[0081] In some embodiments, the culture medium of the present invention comprises a Rho-associated kinase (ROCK) selective inhibitor such as GSK429286A or a functional analog thereof, a protein kinase D (PKD) inhibitor such as CID755673 or a functional analog thereof, a Notch inhibitor such as DAPT or a functional analog thereof, a BMP inhibitor such as LDN193189 or a functional analog thereof, an ATP-citrate lyase inhibitor such as ETC1002 or a functional analog thereof, a Wnt inhibitor such as IWP2 or a functional analog thereof, and a prolyl endopeptidase inhibitor such as Salidroside or a functional analog thereof.

[0082] In some embodiments, the culture medium of the present invention comprises a compound selected from GSK429286A, Y27632, Y39983, Chroman 1, AT13148 and AR13324, a compound selected from CID755673, 3-IN-PP1, CID2011, CRT5, 01-56, 01-41, 01-6, 01-27, 01-38, 01-45 and 01-71, DAPT, LDN193189, ETC1002, IWP2 and Salidroside.

[0083] In some embodiments, the culture medium described herein comprises GSK429286A, CID755673, DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0084] The culture medium of the present invention can also include a basal medium. Suitable basal medium includes, but is not limited to, for example, the William's E culture medium listed in the examples. Other basal culture mediums known in the art for hepatocyte culture can also be applied to the present invention. In some embodiments, the basal medium is LiveRSup human hepatocyte basal medium (Cat#H2C-01, Plastech). In some embodiments, the basal medium is Hepatocyte Maintenance Medium (Cat#CC-3198, Lonza).

[0085] In some embodiments, the culture medium of the present invention does not contain any additional small molecule compounds other than the basal medium and one or more compounds described above. The additional small molecule compounds are small molecule compounds used for hepatocyte culture and / or expansion.

[0086] The concentration of each of the one or more compounds in the culture medium of the present invention can be about 0.01 μM to about 100 μM, such as about 0.01 μM, about 0.1 μM, about 0.5 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 7.5 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM. The preferred concentration is about 0.1 μM to about 20 μM.

[0087] The concentration of GSK429286A or a functional analog thereof in the culture medium of the present invention can be about 0.01 μM to about 100 μM, for example, about 0.01 μM, about 0.1 μM, about 0.5 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 7.5 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM, preferably about 0.1 μM to about 20 μM, and more preferably about 2 μM.

[0088] The concentration of CID755673 or its functional analogue in the culture medium of the present invention can be about 0.01 μM-about 100 μM, for example, about 0.01 μM, about 0.1 μM, about 0.5 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 7.5 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM, preferably about 0.1 μM-about 10 μM, and more preferably about 5 μM.

[0089] The concentration of DAPT or a functional analog thereof in the culture medium of the present invention can be about 0.01 μM to about 100 μM, for example, about 0.01 μM, about 0.1 μM, about 0.5 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 7.5 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM, preferably about 0.1 μM to about 10 μM, and more preferably about 5 μM.

[0090] The concentration of LDN193189 or its functional analogue in the culture medium of the present invention can be about 0.01 μM-about 100 μM, for example, about 0.01 μM, about 0.1 μM, about 0.5 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 7.5 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM, preferably about 0.01 μM-about 5 μM, more preferably about 0.1 μM.

[0091] The concentration of ETC1002 or its functional analogue in the culture medium of the present invention can be about 0.01 μM-about 100 μM, for example, about 0.01 μM, about 0.1 μM, about 0.5 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 7.5 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM, preferably about 0.1 μM-about 10 μM, and more preferably about 5 μM.

[0092] The concentration of IWP2 or a functional analog thereof in the culture medium of the present invention can be about 0.01 μM to about 100 μM, for example, about 0.01 μM, about 0.1 μM, about 0.5 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 7.5 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM, preferably about 0.01 μM to about 5 μM, more preferably about 0.5 μM.

[0093] The concentration of salidroside or its functional analogue in the culture medium of the present invention can be about 0.01 μM-about 100 μM, for example, about 0.01 μM, about 0.1 μM, about 0.5 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 7.5 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM, preferably about 0.1 μM-about 10 μM, and more preferably about 2 μM.

[0094] In some embodiments, the culture medium of the present invention further comprises hepatocyte growth factor (HGF) and / or epidermal growth factor (EGF), preferably HGF. HGF and / or EGF, preferably HGF, can further promote the expansion of hepatocytes. The HGF or EGF can be isolated HGF or EGF, or recombinantly produced HGF or EGF.

[0095] The concentration of EGF in the culture medium of the present invention may be about 0.1 ng / ml to about 1000 ng / ml, preferably about 1 ng / ml to about 100 ng / ml, and more preferably about 20 ng / ml. The concentration of HGF in the culture medium of the present invention may be about 0.1 ng / ml to about 1000 ng / ml, preferably about 1 ng / ml to about 100 ng / ml, and more preferably about 40 ng / ml.

[0096] In one aspect, the present invention provides a method for culturing and / or expanding hepatocytes in vitro, the method comprising culturing hepatocytes in the presence of the small molecule compound combination of the present invention or in the culture medium of the present invention.

[0097] The small molecule compound combination, the culture medium or the method of the present invention are particularly suitable for long-term culture and / or expansion of hepatocytes. For example, the small molecule compound combination, the culture medium or the method of the present invention can be used for in vitro culture and / or expansion of hepatocytes for about 1 day to about one year or longer, for example, for culture and / or expansion of hepatocytes for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least half a year, at least one year or even longer.

[0098] The hepatocytes cultured and / or expanded (such as long-term culture and / or expansion) using the small molecule compound combination, the culture medium or the method of the present invention can substantially retain the function of normal hepatocytes. For example, the cultured and / or expanded hepatocytes have morphological characteristics, gene expression profiles, self-renewal ability, glycogen storage ability, albumin and / or urea secretion, hepatocyte marker genes (such as liver function-related genes ABCB11, ABCB4, HNF4A, UGT1A1, PEPCK, ALDOB, OATPB, ABCC2 and SLC10A1, ALB, CYP3A4, BSEP and MRP2, etc., proliferation marker gene Ki67) expression and / or enzyme activity such as CYP450 enzymes (such as CYP1A2, CYP2C9, CYP2D6 and CYP3A4) activity comparable to normal hepatocytes (such as freshly isolated primary hepatocytes, or freshly thawed uncultured frozen hepatocytes). The morphological characteristics, gene expression profile, self-renewal capacity, glycogen storage capacity, albumin and / or urea secretion, hepatocyte marker gene expression and / or enzyme activity such as CYP450 enzyme activity of hepatocytes can be identified according to the methods described in the examples of this application. In some embodiments, the cultured and / or expanded hepatocytes can proliferate in response to regeneration signals such as EGF and / or HGF.

[0099] The number of hepatocytes cultured and / or expanded (such as long-term culture and / or expansion) for a period of time using the small molecule compound combination, the culture medium or the method of the present invention can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500% or more compared to the starting number of hepatocytes at the beginning of culture and / or expansion. The period of time can be about 1 day to about one year or longer, for example, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least half a year, at least one year or even longer.

[0100] In the present invention, the hepatocytes (such as hepatocytes to be cultured and / or expanded in vitro) can be isolated (ex vivo) hepatocytes, preferably isolated hepatocytes. The hepatocytes (such as hepatocytes to be cultured and / or expanded in vitro) can be primary hepatocytes, such as primary hepatocytes.

[0101] The hepatocytes described in the present invention (eg, hepatocytes to be cultured and / or expanded in vitro) may be hepatocytes isolated from a normal or healthy subject (donor), preferably hepatocytes.

[0102] The hepatocytes of the present invention (such as hepatocytes to be cultured and / or expanded in vitro) can also be hepatocytes differentiated from stem cells. The stem cells are, for example, induced pluripotent stem cells (iPSCs). The iPSCs can be induced by transgenics and / or by chemical small molecules. Various methods for obtaining hepatocytes by iPSC differentiation are known in the art, for example, see James L. Corbett et al. Front. Med., 15 November 2019: iPSC-Derived Hepatocytes as a Platform for Disease Modeling and Drug Discovery.

[0103] The hepatocytes of the present invention (e.g., hepatocytes to be cultured and / or expanded in vitro) can be hepatocytes obtained by transdifferentiation from other types of cells. For example, the other types of cells include, but are not limited to, neurons, skeletal muscle cells, fibroblasts, osteoblasts, chondrocytes, adipocytes, endothelial cells, interstitial cells, smooth muscle cells, cardiomyocytes, nerve cells, hematopoietic cells, pancreatic islet cells, or almost any cell in the body. Various methods for obtaining hepatocytes by transdifferentiation are known in the art, for example, see Huang, P. et al. Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. Cell Stem Cell 14, 370–384 (2014); Du, Y. et al. Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming. Cell Stem Cell 14, 394–403 (2014); Xie, B. et al. Cell Res. 2019 Sep; 29 (9): 696-710. A two-step lineage reprogramming strategy to generate functionally competent human hepatocytes from fibroblasts.

[0104] The hepatocytes described in the present invention (such as hepatocytes to be cultured and / or expanded in vitro) can also be hepatocytes in isolated liver tissue or artificially prepared liver organoids.

[0105] In the present invention, the hepatocytes (such as hepatocytes to be cultured and / or amplified in vitro) can be derived from mammals or non-mammals. In some embodiments of the present invention, the hepatocytes are derived from non-human mammals. In some embodiments of the present invention, the hepatocytes are derived from mice such as mice or rats or non-human primates. In some preferred embodiments of the present invention, the hepatocytes are derived from humans.

[0106] The hepatocytes described in the present invention (such as the hepatocytes to be cultured and / or expanded in vitro) can be freshly isolated or prepared primary hepatocytes, or can be cryopreserved hepatocytes.

[0107] In some embodiments of the various aspects of the invention, the starting concentration of the hepatocytes is about 1×10 4 cells / cm 2- Approximately 1×10 6 cells / cm 2 , preferably about 1×10 5 cells / cm 2 .

[0108] In addition to the small molecule compound combination of the present invention or the culture medium of the present invention, conditions suitable for culturing hepatocytes, such as temperature, atmosphere, humidity, etc., are readily available to those skilled in the art. Suitable culture conditions are, for example, stable temperature, stable CO2 levels, and high relative humidity. In some specific embodiments, suitable culture conditions include about 37°C, about 5% CO2, and about 95% relative humidity.

[0109] In one aspect, the present invention provides hepatocytes cultured and / or expanded by the small molecule compound combination, the culture medium or the method of the present invention.

[0110] In one aspect, the present invention provides the use of hepatocytes cultured and / or expanded by the small molecule compound combination, culture medium or method of the present invention for screening candidate drugs for treating liver diseases, and / or for testing the pharmacological properties (e.g., drug toxicity) of candidate drugs.

[0111] Liver disease described herein can be various liver diseases, including but not limited to acute liver failure, chronic liver failure, liver fibrosis, liver cirrhosis, liver cancer (primary liver cancer or metastatic liver cancer), toxic liver injury, biliary cirrhosis, primary sclerosing cholangitis, chronic autoimmune hepatitis, fatty liver, alcoholic liver disease, non-alcoholic fatty liver, liver injury related to pathogen infection (such as parasitic infection or hepatitis virus infection, such as hepatitis A, hepatitis B, hepatitis C, etc.), hereditary liver disease, drug-induced liver injury, liver injury caused by liver resection, and impaired function of transplanted liver. In some specific embodiments of various aspects of the present invention, the liver disease is metabolic dysfunction-related fatty liver (MAFLD). In some specific embodiments of various aspects of the present invention, the liver disease is acute liver failure caused by acetaminophen (APAP). In some specific embodiments of various aspects of the present invention, the liver disease is cholestasis, such as cholestasis caused by drugs (such as cyclosporine A or troglitazone). In some specific embodiments of various aspects of the present invention, the liver disease is caused by drug-induced liver injury, such as drug-induced liver injury caused by a substance selected from the following: Amiodarone, Bosentan, Diclofenac, Nefazodone, Paracetamol, Perhexiline, Tolcapone, Troglitazone, or Nitrofurantoin (NFT).

[0112] In one aspect, the present invention provides the use of hepatocytes cultured and / or amplified by the small molecule compound combination, the culture medium or the method of the present invention, which is used to establish a liver disease model. In some embodiments, the disease model is used to screen candidate drugs for treating liver disease, and / or to test the pharmacological properties (e.g., drug toxicity) of candidate drugs.

[0113] In one aspect, the present invention provides a method for preparing a bioartificial liver, the method comprising: culturing hepatocytes in a bioreactor in the presence of the small molecule compound combination of the present invention or in the culture medium of the present invention. In some embodiments, the bioreactor comprises microcarriers.

[0114] In one aspect, the present invention provides a bioartificial liver prepared by the method of the present invention.

[0115] In one aspect, the present invention provides use of a bioartificial liver prepared by the method of the present invention for treating a liver disease in a subject, or for screening candidate drugs for treating a liver disease, or for testing the pharmacological properties (e.g., drug toxicity) of candidate drugs for treating a liver disease.

[0116] In one aspect, the present invention provides the use of hepatocytes cultured and / or expanded by the small molecule compound combination, the culture medium or the method of the present invention in the preparation of a medicament. In some embodiments, the medicament is used to treat liver disease in a subject. In some embodiments, the cultured and / or expanded hepatocytes are used to treat liver disease by hepatocyte transplantation.

[0117] In one aspect, the present invention provides a pharmaceutical composition comprising hepatocytes cultured and / or expanded by the small molecule compound combination, the culture medium or method of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is used to treat liver disease in a subject.

[0118] As used herein, a "subject" may be a mammal or a non-mammal. The subject may be a human, or a non-human mammal such as a mouse or a rat or a non-human primate, preferably a human.

[0119] As used herein, "pharmaceutically acceptable carrier" refers to a substance that facilitates the administration of an active substance to a subject and the absorption of the subject, which can be included in the pharmaceutical composition of the present invention without causing significant toxic side effects to the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, sucrose, glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, Ringer's solution, alcohol, oil, gelatin, carbohydrates such as lactose, straight chain starch or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone and coloring agents, etc.

[0120] In one aspect, the present invention provides a method for establishing a disease model of metabolic dysfunction associated fatty liver (MAFLD) and / or screening a drug for treating MAFLD, the method comprising:

[0121] i) providing hepatocytes such as freshly isolated primary hepatocytes or thawed cryopreserved hepatocytes;

[0122] ii) culturing the hepatocytes for a first period of time in the presence of the small molecule compound combination of the present invention or in the culture medium of the present invention;

[0123] iii) treating the hepatocytes obtained in ii) with fatty acids such as palmitic acid (PA) and oleic acid (OA) for a second period of time;

[0124] iv) harvesting the hepatocytes obtained in iii) as a disease model of MAFLD.

[0125] In some embodiments, the first period of time is at least 18 days, at least one month or longer. In some embodiments, the second period of time is at least 72 hours or longer.

[0126] In some embodiments, wherein in iii), the hepatocytes obtained in ii) are treated with 10 μM PA and 5 μM OA for a second period of time. In some embodiments, wherein in iii), the hepatocytes obtained in ii) are treated with 50 μM PA and 25 μM OA for a second period of time.

[0127] In some embodiments, the treatment of step iii) is carried out in the presence of the small molecule compound combination of the present invention or in the culture medium of the present invention. For example, step iii) includes adding fatty acids such as palmitic acid (PA) and oleic acid (OA) to the culture obtained in step ii) to treat the second time period.

[0128] In some embodiments, the method further comprises

[0129] v) treating the hepatocytes of the disease model of MAFLD obtained in step iv) with a candidate drug, and optionally evaluating the effectiveness of the candidate drug for treating MAFLD. In some embodiments, the effectiveness of the candidate drug is evaluated by changes in the expression of fatty acid synthesis-related genes, changes in the expression of oxidation-related genes, changes in the expression of inflammation-related genes, changes in the expression of liver function-related genes, reduction of inflammation, and / or reduction of fatty acid accumulation. Specific evaluation methods are, for example, referring to the present application examples.

[0130] In one aspect, the present invention provides a method for establishing a disease model of acute liver failure caused by acetaminophen (APAP) and / or screening a drug for treating acute liver failure caused by acetaminophen (APAP), the method comprising:

[0131] i) providing hepatocytes such as freshly isolated primary hepatocytes or thawed cryopreserved hepatocytes;

[0132] ii) culturing the hepatocytes for a first period of time in the presence of the small molecule compound combination of the present invention or in the culture medium of the present invention;

[0133] iii) treating the hepatocytes obtained in ii) with acetaminophen (APAP) for a second period of time;

[0134] iv) harvesting the hepatocytes obtained in iii) as a disease model of acute liver failure induced by acetaminophen (APAP).

[0135] In some embodiments, the first period of time is at least one month, such as at least two months or longer. In some embodiments, the second period of time is at least 16 hours or longer.

[0136] In some embodiments, wherein in iii), the hepatocytes obtained in ii) are treated with about 10 to about 20 mM APAP for a second period of time.

[0137] In some embodiments, the treatment in step iii) is performed in the presence of the small molecule compound combination of the present invention or in the culture medium of the present invention. For example, step iii) comprises adding APAP to the culture obtained in step ii) to treat the second period of time.

[0138] In some embodiments, the method further comprises

[0139] v) treating the hepatocytes obtained in step iv) as a disease model of acute liver failure caused by acetaminophen (APAP) with a candidate drug, and optionally evaluating the efficacy of the candidate drug in treating acute liver failure caused by acetaminophen (APAP). In some embodiments, the efficacy of the candidate drug is evaluated by AST (aspartate aminotransferase) level, DNA damage, apoptosis and / or mitochondrial membrane potential. For specific evaluation methods, see, for example, the examples of the present application.

[0140] In one aspect, the present invention provides a method for identifying the hepatotoxicity of a given drug, the method comprising:

[0141] i) providing hepatocytes such as freshly isolated primary hepatocytes or thawed cryopreserved hepatocytes;

[0142] ii) culturing the hepatocytes for a first period of time in the presence of the small molecule compound combination of the present invention or in the culture medium of the present invention;

[0143] iii) treating the hepatocytes obtained in ii) with the given drug for a second period of time; and

[0144] iv) evaluating the toxicity of the given drug to hepatocytes.

[0145] In some embodiments, the first time period is any time period, such as at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 18 days, to 20 days, or at least one month, such as at least 40 days or longer. In some embodiments, the second time period is at least 1 day, at least 3 days, at least 5 days, or at least 15 days or longer.

[0146] In some embodiments, the treatment of step iii) is carried out in the presence of the small molecule compound combination of the present invention or in the culture medium of the present invention. For example, step iii) includes adding the given drug to the culture obtained in step ii) to treat the second time period.

[0147] In some embodiments, the toxicity of the given drug to hepatocytes is evaluated by detecting bile secretion of hepatocytes, viability of hepatocytes and / or AST levels. In some embodiments, the toxicity is cholestatic toxicity. In some embodiments, the given drug causes chronic liver damage.

[0148] In one aspect, the present invention provides a method for establishing a drug-induced liver injury model caused by a given drug and / or screening a drug for treating drug-induced liver injury caused by a given drug, the method comprising:

[0149] i) providing hepatocytes such as freshly isolated primary hepatocytes or thawed cryopreserved hepatocytes;

[0150] ii) culturing the hepatocytes for a first period of time in the presence of the small molecule compound combination of the present invention or in the culture medium of the present invention;

[0151] iii) treating the hepatocytes obtained in ii) with the given drug for a second period of time; and

[0152] iv) harvesting the hepatocytes obtained in iii) as a disease model of drug-induced liver injury caused by the given drug.

[0153] In some embodiments, the first time period is any time period, such as at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 18 days, to 20 days, or at least one month, such as at least 40 days or longer. In some embodiments, the second time period is at least 1 day, at least 3 days, at least 5 days, or at least 15 days or longer.

[0154] In some embodiments, the treatment of step iii) is carried out in the presence of the small molecule compound combination of the present invention or in the culture medium of the present invention. For example, step iii) includes adding the given drug to the culture obtained in step ii) to treat the second time period.

[0155] In some embodiments, the method further comprises

[0156] v) treating the hepatocytes obtained in step iv) as a disease model of drug-induced liver injury caused by the given drug with the candidate drug, and optionally evaluating the efficacy of the candidate drug in treating the disease model of drug-induced liver injury caused by the given drug. In some embodiments, the efficacy of the candidate drug is evaluated by bile secretion, viability of hepatocytes and / or AST levels. For specific evaluation methods, see, for example, the examples of this application.

[0157] In some embodiments of various aspects of the invention, the given drug is a clinically used drug, such as a drug used to treat infection (bacterial, viral or parasitic), cancer, genetic disease, autoimmune disease, inflammatory disease.

[0158] In addition to the above, this application also provides at least the following implementation schemes:

[0159] Embodiment 1. A small molecule compound combination for in vitro culture and / or expansion of hepatocytes, the combination comprising one or more compounds selected from GSK429286A or a functional analogue thereof, CID755673 or a functional analogue thereof, DAPT or a functional analogue thereof, LDN193189 or a functional analogue thereof, ETC1002 or a functional analogue thereof, IWP2 or a functional analogue thereof, and Salidroside or a functional analogue thereof.

[0160] Embodiment 2. The combination of embodiment 1, wherein the combination comprises one or more compounds selected from GSK429286A, CID755673, DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0161] Embodiment 3. The combination of embodiment 1, wherein the combination comprises GSK429286A or a functional analogue thereof, CID755673 or a functional analogue thereof, and, optionally, one or more compounds selected from DAPT or a functional analogue thereof, LDN193189 or a functional analogue thereof, ETC1002 or a functional analogue thereof, IWP2 or a functional analogue thereof, and Salidroside or a functional analogue thereof.

[0162] Embodiment 4. The combination of embodiment 3, wherein the combination comprises GSK429286A, CID755673, and optionally, one or more compounds selected from DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0163] Embodiment 5. The combination of embodiment 3, wherein the combination comprises GSK429286A or a functional analogue thereof and CID755673 or a functional analogue thereof.

[0164] Embodiment 6. The combination of embodiment 5, wherein the combination comprises GSK429286A and CID755673.

[0165] Embodiment 7. The combination of embodiment 1, wherein the combination comprises GSK429286A or a functional analogue thereof, CID755673 or a functional analogue thereof, DAPT or a functional analogue thereof, LDN193189 or a functional analogue thereof, and optionally, one or more compounds selected from ETC1002 or a functional analogue thereof, IWP2 or a functional analogue thereof, and Salidroside or a functional analogue thereof.

[0166] Embodiment 8. The combination of embodiment 7, wherein the combination comprises GSK429286A, CID755673, DAPT, LDN193189, and optionally, one or more compounds selected from ETC1002, IWP2, and Salidroside.

[0167] Embodiment 9. The combination of embodiment 7, wherein the combination comprises GSK429286A or a functional analogue thereof, CID755673 or a functional analogue thereof, DAPT or a functional analogue thereof, and LDN193189 or a functional analogue thereof.

[0168] Embodiment 10. The combination of embodiment 9, wherein the combination comprises GSK429286A, CID755673, DAPT, and LDN193189.

[0169] Embodiment 11. The combination of embodiment 1, wherein the combination comprises GSK429286A or a functional analogue thereof, CID755673 or a functional analogue thereof, DAPT or a functional analogue thereof, LDN193189 or a functional analogue thereof, ETC1002 or a functional analogue thereof, IWP2 or a functional analogue thereof, and Salidroside or a functional analogue thereof.

[0170] Embodiment 12. The combination of embodiment 11, wherein the combination comprises GSK429286A, CID755673, DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

[0171] Embodiment 13. A culture medium for in vitro culture and / or expansion of hepatocytes, the culture medium comprising the small molecule compound combination according to any one of Embodiments 1-12.

[0172] Embodiment 14. The culture medium of embodiment 13, further comprising a basal culture medium such as William's E medium, LiveRSup human hepatocyte basal culture medium, or Hepatocyte Maintenance Medium.

[0173] Embodiment 15. The culture medium of embodiment 13 or 14, further comprising hepatocyte growth factor (HGF) and / or epidermal growth factor (EGF), preferably HGF.

[0174] Embodiment 16. The culture medium of any one of embodiments 13-15, wherein the concentration of each of the one or more compounds in the culture medium can be about 0.01 μM to about 100 μM.

[0175] Embodiment 17. A method for culturing and / or expanding hepatocytes in vitro, the method comprising culturing hepatocytes in the presence of the small molecule compound combination of any one of embodiments 1-12 or in the culture medium of any one of embodiments 13-16.

[0176] Embodiment 18. The method of embodiment 17, wherein the hepatocytes are cultured for about 1 day to about one year or longer.

[0177] Embodiment 19. The method of embodiment 17 or 18, wherein the hepatocytes are of human origin.

[0178] Embodiment 20. The method according to any one of Embodiments 17 to 19, wherein the hepatocytes may be freshly isolated or prepared primary hepatocytes, or may be cryopreserved hepatocytes.

[0179] Embodiment 21. Hepatocytes cultured and / or expanded by the method of any one of embodiments 17-20.

[0180] Embodiment 22. Use of the hepatocytes of Embodiment 21 for screening candidate drugs for treating liver diseases, and / or for testing the pharmacological properties of drugs (eg drug toxicity).

[0181] Embodiment 23. The use of embodiment 21, wherein the liver disease is selected from acute liver failure, chronic liver failure, liver fibrosis, cirrhosis, liver cancer (primary liver cancer or metastatic liver cancer), toxic liver injury, biliary cirrhosis, primary sclerosing cholangitis, chronic autoimmune hepatitis, fatty hepatitis, alcoholic liver disease, non-alcoholic fatty hepatitis, non-alcoholic fatty liver, liver damage associated with pathogen infection (e.g., parasitic infection or hepatitis virus infection, such as hepatitis A, hepatitis B, hepatitis C, etc.), hereditary liver disease, drug-induced liver damage, liver damage caused by liver resection surgery, and impaired function of a transplanted liver.

[0182] Embodiment 24. The use according to embodiment 21, wherein the liver disease is metabolic dysfunction associated fatty liver disease (MAFLD).

[0183] Embodiment 25. The use of embodiment 21, wherein the liver disease is acute liver failure caused by acetaminophen (APAP).

[0184] Embodiment 26. The use of embodiment 21, wherein the pharmacological property of the drug (eg, drug toxicity) is induction of cholestasis. Example

[0185] The present invention will be further described below by way of examples, but the present invention is not limited to the described examples.

[0186] Materials and methods

[0187] Isolation and cell culture of human hepatocytes Primary human hepatocytes were isolated by a standard two-step EGTA / collagenase P perfusion procedure and frozen for further use. For cell culture experiments, cryopreserved human hepatocytes were thawed in hepatocyte thawing medium (K8000, XENOTECH), centrifuged at 100 × g for 10 min, and the supernatant discarded. The cells were resuspended in plating medium (K8200, XENOTECH) and plated at 1-2 × 10 5 cells / cm 2 The cells were plated at a density of 1:1 in 96-well or 24-well rat tail collagen I (A1048301, Gibco) coated plates. Six hours after plating, the medium was replaced with William's E medium, with or without X5C, or with LiveRSup human hepatocyte basal medium (HHBCM) (Cat# H2C-01, Plastech), with or without 7C or 2C chemical combinations. X5C: SB431542 (SB, 10μM), Forskolin (FSK, 20μM), IWP2 (0.5μM), DAPT (5μM) and LDN193189 (LDN, 0.1μM); 7C: ETC-1002 (ETC, 5μM), GSK4 29286A (2μM), Salidroside (2μM), CID755673 (5μM), IWP2 (0.5μM), DAPT (5μM) and LDN (0.1μM); 2C: GSK429286A (2μM) and CID755673 (5μM).

[0188] Immunofluorescence:The cells were fixed in 4% paraformaldehyde (PFA) at room temperature for 15 minutes, and then washed 3 times with PBS. Then, the cells were permeabilized and blocked at room temperature for 1 hour with PBS containing 0.2% Triton X-100 and 3% donkey serum, and incubated with the primary antibody at 4°C overnight. After washing 3 times with PBS, the cells were incubated with the secondary antibody at 37°C for 1 hour. Hoechst33342 (Yeasen, catalog: 40732ES03) was used to stain the nuclei for 5 minutes, and then washed 3 times with PBS. Finally, the fluorescence images were taken with the PerkinElmer Opera Phoenix / Harmony system.

[0189] EdU, PI and JC-1 staining : To assess cell proliferation capacity, 4 μM 5-ethynyl-2'-deoxyuridine (EdU) was added to the culture medium and incubated for 12 hours. Subsequently, the proliferating cells were visualized using the Click-iT reaction mixture in the EdU Cell Proliferation Imaging Kit (Orange Fluorescence) (KTA2031, Abbkine). Propidium iodide (P4170, Sigma-Aldrich) staining: PHH was incubated with 3 μM dye for 20 minutes, followed by Hoechst staining. JC-1 staining: PHH was incubated with JC-1 dye (T3168, Thermo Fisher) in a 5 mg stock solution in 12 ml DMSO and in a working dilution of 52 μl stock solution in 1 ml PBS for 2 hours, followed by Hoechst staining. Fluorescence images were acquired using the PerkinElmer OperaPhenix / Harmony system.

[0190] Acute and chronic hepatotoxicity induced by APAP : To induce hepatotoxicity, PHH were incubated with 10 or 20 mM APAP for 16 hours. DNA damage levels, apoptosis, and changes in mitochondrial membrane potential were assessed by staining with anti-γH2AX, PI, or JC-1 according to the methods described above. To rescue damaged PHH, NAC, 4MP, or solvent (control) was added to the culture medium after treatment with 10 mM APAP for another 48 hours.

[0191] Cell viability assayPHH, HepG2, or Huh7 were seeded at 50,000 cells per well in 96-well plates in LiveRSup HHBCM medium or DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. To induce hepatotoxicity, cells were then treated with DILI or non-DILI compounds dissolved in DMSO or water. Compound concentration-response curves were performed with a maximum concentration of 300 μM, 800 μM, 1,000 μM, or 3,000 μM, including 6 or 8 concentration points, with a final concentration of 0.1% DMSO. The 50 μL / mL PBS was used according to the manufacturer's instructions. The luminescent cell viability assay (G7571, Promega) measures ATP levels to assess cell viability in culture.

[0192] AST, human ALB and urea secretion assay :ASTL, cobas c, Integra-Regents, kit (20764949322, Roche) were used to measure AST in cell supernatants according to the manufacturer's instructions. Human ALB and urea secreted by PHH were determined using human albumin ELISA kit (ab108788, abcam) or QuantiChromTM urea assay kit (DIUR-100), respectively, and the culture medium was harvested and diluted 1:200 after 48 hours of cultivation. The corresponding OD450 or OD520 values ​​in different culture media were then used to calculate ALB and urea concentrations and compared with the standard curve.

[0193] Bile acid deficiency (cholestasis) model CellTracker Green CMFDA dye (C2925, Thermofisher) was used to observe hepatic cholestasis. PHH were incubated with 4 μM CMFDA diluted in culture medium for 30 minutes and then stained with Hoechst. Melatonin (S1204, Selleck) was used as a non-toxic control, and treatment with cholestatic drugs cyclosporine A (S2286, Selleck) and troglitazone (HY-50935, MCE) for 24 hours effectively reduced the accumulation of CMF in PHH. Images were captured using the PerkinElmer Opera Phoenix / Harmony system, and bile ducts were counted and normalized for the number of nuclei using the ImageJ particle counting module.

[0194] Hepatitis induced by hepatic lipid accumulation in the MAFLD modelPalmitic acid (PA) and oleic acid (OA) were prepared at a stock concentration of 15 mM for induction of hepatocyte inflammation and lipid accumulation in the MAFLD model. After 18 days of culture, 10 μM PA and 5 μM OA (10:5) or 50 μM PA and 25 μM OA (50:25) were added to the PHH medium for 3 days and treated with or without 30 μM ETC for an additional 16 hours. Wash 3 times with HBSS (Ca2+ / Mg2+). BODIPY TM Neutral lipids were visualized using 493 / 503 (D-3922, Thermo Fisher) or Oil Red (G1262, Solarbio).

[0195] PAS staining and ICG uptake experiments : Cells were plated onto glass coated with rat tail collagen I and cultured in the specified medium. Staining was performed using a periodic acid Schiff kit (PAS; Sigma, catalog: 395B-1KT) according to the manufacturer's instructions, or treated with 1 mg / ml ICG solution (Sigma, catalog: 1340009) at 37°C for 1 hour. After washing 3 times with PBS, absorption images were captured using a ZEISS microscope.

[0196] Detection of CYP enzyme activity : CYP activity was measured by incubating primary human hepatocytes cultured in different media in 48-well or 24-well culture dishes with a mixture of CYP-specific substrates. Products were quantified using liquid chromatography-tandem mass spectrometry (LC-MS / MS) to determine specific products (https: / / www.fda.gov / drugs / drug-interactions-labeling / drug-development-and-drug-interactions-table-substrates-inhibitors-and-inducers).

[0197] RT-qPCR Total RNA was extracted using the EasyPure RNA kit from TransGen Biotech and reverse transcribed into cDNA using TransScript One-step gDNA Removal and cDNA SynthesisSuperMix also from TransGen Biotech. RT-qPCR was then performed using the Quantagene q225 system from KUBO technology and 2×T5 Fast qPCR Mix from TSINGKE Biological Technology.

[0198] RNA-seq :The cells were digested with trypsin-EDTA, and RNA was extracted and genomic DNA was removed using RaPure Total RNA Micro Kit (Magen) and DNase I. Then, the samples that passed the quality inspection were sent to Novogene for RNA sequencing. The RNA-seq matrix of the samples was evaluated using MATLAB R2019a. For PCA of bulk RNA-seq data under different conditions, genes with a mean value less than 1 or a coefficient of variation less than 0.1 after normalization to log2FPKM+1 were excluded. The z-scores of gene expression across samples were used as data input to the pca function, and clustering and heat map visualization were performed using the clustergram function.

[0199] Single-cell RNA-seq : 1×10 5 Single cell suspensions were prepared in PBS (HyClone) at a density of 10 cells / ml and loaded onto the microfluidic device. Program (SingleronBiotechnologies), using RNA-seq libraries were created using the Single Cell RNA Library Kit (Singleron Biotechnologies). Individual libraries were diluted to a concentration of 4 nM and then pooled and sequenced on an Illumina HiSeq X using 150 bp end reads.

[0200] Raw reads were processed using CeleScope v1.12.0 (Singleron Biotechnologies) with default parameters to generate gene expression profiles.

[0201] Briefly, barcodes and UMIs were extracted and corrected from R1 reads. Adapter sequences and polyA tails were trimmed from R2 reads. Trimmed R2 reads were aligned to the GRCh38 (hg38) transcriptome using STAR (v2.6.1a). Uniquely mapped reads were then assigned to exons using FeatureCounts (v2.0.1). To generate a gene expression matrix for further analysis, successfully mapped reads with the same cell barcode, UMI, and gene were grouped together.

[0202] Scanpy v1.8.2 under Python 3.9.10 was used for quality control, dimensionality reduction, and clustering. For each sample dataset, the expression matrix was filtered using the following criteria: 1) exclude cells with gene counts less than 200 or the top 2% of gene counts; 2) exclude cells in the top 2% of UMI counts; 3) exclude cells with mitochondrial content >30%; 4) exclude genes expressed by less than 5 cells. After filtering, a total of 52,601 cells were retained for downstream analysis, with an average of 1,095 genes and 3,477 UMIs per cell. The raw count matrix was normalized by the total counts per cell and log-transformed into a standardized data matrix. The top 2,000 variable genes were selected by setting flavor = 'seurat'. Principal component analysis (PCA) was performed on the scaled variable gene matrix. The first 13 principal components were used for clustering and dimensionality reduction. Cells were grouped into 18 clusters using the Louvain algorithm with a resolution parameter of 1.2. Cell clusters were visualized using uniform manifold approximation and projection (UMAP). Harmony was used to visualize the cell clusters. 23 v0.0.5, using the first 13 principal components in PCA to eliminate batch effects between samples. The batch effects between samples were eliminated using the first 13 principal components in PCA by Harmony23 v0.0.5.

[0203] To identify differentially expressed genes (DEGs), scanpy.tl.rank_genes_groups() function based on Wilcoxon rank sum test was used, using default parameters, and genes expressed in more than 10% of the cells in any compared cell group and with an average logarithm (fold change) of more than 0.25 were selected as DEGs. The adjusted p value was calculated using the Benjamini-Hochberg correction. A value of 0.05 was used as the standard for evaluating statistical significance.

[0204] Using Cell-ID 24Cell-ID is a multivariate method. It extracts gene signatures for each individual cell and performs cell identity identification using the hypergeometric test (HGT). Dimensionality reduction of the normalized gene expression matrix is ​​performed using multiple correspondence analysis, where both cells and genes are projected into the same low-dimensional space. Then, the gene ranking for each cell is calculated to obtain the most characteristic gene set for that cell. HGT is performed on these gene sets against the liver reference in the SynEcoSys database, which contains characteristic genes for all cell types. The identity of each cell is determined as the cell type with the minimum HGT p-value. To annotate the clusters, the frequency of each cell type in each cluster is calculated, and the cell type with the highest frequency is selected as the identity of the cluster. For each cluster, the cell type is identified based on the canonical gene expression in the SynEcoSysTM reference database. To obtain a high-resolution map of liver cells, cells from a specific cluster are extracted and re-clustered for more detailed analysis using the same procedure as above. The cluster resolution was set to 0.6.

[0205] For RNA velocity, BAM files containing hepatocytes and the reference genome GRCh38 (hg38) were used with velocity (v0.17.17) in Python using default parameters. 24 and scVelo (v 0.2.4) 25 For consistency in visualization, the results were projected onto the UMAP plot of the Seurat cluster analysis.

[0206] Data processing and analysis Data are presented as mean ± SD of at least three independent experiments. Statistical analysis was performed using GraphPad Prism 9.0.0.121 (GraphPad Software, San Diego, CA). Unpaired Student's t-test was used to compare significant differences between two groups, while one-way ANOVA (ANOVA) was used to compare significant differences between more than three groups.

[0207] Example 1. Combination of novel compounds maintains PHH function and self-renewal

[0208] Since factors involved in establishing a specific cell type are always associated with the maintenance of that cell type 14,15Therefore, the inventors speculated that small molecules that regulate the fate of hepatocytes may be beneficial to the maintenance of hepatocyte function and self-renewal in vitro. On this basis, the inventors developed a new combination of seven small molecules (7C: DAPT, IWP2, LDN193189, ETC1002, GSK429286A, Salidroside and CID755673), and compared the effects of 7C and X5C (SB431542, Forskolin, DAPT, IWP2 and LDN193189) in maintaining the function and self-renewal of human hepatocytes.

[0209] Then 7C or X5C was added to the culture medium of primary human hepatocytes (PHH) and cultured for 18 days ( Figure 6 a), the results show that for 1×10 5 Pieces / cm 2 The combination of these two small chemical molecules promoted the maintenance of PHH in vitro when cultures were started at a starting cell density of 100 μg / mL or greater. Compared with cultures treated with X5C, the maintenance of PHH at day 9 ( Figure 1 a) and day 18 ( Figure 6 a), PHHs cultured with 7C had higher cell numbers, clearer hepatocyte morphology, and increased glycogen storage. In addition, at day 18, the proportion of human hepatocytes expressing ALB in 7C-treated cells was significantly higher than that in cells cultured with X5C ( Figure 6 Surprisingly, very few Ki67-expressing cells were detected in X5C-cultured PHHs, indicating that they lacked the ability to self-renew, whereas approximately 10%-15% of 7C-treated PHHs were Ki67-positive, indicating that these cells were able to proliferate ( Figure 6 be). EdU staining further indicated that chromatin duplication and cytoplasmic division occurred ( Figure 1 b). In addition, cells labeled with EdU can also express CYP3A4 and MRP2 ( Figure 6 f), indicating that the expanded cells retain hepatocyte function.

[0210] These results indicate that the addition of seven small molecules promoted the maintenance and expansion of human hepatocytes in vitro.

[0211] Example 2. Optimized small molecule combination capable of maintaining PHH identity and self-renewal

[0212] To further identify small molecules that play a key role in the maintenance and self-renewal of human hepatocytes, the inventors subtracted each small molecule from 7C and then performed culture tests on human hepatocytes to quantify PHH proliferation and functional gene expression ( Figure 1 c, d).

[0213] The results showed that the number of cells decreased significantly when GSK was subtracted, suggesting that GSK is involved in maintaining proliferation capacity ( Figure 1 c, d). Expression analysis of ALB, CYP3A4, MRP2, and Ki67 also supports the involvement of GSK and CID in promoting self-renewal and maintaining cell function ( Figure 1 c, d); DAPT and LDN also affected the expression of some functional genes, although to a lower extent ( Figure 1 c, d).

[0214] Therefore, the inventors generated a 4C combination, including the above four key small molecules GSK, CID, DAPT and LDN, and tested its effect on isolated PHH. In subsequent screening, based on the expression of hepatocyte function genes such as ALB, CYP3A4, BSEP and MRP2, and the expression of the dedifferentiation marker gene KRT7 ( Figure 7 ad), confirming that the 2C combination (GSK and CID) can maintain PHH identity. Ki67 and EdU staining confirmed that GSK and CID regulate the self-renewal process of human hepatocytes ( Figure 1 c, d, and Figure 7 a, b, and d). It is noteworthy that almost all cells expressing Ki67 are not KRT7-positive cells ( Figure 7 a), indicating that cell expansion is not dependent on dedifferentiation, but rather on hepatocyte self-renewal. Additional CYP enzyme activity experiments showed that LDN and DAPT had no significant effect on PHH function or marker gene expression ( Figure 7 d, e). Interestingly, a large proportion of human hepatocytes cultured long-term with 7C or 4C were able to be re-plated and grown after digestion and passaging, which was difficult to achieve in previous mature hepatocyte culture protocols. However, only a relatively small number of re-plated cells were able to continue to expand.

[0215] In addition, it was found that human hepatocytes cultured in 2C showed the same proportion of Ki67 and EdU positive cells, as well as ALB and MRP2 positive cells compared with human hepatocytes cultured in 4C ( Figure 8 ad). The presence of Ki67-positive and EdU-labeled PHHs indicates that 2C is sufficient for these cells to complete the cell cycle ( Figure 8 ad), maintaining self-renewal through expansion. Therefore, 2C is the minimum combination to maintain functional hepatocyte self-renewal.

[0216] Example 3: Human hepatocytes cultured for a long time with 7C or 2C have metabolic capacity and CYP450 enzyme activity

[0217] After one month of incubation with 7C or 2C, PHH retained CYP1A2, CYP2C9, CYP2D6, and CYP3A4 activities similar to those of fresh PHH ( Figure 2 a). In addition, PHH treated with 7C or 2C continued to secrete albumin and urea, indicating that they were mature cells ( Figure 2 b, c).

[0218] In addition, compared with the control group and X5C group, the 7C and 2C groups cultured for 2 months had strong abilities to store glycogen and absorb ICG ( Figure 2 d), indicating that the combination of these two small chemical molecules can achieve long-term culture of human hepatocytes while maintaining liver function.

[0219] In PHHs cultured with 7C or 2C for 30 or 90 days, the expression of hepatocyte functional genes such as ALB, HNF4A, UGT1A1, PEPCK, ALDOB, and OATPB was similar to or even higher than that of fresh controls or PHHs cultured for 48 hours ( Figure 2 e). Notably, expression of SLC10A1, encoding the HBV receptor NTCP, persisted for up to 90 days after culture ( Figure 2 e), indicating that these long-term cultured PHHs have the potential to serve as platforms for HBV infection and replication. Thus, PHHs cultured with 7C or 2C exhibited CYP enzyme activities close to those of fresh PHHs and retained their abilities to store glycogen and ICG uptake as well as functional gene expression.

[0220] The inventors then attempted to investigate the mechanism of action of the two compounds, GSK and CID. They replaced them with compounds targeting ROCK and PKD1 / 2 / 3, respectively, for long-term PHH culture. The results showed that most compounds with the same targets could replace GSK or CID to promote cell proliferation or function, as shown by Ki67 or MRP2 expression ( Fig. 9 ).

[0221] Example 4: Long-term maintained PHH can respond to regeneration signals of growth factors

[0222] Since human hepatocytes maintained in 2C exhibit self-renewal capacity, they are expected to respond to in vivo liver regeneration signals to promote expansion. In vivo, mitogens such as HGF and EGF are essential for the liver regeneration process, especially after partial hepatectomy. 17 However, their in vitro effects are limited 6 As a test of the hepatocyte regeneration response, the initial cell density was reduced to 3.3 × 10 4 / cm 2 , cells were poorly maintained at this density. However, HGF and EGF signaling significantly enhanced cell maintenance ( Fig.10 a, b). The results showed that the number of cells increased ( Fig.10 c), the proportion of ALB-expressing cells and Ki67-expressing cells increased ( Fig.10 d, e), while HGF also increased the proportion of CYP3A4- and MRP2-expressing cells and decreased the proportion of KRT7-expressing cells ( Fig.10 fh). When HGF and EGF were added, ALB expression increased ( Fig.10 d), but there was no increase in cells expressing Ki67 ( Fig.10 e). Simultaneous addition of HGF and EGF may lead to rapid cell proliferation, filling up the cell growth space, resulting in cell contact inhibition and preventing further cell proliferation. Therefore, this result indicates that 2C-cultured hepatocytes can respond to pro-regenerative signals, especially HGF, which not only promotes human hepatocyte proliferation but also has a promoting effect on the maintenance of liver function.

[0223] Example 5 Evaluation of the treatment of acute liver failure induced by acetaminophen (APAP) in hepatocytes maintained at 2C and 7C

[0224] The use of hepatocytes in vitro to establish an acute liver failure model is conducive to high-throughput drug screening and the study of cytotoxic pathological processes. Since PHHs exhibit liver-related metabolic functions and enzyme activities, the present inventors tested their responses to toxic drugs and therapeutic drugs that induce liver failure ( Figure 3 a). The present inventors found that after 2 months of culture, human hepatocytes cultured in the WE control group and X5C were no longer able to respond to APAP-induced liver injury. After APAP treatment, the AST level in the culture medium of human hepatocytes did not increase significantly ( Figure 3 b). After human hepatocytes cultured in 7C or 2C were treated with APAP, the AST level increased significantly, and AST was mainly distributed in the cytoplasm and mitochondria ( Figure 3 b). In addition, the use of NAC (N-acetylcysteine) and 4MP (4-methylpyrazole), effective rescue drugs for APAP, can significantly reduce the levels of AST in these cells. These indicate that chemically cultured human hepatocytes can be damaged by APAP and then rescued by drug intervention, reversing liver cell damage.

[0225] Interestingly, cells treated with 2C had significantly reduced AST levels after APAP treatment ( Figure 3 b). This finding indicates that the 2C combination can indeed improve liver cell function and reduce liver damage. However, using γH2AX antibody and PI staining to detect cells, it was found that only positive treatment drugs NAC and 4MP could reverse DNA damage in liver cells maintained by 7C or 2C and prevent cell apoptosis ( Figure 3 c and Fig.11ac). JC-1 staining further revealed that mitochondrial damage in cells induced by APAP could be repaired by NAC and 4MP, thereby restoring mitochondrial membrane potential ( Figure 3 d and Fig.11 d). These results indicate that human hepatocytes cultured with 7C or 2C have similar metabolic capacity and cell activity as primary cells, and are able to establish an acute liver failure-induced injury model and respond to toxic induction and drug rescue processes.

[0226] Example 6. 2C promotes the establishment of MAFLD and cholestasis models

[0227] Excessive lipid accumulation can lead to the inability of hepatocytes to metabolize foreign or endogenous molecules, thus inducing MAFLD. To test whether human hepatocytes cultured with 2C can be used to establish a MAFLD model, Figure 3 e) and without 2C treatment ( Figure 3 f) Human hepatocytes cultured for one month were treated with PA (palmitic acid) and OA (oleic acid) (i.e., PO) for 72 hours to induce fat accumulation, mimicking the mild steatosis of MAFLD. After inducing fatty acid accumulation, treatment of 2C-cultured cells with the ATP-citrate lyase (ACLY) inhibitor ETC promoted the reduction of intracellular fat accumulation, according to the reported mechanism of action of ETC 17 , possibly by increasing fatty acid oxidation ( Figure 3 eh).

[0228] Relative expression analysis by RT-qPCR revealed that in cultured cells treated with 2C, genes related to fatty acid synthesis (SREBF1), oxidation (CPT1A), and inflammation (NFKB1 / HLA-A / TGFB1 / IFNGR2 / TNF / IFNAR1) were upregulated, whereas genes related to liver function (ALB / HNF4A / UGT1A1 / ALDOB / PEPCK / ABCC2) were downregulated after PO treatment. Figure 3 i and Fig.12 a, b). In the WE (William's E medium) control, no intracellular fatty acid accumulation was observed even after PO induction, and the hepatocytes in the WE control group showed no transcriptional response to PO treatment ( Fig.12 c, d). ETC treatment increased the expression of fatty acid metabolism genes, including those related to lipogenesis (SCD / ACACA / FASN) 18 and the fatty acid transporter CD36 gene, which may function as a protective metabolic sensor in the liver under lipid overload 19 , thereby reducing inflammation and fat accumulation and at least partially rescuing the expression of genes related to liver function ( Figure 3 i and Fig.12a, b). In hepatocytes treated with 2C after fatty acid accumulation, although the expression of genes related to liver function was restored, no increase in fatty acid metabolism genes was detected, while the expression of inflammation-related genes decreased ( Figure 3 i and Fig.12 b). This finding suggests that 2C treatment, which helps maintain hepatocyte fate, has the potential to affect changes in molecular signaling networks, thereby reducing the development of hepatitis without affecting metabolic function. In contrast, PHH treated with 7C showed a similar response to PO induction as PHH treated with 2C, but responded differently to ETC treatment; whereas PHH treated with X5C showed little lipid accumulation after PO treatment ( Fig.12 el). Therefore, these experiments suggest that 2C or 7C can be used to maintain long-term culture of PHHs and to screen and study relevant therapeutic agents that mimic hepatitis induced by lipid accumulation during MAFLD.

[0229] The above data show that PHH cultured for a long time with 7C or 2C express MRP2 ( Figure 1 c and Figure 8 a), which is required for bile acid secretion. CMFDA staining analysis after 40 days of culture to observe the bile secretion process showed that fluorescent 5-chloromethylfluorescein (5-CMF), a fluorescent product of CMFDA cleavage, was only exported by hepatocytes maintained in 2C, while hepatocytes maintained in 7C showed only negligible green fluorescence signals, and no signals were detected in the control and X5C groups ( Figure 3 j and Fig.12 m). Treatment of PHH maintained at 2C or 7C with cyclosporine A (CysA) or troglitazone (Tgz) resulted in a significant reduction in 5-CMF output, whereas control treatment with melatonin had no effect ( Figure 3 km and Fig.12 n, o). In conclusion, PHHs maintained with 2C in long-term culture can respond to drug-induced cholestatic processes, suggesting their potential application in screening cholestatic drug toxicity.

[0230] Example 7: Establishment of drug-induced liver injury model with PHH cultured in 2C

[0231] Accurate assessment of hepatotoxicity is crucial for preclinical studies. Although primary hepatocytes are considered the gold standard for evaluating drug hepatotoxicity, previous studies have reported both false-positive and false-negative results. 20 Increasing the drug treatment time can reduce these errors, but the limited in vitro hepatocyte culture time limits the improvement of accuracy. In this study, 12 standard compounds were tested for their potential to cause drug-induced liver injury (DILI) in 2C cultures, including 8 compounds that have been previously associated with DILI and 4 compounds that have not been associated with DILI. 20By changing the duration of administration, it is possible that small molecules with DILI potential will accumulate toxicity and produce toxic effects over a longer period of time ( Figure 4 a, b). Similar EC50 values ​​were observed on days 3-4 and 17-18. However, as the duration of drug treatment increased, the EC50 values ​​gradually decreased from days 17-18 to days 15-18 and then to days 3-18 ( Fig.13 a). This trend suggests that toxic drugs may not show toxicity under short-term treatment conditions, such as Bosentan and paracetamol. Therefore, it may be difficult to effectively distinguish whether these drugs are hepatotoxic. Importantly, the toxicity of these drugs to hepatocytes was accurately reflected by the extended treatment period of 3-18 days. It was found that the EC50 / Cmax ratio of hepatotoxic small molecules was <20, and the EC50 / Cmax ratio of non-hepatotoxic small molecules was >20 ( Figure 4 c and Fig.13 a, b). This method can accurately reflect the hepatotoxicity of drugs and has broad application prospects in preclinical drug hepatotoxicity evaluation.

[0232] A recent study on neonicotinoid (NEO) residues in human bile found that nitenpyram (NTP) is often present in high concentrations and is not easily degraded. The study also found a positive correlation between individual NEO concentrations and bile acid, total bilirubin, cholesterol, and alkaline phosphatase levels, indicating a potential hepatotoxic risk associated with NEOs such as NTPs. Another recent long-term follow-up study reported that nitrofurantoin (NFT), a synthetic antibiotic commonly used as a first-line treatment for uncomplicated urinary tract infections, may be a risk factor for liver damage. In particular, when NFT is used as a prophylaxis for recurrent urinary tract infections (UTIs) or as a single post-coital dose, the common dose in clinical practice is 50-100 mg per day. In order to study the potential toxicity of long-term exposure to NFP and NTP to PHH, the inventors exposed 2C-cultured PHH to different doses of NTP and NFT ranging from 800 μM to 49 nM for different lengths of time to simulate short-term and long-term exposure to in vivo hepatotoxicity ( Figure 4 a, b). The results showed that cell viability and AST levels indicated that PHH treated with NTP had no toxic effects ( Figure 4 d). Interestingly, the toxic effects of NFT were observed to increase with the duration of treatment. The IC50 values ​​on days 3-4 and 17-18 were similar, 142.5 μM and 196.3 μM, respectively ( Figure 4 e). However, the IC50 values ​​on days 13-18 and 3-18 were much lower, 99.72 μM and 24 μM, respectively ( Figure 4e). These results indicate that the toxic effects of NFT increase with the duration of exposure. However, in HepG2 and Huh7 cells, an incremental relationship between toxicity and time could not be achieved. This is due to the rapid proliferation capacity of cancer cells and different cellular characteristics compared with primary hepatocytes. Therefore, the stability and reliability of test results using these cell lines are much lower than those of primary hepatocytes ( Fig.13 cf). In addition, NFT has been shown to affect bile duct function and may produce toxic effects at high doses and for prolonged exposure ( Figure 4 Collectively, these results suggest that 2C-cultured PHHs can mimic drug-induced liver injury in vivo and could be used to identify toxic factors in chronically exposed hepatocellular carcinomas and rescue drugs in clinical applications.

[0233] Example 8: Single-cell RNA sequencing (scRNA-seq) reveals that 7C and 2C maintain culture by inhibiting PHH dedifferentiation

[0234] To explore the transcriptome profile of PHH, scRNA-seq was performed on fresh PHH and PHH cultured with WE control for 45 days and 7C or 2C for 90 days or 150 days. Figure 5 a). Quality filtering revealed eighteen cell clusters classified into six major cell types: hepatocytes, cholangiocyte-like cells, hepatic stellate cells (HepSCs), endothelial cells (ECs), T cells, and mononuclear phagocytes (MPs) ( Figure 5 b, c and Fig.14 ac). Cholangiocyte-like cells were defined by the expression of KRT7 and KRT19, but not other cholangiocyte marker genes such as MMP7, EPCAM, and CFTR ( Fig.15 d) These cells resemble dedifferentiated hepatocytes rather than native cholangiocytes.

[0235] Each cell type expresses different marker genes and they are not evenly distributed across the sample ( Fig.14 e). Notably, 7C effectively maintained hepatocytes at day 90, while reducing other cell types ( Figure 5 c). In contrast, the proportion of cholangiocyte-like cells in the control group increased significantly and the proportion of hepatocytes decreased on day 45 ( Figure 5 c). The 2C group faithfully maintained all cell types at similar ratios to the fresh PHH group at day 90 ( Figure 5 c).

[0236] Further analysis revealed seven subpopulations of hepatocytes ( Figure 5d). Subgroups 3 and 4 showed higher maturity than other subgroups, as evidenced by the expression of liver function marker genes such as ALB / CYP1A1 / CYP1A2 / FGG / ABCC2 / FABP1 ( Figure 5 e). The overall gene expression profiles of these subpopulations are more similar to adult hepatocytes than to fetal hepatocytes, iPS-derived hepatocytes, or hepatoma cell lines ( Figure 5 f). These subpopulations were highly enriched (>90%) in day 90 7C cultures ( Figure 5 d), while subpopulations 1 and 2 that did not express functional marker genes were highly enriched in WE cultures on day 45 ( Figure 5 d, e). PHH cultured with 2C contained heterogeneous hepatocyte subpopulations similar to those of fresh PHH groups ( Figure 5 d).

[0237] The inventors speculate that the conversion between different subpopulations such as hepatocytes and cholangiocyte-like cells may represent the maintenance state of hepatocytes at this time. To test this hypothesis, the monocle analysis method was used to illustrate the potential and direction of cell transformation after excluding other cells such as EC, MP and T cells ( Figure 5 g, h, and Fig.14 f). Pseudo-temporal analysis reveals the differentiation status of hepatocytes from functional subpopulations 3 and 4 to subpopulations 5, 6, 7 and 1, 2, and then to cholangiocyte-like cells ( Figure 5 g, h, and Fig.15 ad). Expression of immunological (A2M), mesenchymal (ACTB / ACTG), and hepatic (AGXT / AHSG / ALB) marker genes in different cell populations revealed a pattern of increased immunological, epithelial-mesenchymal transition (EMT), and hepatic function. Many other studies have reported loss of cell function during ex vivo hepatocyte culture ( Figure 5 h).

[0238] RNA velocity was also used to analyze the direction of differentiation. Similar to the monocle analysis, RNA velocity revealed the transition from hepatocytes 3 and 4 to hepatocytes 5, 6, 7 and 1, 2, and then to cholangiocyte-like cells ( Figure 5 g and Fig.15 Notably, the transition in the 7C culture at day 90 was limited to functional hepatocytes (subpopulations 3 and 4), whereas the WE culture at day 45 contained mainly transitions between hepatocytes 1 and 2 and cholangiocyte-like cells ( Figure 5 In contrast, there was a rich transition relationship between all cell types in the fresh PHH and the group cultured with 2C for 90 days ( Figure 5 d and Fig.15 eg).

[0239] discuss:

[0240] Long-term culture of PHHs ex vivo is limited by the intrinsic effects of the preservation process, which either prevents proliferation or requires dedifferentiation and loss of function to induce proliferation. Improving the availability and function of culturable hepatocytes, which are often costly and difficult to obtain, could greatly reduce the demand for donor PHHs and improve the economic utility of PHHs. Here, two chemical combinations were developed that could maintain the self-renewal capacity of PHHs ex vivo and significantly extend their culture time while preserving their function. In these 2C- and 7C-maintained PHHs, gene expression, cell viability, metabolic capacity, and enzyme activity after three months of culture were comparable to those of freshly thawed PHHs.

[0241] HepG2 cells have been widely used to model pathogenic conditions such as fatty acid accumulation and hepatitis. 13 This study provides proof-of-concept evidence that long-term cultures of PHHs also respond to fatty acid accumulation, induced hepatitis, and therapeutic effects of drugs. It avoids important background differences between hepatocytes and hepatocellular carcinoma cells, such as abnormal metabolism and transcriptional dysregulation, thereby improving the effectiveness of ex vivo ADME (absorption, distribution, metabolism, and excretion) or toxicity screening.

[0242] Other studies using sandwich cultured hepatocytes have shown that these cells retain sensitivity to apoptosis and polarity phenotypes but do not proliferate in response to mitogenic stimuli 6 In addition, the chemical treatments tested in this study promoted proliferation (i.e., self-renewal) of cryopreserved human hepatocytes in monolayer culture, allowing for testing of the cells' response to liver regenerative factors such as HGF and EGF. Therefore, this system could serve as a valid model for the development of chemical or genetic factors involved in hepatocyte expansion in liver disease research and regenerative medicine. 21 .

[0243] While acute liver injury can be mimicked by short-term treatment of PHHs with potentially toxic substances (e.g., APAP), chronic liver injury caused by substances that require long-term exposure is limited by PHH culture. In order to accurately assess the toxic changes of drugs that require long-term exposure and determine the risks of drugs with potential chronic toxicity, this study developed a PHH-based primary cell model to test different drug exposure durations, which was found to be able to have 100% fidelity through measurements of twelve DILI-related compounds or non-DILI compounds. It was also found that NTP did not exhibit toxic effects on hepatocytes after 15 days of exposure at concentrations ≤800μM. In contrast, the toxic dose of NFT decreased with long-term exposure compared to short-term treatment, indicating that toxic effects can accumulate over time.

[0244] In summary, this study describes a system for long-term ex vivo culture of PHHs by supplementing the culture medium with small molecule formulations. The functionality and self-renewal capacity of PHHs were maintained by applying a cocktail of two (or seven) small molecules over a three-month culture period. These cells can then be used to establish models of fatty acid accumulation hepatitis, drug-induced liver injury, cholestasis, and other potential conditions. With this approach, hepatocytes that need to mimic long-term biological processes in the liver or cannot be used in a timely manner for various experimental reasons can be maintained in an ex vivo state without losing cell fate or function, thereby expanding the potential applications of PHHs and preventing the waste of precious biomaterials. In addition, some other new research advances can be obtained by testing whether these mixtures can also promote hepatocyte differentiation from pluripotent cells, redifferentiation of dedifferentiated hepatocytes, or whether they can enhance hepatocyte culture and maturation into organoids.

[0245] Some small molecule compounds and their structures involved in this article:

[0246]

[0247]

[0248]

[0249]

[0250] References:

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Claims

1. A small molecule compound combination for in vitro culture and / or expansion of hepatocytes, the combination comprising one or more compounds selected from GSK429286A or a functional analogue thereof, CID755673 or a functional analogue thereof, DAPT or a functional analogue thereof, LDN193189 or a functional analogue thereof, ETC1002 or a functional analogue thereof, IWP2 or a functional analogue thereof, and Salidroside or a functional analogue thereof.

2. The combination of claim 1, wherein the combination comprises one or more compounds selected from the group consisting of GSK429286A, CID755673, DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

3. The combination of claim 1, wherein the combination comprises GSK429286A or a functional analogue thereof, CID755673 or a functional analogue thereof, and, optionally, one or more compounds selected from DAPT or a functional analogue thereof, LDN193189 or a functional analogue thereof, ETC1002 or a functional analogue thereof, IWP2 or a functional analogue thereof, and Salidroside or a functional analogue thereof.

4. The combination of claim 3, wherein the combination comprises GSK429286A, CID755673, and, optionally, one or more compounds selected from DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

5. The combination of claim 3, wherein the combination comprises GSK429286A or a functional analogue thereof and CID755673 or a functional analogue thereof.

6. The combination of claim 5, wherein the combination comprises GSK429286A and CID755673.

7. The combination of claim 1, wherein the combination comprises GSK429286A or a functional analogue thereof, CID755673 or a functional analogue thereof, DAPT or a functional analogue thereof, LDN193189 or a functional analogue thereof, and, optionally, one or more compounds selected from ETC1002 or a functional analogue thereof, IWP2 or a functional analogue thereof, and Salidroside or a functional analogue thereof.

8. The combination of claim 7, wherein the combination comprises GSK429286A, CID755673, DAPT, LDN193189, and, optionally, one or more compounds selected from ETC1002, IWP2, and Salidroside.

9. The combination of claim 7, wherein the combination comprises GSK429286A or a functional analogue thereof, CID755673 or a functional analogue thereof, DAPT or a functional analogue thereof, and LDN193189 or a functional analogue thereof.

10. The combination of claim 9, wherein the combination comprises GSK429286A, CID755673, DAPT, and LDN193189.

11. The combination of claim 1, wherein the combination comprises GSK429286A or a functional analogue thereof, CID755673 or a functional analogue thereof, DAPT or a functional analogue thereof, LDN193189 or a functional analogue thereof, ETC1002 or a functional analogue thereof, IWP2 or a functional analogue thereof, and Salidroside or a functional analogue thereof.

12. The combination of claim 11, wherein the combination comprises GSK429286A, CID755673, DAPT, LDN193189, ETC1002, IWP2, and Salidroside.

13. A culture medium for in vitro culture and / or expansion of hepatocytes, the culture medium comprising the small molecule compound combination according to any one of claims 1 to 12.

14. The culture medium of claim 13, further comprising a basal medium such as William's E medium, LiveRSup human hepatocyte basal medium, or Hepatocyte Maintenance Medium (Cat#CC3198, Lonza).

15. The culture medium according to claim 13 or 14, further comprising hepatocyte growth factor (HGF) and / or epidermal growth factor (EGF), preferably HGF.

16. The culture medium of any one of claims 13 to 15, wherein the concentration of each of the one or more compounds in the culture medium may be about 0.01 μM to about 100 μM.

17. A method for culturing and / or expanding hepatocytes in vitro, the method comprising culturing hepatocytes in the presence of the small molecule compound combination according to any one of claims 1 to 12 or in the culture medium according to any one of claims 13 to 16.

18. The method of claim 17, wherein the hepatocytes are cultured for a period of about 1 day to about one year or longer.

19. The method of claim 17 or 18, wherein the hepatocytes are of human origin.

20. The method according to any one of claims 17 to 19, wherein the hepatocytes can be freshly isolated or prepared primary hepatocytes, or can be cryopreserved hepatocytes.

21. Hepatocytes cultured and / or expanded by the method of any one of claims 17 to 20.

22. Use of the hepatocytes according to claim 21 for screening candidate drugs for treating liver diseases, and / or for testing the pharmacological properties of drugs (such as drug toxicity).

23. The method of claim 21, wherein the liver disease is selected from the group consisting of acute liver failure, chronic liver failure, liver fibrosis, cirrhosis, liver cancer (primary liver cancer or metastatic liver cancer), toxic liver injury, biliary cirrhosis, primary sclerosing cholangitis, chronic autoimmune hepatitis, fatty liver disease, alcoholic liver disease, non-alcoholic fatty liver disease, liver damage associated with pathogen infection (e.g., parasitic infection or hepatitis virus infection, such as hepatitis A, hepatitis B, hepatitis C, etc.), hereditary liver disease, drug-induced liver damage, liver damage caused by liver resection surgery, and impaired function of a transplanted liver.

24. The use of claim 21, wherein the liver disease is metabolic dysfunction associated fatty liver disease (MAFLD).

25. The use of claim 21, wherein the liver disease is acetaminophen (APAP)-induced acute liver failure.

26. The use of claim 21, wherein the pharmacological property of the drug (e.g., drug toxicity) is induction of cholestasis.

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