Methods of Producing and Using Liver Cells

Abstract

Methods for making and using many different types of liver cells are provided. The present invention provides a method for expanding hepatoblasts, comprising culturing the hepatoblasts in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or its equivalent, and a method for obtaining mature hepatocytes, comprising culturing the hepatoblasts in the presence of thyroid hormone or a thyroid hormone receptor agonist.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Application No. 62 / 857,180, filed June 4, 2019.

[0002] Technical Field FIELD OF THE DISCLOSURE The present disclosure relates generally to stem cells, and more particularly to the expansion and differentiation of stem cells. [Background technology]

[0003] background The liver is the largest solid organ and largest gland in the human body. It is classified as part of the digestive system and performs over 500 essential functions, including detoxification, protein synthesis, and the production of enzymes that aid in food digestion. Despite the liver's regenerative capabilities, a diseased or dysfunctional liver can be dangerous or even fatal. Cell therapy is a viable alternative but requires the ability to generate large numbers of multiple, different types of liver cells. Furthermore, the ability to generate multiple, different types of liver cells allows for advances in research. Summary of the Invention

[0004] overview The present disclosure describes methods for making and using many different types of liver cells.

[0005] In one aspect, a method for expanding hepatoblasts is provided. Such a method typically comprises culturing hepatoblasts in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or its equivalent.

[0006] In some embodiments, the activator of Wnt pathway is CHIR99021, CHIR98014, BIO, (potent) GSK-3 inhibitor, or natural Wnt agonist, such as Wnt3. In some embodiments, the TGF-β receptor inhibitor is SB431542, A83-01, or ALK4 and / or ALK7 inhibitor (e.g., SB525334, SB505124, etc.). In some embodiments, FGF19 or its equivalent is a modified form of FGF19 called NGM282. In some embodiments, the method is carried out under hypoxic conditions.

[0007] In another aspect, provided is the method for expanding hepatoblasts.Such method typically comprises culturing hepatoblasts under hypoxic conditions.In some embodiments, such method further comprises culturing hepatoblasts under the presence of Wnt pathway activator, TGF-β receptor inhibitor and FGF19 or its equivalent.

[0008] In another aspect, a method for expanding hepatoblasts is provided. Such a method typically comprises culturing hepatoblasts under hypoxic conditions in the presence of a Wnt pathway activator, a TGFβ inhibitor, FGF19, or an equivalent thereof. In some embodiments, when cultured under ambient O2 conditions, the number of hepatocytes is increased by about 100-fold to about 400-fold within 3 to 5 passages. In some embodiments, when cultured under hypoxic conditions, the number of hepatocytes is increased by about 75-fold to about 1000-fold within 3 to 5 passages. In some embodiments, an inhibitor of Notch signaling can be used in the culture to maintain hepatoblast characteristics.

[0009] In another aspect, a method for obtaining mature hepatocytes is provided. Such a method typically comprises culturing hepatoblasts in the presence of thyroid hormone or thyroid hormone receptor agonist. In some embodiments, the thyroid hormone is triiodothyronine or thyroxine. In some embodiments, the thyroid hormone receptor agonist is GC-1. In some embodiments, the hepatoblasts are cultured as a monolayer. In some embodiments, the hepatoblasts are cultured as aggregates (with thyroid hormone added; it also functions in aggregates). In some embodiments, the hepatoblasts are cultured in the absence of cAMP. In some embodiments, mature hepatocytes express little or no alpha-fetoprotein (AFP). In some embodiments, mature hepatocytes express albumin. An inhibitor of Notch signaling can be used in the culture to maintain hepatocyte characteristics.

[0010] In some aspects, a method for producing zone 1 hepatocytes is provided. Such a method typically comprises culturing hepatoblasts in the presence of a Wnt pathway inhibitor. In some embodiments, the Wnt pathway inhibitor is XAV939, IWP2, IWP4, or ICRT14. In some embodiments, the hepatoblasts are cultured in monolayer or aggregate.

[0011] In another aspect, the present invention provides a method for producing zone 3 hepatocytes.Such method typically comprises culturing hepatoblasts in the presence of an activator of Wnt pathway.In some embodiments, hepatoblasts are cultured in monolayer or aggregate.

[0012] In some aspects, provide a method for producing cholangiocytes.Such method typically comprises culturing hepatoblasts in the presence of retinoic acid, retinol or RA receptor agonist.In some embodiments, cholangiocytes are identified based on the presence of cystic fibrosis transmembrane conductance regulator (CFTR) protein.In some embodiments, cholangiocytes are identified based on the binding to DHC5-4D9 antibody.

[0013] In another aspect, provided is the method for producing liver organoid.Such method typically comprises the step of combining mesothelial cell (US20160215263) and hepatoblast under the condition of promoting hepatic organoid self-organization.In some embodiments, such method further comprises the step of expanding hepatoblast under the presence of Wnt pathway activator, TGFβ inhibitor and FGF19.

[0014] In another aspect, provide the method for producing stellate cell.This method typically comprises culturing the liver organoid as described herein under the condition that produces stellate cell.

[0015] In another aspect, there is provided a method for treating a liver disease (e.g., cholangiopathy, including but not limited to, bile duct disease or hepatocellular carcinoma) in a subject. Such a method typically includes transplanting a composition comprising cholangiocytes into the subject.

[0016] In another aspect, provided is a method for treating a subject with liver disease.This method typically comprises: transplanting the composition comprising the hepatoblasts that are expanded using any of the methods described herein; transplanting the composition comprising the hepatocytes that are matured using any of the methods described herein; transplanting the composition comprising the zone 1 hepatocytes that are produced using any of the methods described herein; transplanting the composition comprising the zone 3 hepatocytes that are produced using any of the methods described herein; transplanting the composition comprising the bile duct cells that are produced using any of the methods described herein; transplanting the composition comprising the liver organoid as described herein into subject; and / or transplanting the composition comprising the stellate cells that are produced using any of the methods described herein.In some embodiments, the composition further comprises epithelial cells.In some embodiments, the method further comprises monitoring the subject for albumin level.In some embodiments, the method further comprises monitoring the subject for one or more liver enzymes (total bilirubin, aspartate transaminase (AST), alanine transaminase (ALT) or gamma-glutamyltransferase (GTP)) levels. In some embodiments, the transplant is performed directly into the liver or heterotopically into the abdomen.

[0017] In another aspect, provide a method for culturing liver cells.Such method typically comprises culturing liver cells on a substrate under conditions that liver cells grow as a monolayer.In some embodiments, the method further comprises culturing liver cells as a monolayer and then culturing them as aggregates.In some embodiments, the number of liver cells that arise from the monolayer is at least 10 times (for example, 15 times, 20 times) greater than the number of liver cells that arise from the culture of aggregated cells.

[0018] In yet another aspect, the present invention provides a method for cryopreserving liver cells.This method typically comprises the following steps: culturing liver cells in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or its equivalent for at least 3 days; and cryopreserving the cultured liver cells.This method can further comprise thawing the cryopreserved liver cells, and culturing the thawed liver cells in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or its equivalent.

[0019] In yet another aspect, provided is the method for recovering cryopreserved liver cells.This method typically comprises the steps of thawing cryopreserved liver cells; and culturing the thawed liver cells in the presence of Wnt pathway activator, TGFβ inhibitor and FGF19 or equivalent.This method can further comprise the steps of culturing liver cells in the presence of Wnt pathway activator, TGFβ inhibitor and FGF19 or equivalent for at least 3 days before cryopreserving liver cells.

[0020] In some embodiments, cryopreservation comprises freezing the liver cells at -80°C in a medium comprising DMSO, FSC and DMEM / F12. In some embodiments, thawing comprises heating the liver cells to 37°C for about 5 minutes. In some embodiments, the liver cells are hepatoblasts.

[0021] In another aspect, the present invention provides a method for screening therapeutic compounds for cystic fibrosis and / or cilia-related diseases.Such method typically comprises contacting bile duct cells with a test compound and determining the presence or absence of CFTR function.Generally, the presence or absence of CFTR function indicates that the test compound is therapeutic for cystic fibrosis and / or cilia-related diseases.

[0022] [The present invention 1001] 1. A method for expanding hepatoblasts, comprising: Culturing hepatoblasts in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or an equivalent thereof. A method comprising: [The present invention 1002] 1001. The method of claim 1001, wherein the activator of the Wnt pathway is CHIR99021, CHIR98014, BIO, a GSK-3β inhibitor, or a natural Wnt agonist, such as Wnt3. [The present invention 1003] The method of claim 1001 or 1002, wherein the TGF-β receptor inhibitor is SB431542, A83-01, or an ALK4 and / or ALK7 inhibitor. [The present invention 1004] The method of any one of claims 1001 to 1003, wherein the FGF19 or its equivalent is NGM282. [The present invention 1005] The method according to any one of claims 1001 to 1004, which is carried out under hypoxic conditions. [The present invention 1006] 1. A method for expanding hepatoblasts, comprising: Culturing hepatoblasts under hypoxic conditions A method comprising: [The present invention 1007] Culturing hepatoblasts in the presence of a Wnt pathway activator, a TGF-β receptor inhibitor, and FGF19 or an equivalent thereof. The method of the present invention 1006 further comprising: [The present invention 1008] 1. A method for expanding hepatoblasts, comprising: Culturing hepatoblasts under hypoxic conditions in the presence of a Wnt pathway activator, a TGFβ inhibitor, FGF19 or an equivalent thereof. A method comprising: [The present invention 1009] 1005. The method of any of claims 1001 to 1004, wherein the number of hepatocytes is increased by about 100-fold to about 400-fold within 3 to 5 passages when cultured under ambient O 2 conditions. [The present invention 1010] 9. The method of any one of claims 1005 to 1008, wherein the number of hepatocytes is increased by about 75 to about 1000 fold within 3 to 5 passages when cultured under hypoxic conditions. [The present invention 1011] 1. A method for obtaining mature hepatocytes, comprising: Culturing hepatoblasts in the presence of thyroid hormone or a thyroid hormone receptor agonist A method comprising: [The present invention 1012] The method of claim 1011, wherein the thyroid hormone is triiodothyronine or thyroxine. [The present invention 1013] The method of claim 1011, wherein the thyroid hormone receptor agonist is GC-1. [The present invention 1014] The method of any one of claims 1011 to 1013, wherein the hepatoblasts are cultured as a monolayer. [The present invention 1015] 10. The method of any one of claims 1011 to 1014, wherein the hepatoblasts are cultured as aggregates. [The present invention 1016] The method of any of claims 1011 to 1015, wherein the hepatoblasts are cultured in the absence of cAMP. [The present invention 1017] 17. The method of any of claims 1011 to 1016, wherein the mature hepatocytes express little or no alpha-fetoprotein (AFP). [The present invention 1018] The method of any one of claims 1011 to 1017, wherein the mature hepatocytes express albumin. [The present invention 1019] 1. A method for producing zone 1 hepatocytes, comprising: Culturing hepatoblasts in the presence of an inhibitor of the Wnt pathway A method comprising: [The present invention 1020] The method of claim 1019, wherein the inhibitor of the Wnt pathway is XAV939, IWP2, IWP4, or ICRT14. [The present invention 1021] The method of claim 1019 or claim 1020, wherein the hepatoblasts are cultured in monolayer or in aggregates. [The present invention 1022] 1. A method for producing zone 3 hepatocytes, comprising: Culturing hepatoblasts in the presence of an activator of the Wnt pathway. A method comprising: [The present invention 1023] The method of claim 1022, wherein the hepatoblasts are cultured in monolayer or in aggregates. [The present invention 1024] The method of any of claims 1019 to 1023, wherein the hepatoblasts are cultured in the presence of a NOTCH inhibitor. [The present invention 1025] 1. A method for producing cholangiocytes, comprising: Culturing hepatoblasts in the presence of retinoic acid, retinol, or an RA receptor agonist A method comprising: [The present invention 1026] The method of claim 1025, wherein the cholangiocytes are identified based on the presence of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. [The present invention 1027] The method of claim 1025 or claim 1026, wherein the cholangiocytes are identified based on binding to the DHC5-4D9 antibody. [The present invention 1028] 1. A method for producing liver organoids, comprising: Combining mesothelial cells and hepatoblasts under conditions that promote their self-organization into liver organoids A method comprising: [The present invention 1029] Expanding hepatoblasts in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or an equivalent thereof. The method of the present invention 1028 further comprising: [The present invention 1030] 1. A method for producing stellate cells, comprising: Culturing the liver organoids produced using the method of the present invention 1028 under conditions that produce stellate cells. A method comprising: [The present invention 1031] 1. A method of treating liver disease in a subject, comprising: Transplanting a composition comprising cholangiocytes into a subject, wherein the cholangiocytes are produced by the method of the present invention. A method comprising: [The present invention 1032] The method of claim 1031, wherein the liver disease is cholangiopathy, specifically bile duct disease or cholangiopathia. [The present invention 1033] 1. A method of treating a subject having liver disease, comprising implanting a composition into the subject, The composition comprises: Hepatoblasts expanded using any of the methods of inventions 1001 to 1010; 10 to 10 hepatocytes matured using any of the methods of the present invention; Zone 1 hepatocytes produced using the method of any of the inventions 1019 to 1021 or 1024; Zone 3 hepatocytes produced using any of the methods of inventions 1022 to 1024; Bile duct cells produced using any of the methods of the present invention 1025 to 1027; Liver organoids produced using the method of invention 1028 or 1029; and / or Stellate cells prepared using any of the methods of the present invention 1030 to 1032. A method comprising: [The present invention 1034] The method of any one of claims 1031 to 1033, wherein the composition further comprises epithelial cells. [This invention 1035] Monitoring the subject for albumin levels Any of the methods of inventions 1031 to 1034, further comprising: [The present invention 1036] monitoring the subject for levels of one or more liver enzymes Any of the methods of inventions 1031 to 1035, further comprising: [This invention 1037] 1037. The method of any one of claims 1031 to 1036, wherein the transplantation is performed directly into the liver or ectopically into the abdomen. [The present invention 1038] 1. A method for culturing liver cells, comprising: culturing the liver cells on the substrate under conditions in which the liver cells grow as a monolayer; A method comprising: [This invention 1039] Culturing liver cells as a monolayer and then as aggregates The method of the present invention 1038 further comprising: [The present invention 1040] 1039. The method of claim 1038 or 1039, wherein the number of liver cells resulting from the monolayer is at least 10 times greater than the number of liver cells resulting from the culture of aggregated cells. [The present invention 1041] 1. A method for cryopreserving liver cells, comprising: Culturing liver cells in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or an equivalent thereof for at least 3 days; Cryopreserving the cultured liver cells A method comprising: [The present invention 1042] thawing the cryopreserved liver cells; and Culturing the thawed liver cells in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or an equivalent thereof. The method of the present invention 1041 further comprising: [This invention 1043] 1. A method for recovering cryopreserved liver cells, comprising: thawing the cryopreserved liver cells; and Culturing the thawed liver cells in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or an equivalent thereof. A method comprising: [This invention 1044] Culturing the liver cells in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or an equivalent thereof for at least 3 days prior to cryopreserving the liver cells. The method of the present invention 1043 further comprising: [This invention 1045] The method of any of claims 1041 to 1044, wherein the cryopreservation comprises freezing the liver cells at -80°C in a medium containing DMSO, FSC, and DMEM / F12. [The present invention 1046] 1046. The method of any one of claims 1041 to 1045, wherein the thawing step comprises heating the liver cells to 37°C for about 5 minutes. [This invention 1047] The method of claim 1047, wherein the liver cells are hepatoblasts. [This invention 1048] 1. A method of screening for therapeutic compounds for cystic fibrosis and / or ciliopathies, comprising: contacting the bile duct cells prepared using any of the methods of the present inventions 1025 to 1027 with a test compound; and Determining the presence or absence of CFTR function Including, The method wherein the presence or absence of CFTR function is indicative of a test compound that is therapeutic for cystic fibrosis and / or a ciliary-associated disorder. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the method and composition belong.Methods and materials similar to or equivalent to those described herein can be used in the implementation or testing of the method and composition, and suitable methods and materials are described below.In addition, materials, methods and examples are only illustrative and are not intended to be limiting.All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. [Brief explanation of the drawings]

[0023] [Figure 1] 1 is a schematic diagram showing the different cell types in the adult liver. [Figure 2A] 1 shows the expansion of hPSC-derived hepatoblast populations. [Figure 2B]This indicates that hepatoblasts within the expanded population retained their differentiation potential, and cells from passage 3 onwards differentiated along both hepatocyte and cholangiocyte fates. [Figure 3A] FIG. 1 is a schematic diagram showing that hepatoblasts can be cultured under hypoxic conditions in the presence of three pathway modulators and serially expanded for a total of 10 passages. [Figure 3B] 1 is a graph showing the fold expansion when hepatoblasts are cultured under hypoxic conditions in the presence of three pathway regulators. [Figure 3C] 10 is a plot showing the distribution of AFP+ ALB+ expressing cells generated under ambient or hypoxic O2 conditions. [Figure 4] We show that thyroid hormone (T3) promotes the maturation of hPSC-derived hepatocytes. [Figure 5] We show that the Wnt signaling pathway regulates zonation of hPSC-derived hepatocytes. [Figure 6A] 1 is a schematic diagram showing differentiation of progenitor cells into zone 1 and zone 3-like cells. [Figure 6B] 1 shows that hepatoblasts cultured under monolayer conditions expressed albumin and suppressed AFP in the monolayer. [Figure 6C] Graph of qPCR analysis. [Figure 6D] FIG. 1 is a schematic diagram showing the estimated number of differentiated zone 1 and zone 3-like hepatocytes from a single ES cell following the expansion protocol described herein. [Figure 7] We show that retinoic acid signaling promotes the generation of CFTR-expressing cholangiocytes (bile duct cells) in monolayer culture. [Figure 8] We demonstrate the identification of a signaling pathway that promotes the development of functional cholangiocytes, ciliated cholangiocytes, in monolayer culture. [Figure 9] 1 shows the characteristics of NFR-induced cholangiocytes. [Figure 10] Figure 1 shows that hPSC-derived mesothelial cells support hepatoblast function in vitro. [Figure 11] Shows engraftment of hPSC-derived cholangiocytes. [Figure 12] Shows subcutaneous (heterotopic) transplantation and engraftment of liver organoids. [Figure 13] Shows intraperitoneal (heterotopic) transplantation and engraftment of hepatic organoids. [Figure 14] A shows representative flow cytometry analysis of ALB and AFP expression in hepatoblast populations after 8 days of culture of thawed cryopreserved cells. B shows the percentage of ALB- and AFP-positive cells in expanded hepatoblast populations after 8 days of culture of thawed cryopreserved cells ("-", cryopreserved without expansion; "+", expanded population cryopreserved). C shows a graph showing the fold expansion of hepatoblast populations after 8 days of culture of cryopreserved cells. Numbers are compared to the number of cells plated immediately after thawing ("-", cryopreserved without expansion; "+", expanded population cryopreserved). D shows representative flow cytometry analysis of ALB and AFP expression in zone 1 and zone 3 hepatoblasts generated from cryopreserved hepatoblasts. [Figure 15] A shows a scheme and timeline for hepatoblast expansion and zone maturation prior to heterotopic transplantation under the kidney capsule in NSG mice. B is a graph showing human albumin levels in mouse serum 4 weeks after engraftment of the indicated populations. Aggregates of the indicated populations were transplanted into the kidney capsule of NSG mice. Zone 1 / Zone 3: Equal numbers of Zone 1 and Zone 3 aggregates were mixed and allowed to engraft. Data are presented as mean + / - SEM, * indicates P<0.05, *** indicates P<0.0001. Statistical analysis: One-way ANOVA. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description The adult liver is a complex tissue containing multiple cell types of both endodermal and mesodermal origin, including hepatocytes, cholangiocytes, hepatic sinusoidal endothelial cells, hepatic stellate cells, and Kupffer cells. Figure 1 is a schematic diagram showing the various cell types in the adult liver. To be able to generate functional liver tissue derived from human pluripotent stem cells (hPSCs) in vitro or in vivo, it will likely be necessary to include most, if not all, of these cell types in an artificial construct. This disclosure describes methods for producing the large number of liver cells shown in Figure 1 and further describes the numerous ways in which such liver cells can be used.

[0025] Hepatocytes and cholangiocytes Hepatocytes comprise the liver parenchyma and account for approximately 75% of the total cell population within this organ. These cells perform over 3,000 essential functions in the body, which involve different enzymatic reactions occurring simultaneously. To achieve this, hepatocytes with different functions are compartmentalized into different zones of the parenchyma. Recent single-cell RNA-SEQ studies have shown that approximately half of the genes expressed in mouse hepatocytes are zoned. The region surrounding the portal vein is known as zone 1, and hepatocytes within this region ("zone 1 hepatocytes") are primarily responsible for gluconeogenesis and urea synthesis. In contrast, the region around the central vein is known as zone 3, and hepatocytes within this region ("zone 3 hepatocytes") are responsible for xenobiotic metabolism.

[0026] As described herein, the strategy for generating functional hepatocytes from hPSCs involves specific steps that recapitulate key stages of liver development in the early embryo, including the induction of appropriate liver progenitor cells (hepatoblasts) and their maturation into hepatocytes with zonal functional heterogeneity. Using this approach, new insights into liver development from hPSCs have emerged, enabling the induction of cells that exhibit distinct characteristics of primary human hepatocytes with a zonal distribution. These advances make it possible to generate hPSC-derived populations that contain the functional heterogeneity of primary hepatocytes that constitute the portal-to-central vein axis of the liver.

[0027] In addition to hepatocytes, cholangiocytes also play an important role in liver function by forming bile ducts that transport bile acids. Furthermore, cholangiocytes also modify bile acids as they flow through the ducts. Although cholangiocytes account for only 5% of the total liver mass, they are directly involved in numerous diseases that can lead to liver failure. Liver diseases associated with bile duct failure are the primary cause of 80% of pediatric liver transplants. Over the past decade, numerous groups have invested significant time and effort in generating hepatocyte-like and cholangiocyte-like cells from human pluripotent stem cells (hPSCs). Despite this, the generation of functional, mature, zoned hepatocytes and mature, ciliated cholangiocytes has not previously been achieved.

[0028] As described herein, RA signaling was identified as a regulator of early cholangiocyte specification, and the combination of BMP inhibition, Rho kinase, and cAMP signaling was identified in the maturation of hPSC-derived cholangiocytes. Stepwise manipulation of these pathways promoted the efficient generation of functional CFTR-positive ciliated cholangiocytes from hPSCs. Furthermore, cholangiocyte monolayers efficiently generated cholangiocyte cysts and organoids.

[0029] The present disclosure describes a method for generating functional hepatocytes and functional bile duct cells, which can be carried out in either monolayer format or aggregate / organoid format.The present disclosure also describes a method for expanding hepatocyte (heptatoblast) population under conditions that maintain their differentiation capacity, and generating functional hepatocytes and bile duct cells, which can be carried out in either monolayer format or aggregate / organoid format.It will be understood that any of the liver cells described herein can be grown as aggregates before and / or after growing these liver cells in monolayer.When cells (e.g., hepatocytes) are grown in monolayer, inhibiting both Notch signaling and TGF-β signaling can improve the quality of maturation (e.g., compared to the maturation of the same type of cells grown in 3D culture).As described herein, the number of liver cells that can be obtained by culturing in monolayer can be at least 10 times (e.g., at least 15 times, at least 20 times) greater than the number of liver cells that can be obtained by culturing in aggregate. Furthermore, the method described herein, together with manipulation of Wnt signaling for hepatic zonation, results in a significant reduction of AFP-positive cells under liver maturation conditions compared to a previously published method (Ogawa et al., 2013, Development, 140(15):3285-96).

[0030] Method for expanding liver progenitor cells Described herein are a number of different methods for expanding hepatoblasts. In some embodiments, hepatoblasts can be significantly expanded in number by culturing the cells in the presence of a cell expansion cocktail. As described herein, the cell expansion cocktail typically contains a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or its equivalent. These culture conditions allow for continuous expansion of hepatoblasts, with each expansion resulting in a 6- to 8-fold increase in cell number. Hepatoblast populations can be continuously expanded for at least 10 passages while maintaining the functional characteristics of hepatic progenitor cells (e.g., greater than 90% of the cells express both ALB and AFP).

[0031] The activator of Wnt pathway is known or can be identified by those skilled in the art.Representative activator of Wnt pathway includes but is not limited to CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile; TOCRIS), CHIR98014 (N6-[2-[[4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine; TOCRIS), BIO ((2'Z,3'E)-6-bromoindirubin-3'-oxime; TOCRIS), any number of (potent) GSK-3β inhibitors or natural Wnt agonists, such as Wnt3. TGF-β receptor inhibitors are known or can be identified by those skilled in the art.Representative TGF-β receptor inhibitors include, but are not limited to, SB431542, A83-01, other TGFβ receptor inhibitors, or ALK4 and / or ALK7 inhibitors (for example, SB525334, SB505124, etc.).FGF19 is known in the art.For example, see GI accession number 37181724 for the protein sequence of human FGF19.In addition, equivalents of FGF19 are known, and include, for example, the modified form called NGM282.

[0032] Additionally or alternatively, hepatoblasts can be significantly increased in number by culturing cells under hypoxic conditions.Hypoxic conditions are known in the art.In terms of cell culture, ambient oxygen (O2) conditions generally refer to the oxygen level in culture of about 20% O2 (for example, about 18%, 20%, 22.5% or 25% O2), while hypoxic conditions generally refer to the oxygen level in culture of less than about 20% O2 (for example, about 15%, 10%, 5% or 2.5% O2).

[0033] As described herein, hepatoblasts can be expanded to very large numbers by culturing the cells under hypoxic conditions in the presence of a cell expansion cocktail. Based on the preliminary results presented herein, it is predicted that hepatocytes can be expanded about 100-fold to about 400-fold within 3-5 passages when cultured under ambient O2 conditions in the presence of a cell expansion cocktail, and about 75-fold to about 1000-fold when cultured under hypoxic conditions in the presence of a cell expansion cocktail.

[0034] Methods for generating liver cells Also described herein are methods for producing multiple different types of liver cells.For example, methods for producing mature liver cells are described, including zone 1-like hepatocytes and zone 3-like hepatocytes, and methods for producing bile duct cells are also described.

[0035] In some embodiments, mature hepatocytes can be obtained by culturing hepatoblasts in the presence of thyroid hormone or thyroid hormone receptor agonist.Mature hepatocytes are generally characterized as hepatocytes that express albumin and express little or no (detectable) alpha-fetoprotein (AFP).Thyroid hormones are known in the art, as are thyroid hormone receptor agonists.Representative thyroid hormones include, but are not limited to, triiodothyronine or thyroxine, while a representative thyroid hormone receptor agonist is GC-1.Notably, by culturing hepatoblasts in the absence of little or no cAMP, an increased number of mature hepatocytes can be obtained.

[0036] As described herein, hepatoblast zonation can be promoted by manipulating Wnt signaling in cells along with thyroid hormone treatment. Zone 1 hepatocytes (or zone 1-like hepatocytes) can be obtained by culturing hepatoblasts in the presence of a Wnt pathway inhibitor. Wnt pathway inhibitors are known or can be identified by those skilled in the art; representative Wnt pathway inhibitors include, but are not limited to, XAV939, IWP2, IWP4, or ICRT14 (see, for example, selleckchem.com / Wnt on the World Wide Web). Zone 3 hepatocytes (or zone 3-like hepatocytes) can be obtained by culturing hepatoblasts in the presence of a Wnt pathway activator. Wnt pathway activators are discussed herein and include, but are not limited to, CHIR99021, CHIR98014, BIO, GSK-3 inhibitors, and natural Wnt agonists (e.g., Wnt3). Zone 3 hepatocytes express multiple CYP enzymes, including but not limited to CYP2C9, CYP2D6, and CYP3A4, which are highly expressed in pericentral hepatocytes in the hepatic lobule, while zone 1 hepatocytes express PCK, G6P, TAT, and CPS1, which are highly expressed in periportal hepatocytes (zone 1) in the hepatic lobule.

[0037] In some embodiments, cholangiocytes can be obtained by culturing hepatoblasts in the presence of retinoic acid, retinol, or RA receptor agonists.It will be appreciated that cholangiocytes can be identified based on the expression of cystic fibrosis transmembrane conductance regulator (CFTR) protein, and can also be identified based on binding to DHC5-4D9 antibody (Millipore Sigma: MABS2040-100 μg; anti-Hpd3 antibody, clone DHIC-4D9).

[0038] In some embodiments, liver organoid can be obtained by combining mesothelial-like cell and hepatoblast under the condition of promoting self-organization into liver organoid.Suitable mesothelial-like cell can be produced, for example, by following the protocol used for producing epicardial cells in US 2016 / 0215263.In some embodiments, hepatic stellate-like cell can be obtained from liver organoid (for example, by culturing liver organoid described herein under the condition that hepatic stellate cell-like cell is spontaneously produced in 3D liver organoid in the presence of Wnt agonist, TGFβ inhibitor and FGF19 or equivalent, and then maintained under liver maturation conditions with the operation of hepatic zonation).

[0039] cryopreservation Liver cells (e.g., hepatoblasts) such as those described herein can be cryopreserved, and the expansion cocktail (i.e., Wnt pathway activator, TGFβ inhibitor, and FGF19 or its equivalent) and expansion conditions described herein can be used after cryopreservation and subsequent thawing to improve cell recovery and maintenance.The use of the expansion cocktail and related conditions described herein after cryopreservation can make more than 85% of cells viable after thawing, and notably, these cells generally maintain the characteristics of hepatic progenitor cells.

[0040] Prior to freezing liver cells, such cells can be cultured in an Expansion Cocktail under the expansion conditions described herein. Culturing cells in an Expansion Cocktail prior to freezing can also be used to improve the ability of cells to recover and expand after cryopreservation of the cells.

[0041] As used herein, cryopreservation refers to freezing cells (e.g., at -80°C) in a medium containing DMSO, FSC, and DMEM / F12, while thawing can be accomplished by gently heating the cells for about 5 minutes (e.g., at 37°C).

[0042] treatment method Any of the liver cells described herein (for example, expanded hepatoblasts, mature hepatocytes, zone 1 hepatocytes, zone 3 hepatocytes, bile duct cells, liver organoids, stellate cells, and their combinations) can be used therapeutically to treat a number of different liver diseases.In relation to cell therapy, it will be understood that administration generally refers to the introduction of cells into subject (for example, by transplantation).When introducing liver cells into subject, transplantation can be carried out directly into liver or heterotopically to liver (for example, intraperitoneally).

[0043] In some embodiments, for example, a composition comprising cholangiocytes produced using the methods described herein can be transplanted into a subject with a liver disease (e.g., cholangiopathy, e.g., bile duct disease or cholangiocytosis). In addition to introducing any of the liver cells described herein into a subject, it will be understood that non-liver cells can also be introduced into a subject as part of the transplant. Non-limiting examples of non-liver cells include, for example, epithelial cells.

[0044] As used herein, subject generally refers to humans, but can also refer to any other type of animal (e.g., mammals or non-mammals; for example, companion animals, agricultural animals or livestock, exotic animals).After transplantation, to determine the health and functionality of transplanted cells, the subject is often monitored for the products or by-products of transplanted cells.For example, the subject receiving liver cells can be monitored for albumin levels and / or the levels of one or more liver enzymes (e.g., total bilirubin, aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyltransferase (GTP), or a combination thereof).

[0045] As described herein, mature cholangiocytes produced in either monolayer or 3D culture formats can engraft and form duct-like structures at both intrahepatic and extrahepatic sites, providing a platform for the development of novel therapeutic applications for the treatment of cholestatic diseases. Those skilled in the art will understand that "treating" or "treatment" typically refers to reducing, improving, or alleviating a disease, the effects of a disease, or one or more symptoms associated with a disease.

[0046] Drug Screening and Laboratory Methods Any of the liver cells described herein can be used in drug screening protocols.For example, the bile duct cells described herein can be used to screen compounds that can show therapeutic effects in the treatment of cystic fibrosis and / or cilia-related diseases.For example, such a method typically includes contacting liver cells with a test compound and determining the presence or absence or amount of one or more " markers ".As used herein, " marker " can refer to the specific functionality of protein or cell, or " marker " can refer to the expression of specific sequence.For example, such a method typically includes contacting bile duct cells with a test compound and determining the presence or absence of CFTR function (for example, chloride channel function).It will be understood that the presence or absence of CFTR function indicates that the test compound can show therapeutic effects in the treatment of cystic fibrosis and / or cilia-related diseases.

[0047] The cells described herein can be evaluated, for example, using a FLIPR assay (fluorescence-based plate reader assay), where a membrane potential dye can be used to measure apical chloride conductance, which indicates CFTR function. The cells described herein can be evaluated for Z-prime scores to determine quality control, and as determined herein, the Z-prime score for the cholangiocytes described herein is 0.63, indicating that these cells are excellent candidates for CFTR drug screening.

[0048] Because the mature cholangiocytes described herein (i.e., produced in either monolayer or 3D culture formats) are functional, they can be used in high-throughput drug screening assays, for example, to measure CFTR function. Such cells can also be used in assays to examine or determine, for example, chemosensing and / or mechanosensing activity (based on primary cilia movement).

[0049] In accordance with the present invention, conventional molecular biology, microbiology, biochemistry, and recombinant DNA techniques within the skill of the art can be used. Such techniques are fully explained in the literature. The present invention is further described in the following examples, but these examples do not limit the scope of the methods and compositions described in the claims. [Example]

[0050] Example 1A - Experimental Materials and Methods for Expanding Liver Progenitor Cells Expansion of hepatoblasts Day 27 hepatoblasts were dissociated as single cells using TrypLE (Thermo Fisher Scientific) and plated at a density of 200,000 cells per well onto 2.5% Matrigel-coated wells (12-well plates) in DMEM / F12 (50:50) medium supplemented with 0.2% BSA, 1% vol / vol ITS-X, ascorbic acid, 1% vol / vol chemically defined lipid mix medium (Thermo Fisher Scientific), 0.5% vol / vol B27, glutamine, MTG, Dex (40 ng / ml), CHIR99021 (1 μM), SB431542 (6 μM), and FGF19 (50 ng / ml). The medium was changed every 2 or 3 days. Cell cultures could be maintained in either ambient (5% CO2, 20% O2, 90% N2) or low-O2 incubators (5% CO2, 5% O2, 90% N2). Plated hepatoblasts were expanded and reached full confluence within 6–10 days. Expanded hepatoblasts could also be expanded by additional passages with single-cell dissociation using TrypLE (Thermo Fisher Scientific). Compared to cultures in ambient O2 incubators, expanded hepatoblasts in low-O2 incubators could be further expanded for up to 10 passages with over 90% expressing both ALB and AFP. Expanded hepatoblasts could also differentiate into zone 1 / 3 hepatocyte-like cells and cholangiocytes after the monolayer protocol described above.

[0051] Example 1B - Experimental results on expansion of liver progenitor cells Figure 2A shows the expansion of a population of hPSC-derived hepatoblasts. To be able to generate sufficient numbers of hPSC-derived hepatocytes for cell-based therapy, it would be advantageous to expand and cryopreserve a bipotential hepatoblast population. To achieve this, hepatoblasts were cultured in a combination of a Wnt signaling agonist (CHIR), a TGFβ signaling antagonist (SB431542), and FGF19 or its equivalent. Activation / inhibition of these pathways plays a role in liver regeneration and promotes hepatocyte proliferation in normal and precancerous livers. When cultured under these conditions, hepatoblasts proliferate and maintain their ALB+ AFP+ profile. Cells were passaged every 6 days for a total of three times, resulting in a total expansion of 160-fold; cells appear to lose their proliferative potential after passage 3.

[0052] Figure 2B shows that hepatoblasts within the expanded population retained their differentiation potential, and cells from passage 3 differentiated along both hepatocyte and biliary cell fates. After culture in the presence of T3 and a Wnt agonist or antagonist, cells within the expanded population generated zone 1- and zone 3-like ALB+ AFP- hepatocytes that expressed PCKl and CPT1a (zone 1-like cells) or CYP3A4 and CYP2D6 (zone 3-like cells). Furthermore, cells within the expanded populations from passages 1, 2, and 3 also differentiated along the biliary cell lineage and gave rise to ciliated cells.

[0053] FIG. 3A is a schematic diagram showing that hepatoblasts can be cultured under hypoxic conditions in the presence of three pathway modulators and serially expanded for a total of 10 passages.

[0054] Figure 3B is a graph showing the fold expansion when hepatoblasts are cultured under hypoxic conditions in the presence of three pathway regulators. For example, at passage 5, there was a 388-fold expansion in ambient O2 and a 1076-fold expansion in hypoxic O2 conditions. After 10 passages, there is an estimated 237,404-fold expansion.

[0055] FIG. 3C is a plot showing the distribution of AFP+ ALB+ expressing cells generated under ambient or hypoxic O2 conditions.

[0056] Example 2A - Experimental materials and methods for generating liver cells Hepatocytes and hepatocyte zonation Maintenance of human ES and iPS cells and differentiation into hepatoblasts Human ES / iPS cells were maintained on irradiated mouse embryonic feeder cells in human ES culture medium consisting of DMEM / F12 (50:50: Gibco) supplemented with 20% Knock-Out Serum Replacement as previously described. Prior to endoderm induction in monolayer culture, hES / iPS cells were passaged onto 2.5% Matrigel-coated surfaces (10-fold less than in previous protocols) in 12-well culture dishes at a cell density of 200,000 cells per well for 1 day. To induce endoderm differentiation, cells were cultured in 2.5% Matrigel-coated medium containing glutamine (2 mM), MTG (4.5 x 10E-4 M; Sigma), activin A (100 ng / ml), and CHIR99021 (2 μM) were cultured for 1 day in RPMI-based medium supplemented with glutamine (2 mM), ascorbic acid (50 μg / ml; Sigma), MTG (4.5 x 10E-4 The cells were cultured in RMPI supplemented with ATP (M; Sigma), basic fibroblast growth factor (bFGF, 5 ng / ml), and activin A (100 ng / ml) for the next 2 days, followed by serum-free differentiation (SFD)-based medium containing the same supplements for 4 days. At that time, the medium was changed every 2 days. On day 7, definitive endoderm cells confirmed to be positive for CXCR4 and cKIT by flow cytometry were specified for hepatic fate by culture in H16 DMEM containing bFGF (40 ng / ml) and bone morphogenetic protein (BMP4, 50 ng / ml), supplemented with 1% vol / vol B27 supplement (Invitrogen, A11576SA), ascorbic acid, and MTG. The medium was changed every 2 days from day 7 to day 13. To promote the maturation of hepatoblast populations, cells were cultured for 8 days in a mixture of H16 DMEM / Ham's F12 (3:1) medium containing 0.1% BSA, 1% vol / vol B27 supplement, ascorbic acid, glutamine, MTG, hepatocyte growth factor (HGF, 20 ng / ml), dexamethasone (Dex, 40 ng / ml), oncostatin M (OSM, 20 ng / ml), and CHIR99021 (1 μM). Differentiation, including endoderm induction, hepatic specification, and maturation, from day 0 to day 21, was maintained in a low-O2 incubator in a 5% CO2, 5% O2, 90% N2 environment. On day 21, cells were transferred to an ambient O2 incubator and cultured for 4 days in a mixture of H21 DMEM / Ham's F12 (3:1) with 0.1% BSA, 1% vol / vol B27 supplement, ascorbic acid, glutamine, MTG, HGF (20 ng / ml), Dex (40 ng / ml), and OSM (20 ng / ml). On day 25, cells were cultured for 2 days in DMEM / F12 (50:50) containing 0.2% BSA, 1% vol / vol ITS-X, ascorbic acid, glutamine, MTG, Dex (40 ng / ml), and OSM (5 ng / ml).

[0057] Generation of mature zone 1 and zone 3 hepatocyte-like cells from hPSC-derived hepatoblasts in 3D aggregates Day 27 hepatoblasts were cultured in monolayer in DMEM / F12 (50:50) containing 0.2% BSA, 1% vol / vol ITS-X, ascorbic acid, glutamine, MTG, Dex (40 ng / ml), and OSM (5 ng / ml) for 6 days. Day 33 hepatoblasts were dissociated using type 1 collagenase enzyme to generate small clusters of hepatoblasts. Dissociated small clusters were maintained in low-cluster culture dishes and cultured for 6 days in DMEM / F12 (50:50) medium supplemented with 0.2% BSA, 1% vol / vol ITS-X, ascorbic acid, 1% vol / vol chemically defined lipid mixture (Thermo Fisher Scientific), 0.5% vol / vol B27, glutamine, MTG, Dex (40 ng / ml), and CHIR99021 (1 μM) to promote maturation into 3D aggregates. To induce differentiation of zone 1-like hepatocytes, 3D aggregates were cultured for 18 days in DMEM / F12 (50:50) medium supplemented with 0.2% BSA, 1% vol / vol ITS-X, ascorbic acid, 1% vol / vol chemically defined lipid mixture (Thermo Fisher Scientific), 0.5% vol / vol B27, glutamine, MTG, Dex (40 ng / ml), T3 (triiodothyronine, 40 nM; Sigma), and XAV939 (2 μM). To induce differentiation of zone 3-like hepatocytes, 3D aggregates were cultured for 18 days in DMEM / F12 (50:50) medium supplemented with 0.2% BSA, 1% vol / vol ITS-X, ascorbic acid, 1% vol / vol chemically defined lipid mixture (Thermo Fisher Scientific), 0.5% vol / vol B27, glutamine, MTG, Dex (40 ng / ml), T3 (triiodothyronine, The cells were cultured for 18 days in DMEM / F12 (50:50) medium supplemented with 40 nM (Sigma) and CHIR99021 (1 μM). The medium was changed every 2 or 3 days. Differentiation was maintained in an ambient O2 incubator.

[0058] Generation of mature zone 1 and zone 3 hepatocyte-like cells from hPSC-derived hepatoblasts in monolayer culture To induce differentiation of zone 1- and zone 3-like hepatocytes from day 27 hepatoblasts in monolayer culture conditions, hepatoblasts were directly cultured in DMEM / F12 (50:50)-based maturation medium in the presence of small molecules that activate or inhibit the Wnt signaling pathway. For differentiation into zone 1-like hepatocytes, day 27 hepatoblasts were cultured for 24 days in DMEM / F12 (50:50) medium supplemented with 0.2% BSA, 1% vol / vol ITS-X, ascorbic acid, 1% vol / vol chemically defined lipid mixture (Thermo Fisher Scientific), 0.5% vol / vol B27, glutamine, MTG, Dex (40 ng / ml), T3 (triiodothyronine, 40 nM; Sigma), SB431542 (6 μM), Notch inhibitors: L-685,458 (5 μM) or DAPT (25 μM), and XAV939 (2 μM). To induce differentiation into zone 3-like hepatocytes, day 27 hepatoblasts were cultured for 24 days in DMEM / F12 (50:50) medium supplemented with 0.2% BSA, 1% vol / vol ITS-X, ascorbic acid, 1% vol / vol chemically defined lipid mixture (Thermo Fisher Scientific). Cells were cultured for 24 days in DMEM / F12 (50:50) medium supplemented with 0.5% vol / vol B27 (Sigma-Aldrich Scientific), glutamine, MTG, Dex (40 ng / ml), T3 (triiodothyronine, 40 nM; Sigma-Aldrich), SB431542 (6 μM), Notch inhibitors: L-685,458 (5 μM) or DAPT (25 μM), and CHIR99021 (1 μM). Medium was changed every 2 or 3 days. Differentiation was maintained in an ambient O2 incubator.

[0059] cholangiocytes Cholangiocyte differentiation in monolayer OP9 cells were maintained as previously described. 30-Gy irradiated OP9 cells were plated on 2.5% Matrigel-coated wells (12-well plates) at a concentration of 200,000 cells per well in alpha-modified minimal essential medium (α-MEM) supplemented with glutamine (2 mM) and 20% fetal bovine serum. To induce cholangiocyte differentiation, day 27 hepatoblasts were dissociated using type I collagenase enzyme and then plated on the irradiated OP9 cells. The plated cells were cultured for 4 days in H21 DMEM / Ham's F12 (3:1) medium supplemented with 0.1% BSA, 1% vol / vol B27 supplement, ascorbic acid, glutamine, MTG, HGF (20 ng / ml), and epidermal growth factor (EGF, 50 ng / ml). To induce CFTR expression in cholangiocyte-like cells, after HGF and EGF treatment, the medium was switched to DMEM / F12 medium containing 0.1% BSA, 1% vol / vol B27 supplement, ascorbic acid, glutamine, MTG, and retinoic acid (RA, 1 μM; Sigma; treatment range: 500 nM–2 μM) for an additional 6 days. Similar effects were observed when retinol, AM580 (RA receptor α agonist), or AC55649 (RA receptor β agonist) were used. To promote the maturation of cholangiocytes expressing primary cilia and 4D9, cells were cultured for 12 days in DMEM / F12 medium containing 0.1% BSA, 1% vol / vol B27 supplement, ascorbic acid, glutamine, MTG, noggin (50 ng / ml), ROCK inhibitor Y-27632 (5 μM), and forskolin (FSK, 5 μM). The medium for all steps of cholangiocyte differentiation was changed every two days. Cells were maintained in an ambient O2 incubator.

[0060] Generation of 3D cholangiocyte organoids Day 49 cholangiocytes obtained after monolayer differentiation were dissociated with type I collagenase enzyme. Small clumps of cholangiocytes were then plated onto low-attachment cluster dishes and cultured in the same medium used for monolayer differentiation. 3D cholangiocyte organoids spontaneously formed cyst-like structures within 6 days. Cells were maintained in an ambient O2 incubator.

[0061] Stellate cell generation Stellate cells were obtained from cholangiocyte organoids by culturing them for 6 days in DMEM / F12 medium supplemented with 0.2% BSA, 1% vol / vol ITS-X, ascorbic acid, 1% vol / vol chemically defined lipid mixture (Thermo Fisher Scientific), 0.5% vol / vol B27, glutamine, MTG, Dex (40 ng / ml), CHIR99021 (1 μM), SB431542 (6 μM), and FGF19 (50 ng / ml). After 6 days, CHIR99021, SB431542, and FGF19 were removed from the medium, allowing the cholangiocyte organoids to mature into stellate cells.

[0062] FLIPR membrane potential assay FLIPR membrane potential assays were performed according to a previously described protocol (Ahmadi et al., 2017, "Phenotypic profiling of CFTR modulators in patient-derived respiratory epithelia," Genomic Med., 2:12). This assay can be used to measure apical chloride conductance, which indicates functional activity of CFTR protein in cells. Briefly, day 27 hepatoblasts were dissociated and plated onto 96-well plates with clear bottoms (Corning). After 4 days of culture with HGF and EGF, the cells were treated with different concentrations of retinoic acid for 6 days, including 2 μl of DMSO as a control. Prior to the assay, cells were incubated for 40 min at 37°C in 200 μL NMDG-gluconate buffer (150 mM NMDG-gluconate, 3 mM KCl, 10 mM HEPES, pH 7.35, osmolality 300 mOsm) containing 0.5 mg / mL FLIPR membrane potential dye (Molecular Devices). After the dye loading procedure, cells were transferred to a SpectraMax i3X plate reader (Molecular Devices), and their fluorescence was measured using excitation at 530 nm and emission at 560 nm in well-scanning mode. Baseline fluorescence was measured for 24 min (6 min / read), followed by stimulation of CFTR-mediated chloride flux with forskolin (FSK, 10 μM). After recording membrane potential changes for 24 min, CFTR function was assessed by stimulating CFTR with 10 μM CFTR. inh Inhibition was performed for 18 minutes with -172. Raw data were exported and analyzed using an established platform in Christine Bear's laboratory (The Hospital for Sick Children, Toronto, Canada).

[0063] Example 2B - Experimental results for generating liver cells Hepatocytes and hepatocyte zonation Figure 4 shows that thyroid hormone (T3) promotes the maturation of hPSC-derived hepatocytes. One of the hallmarks of hepatocyte maturation is the downregulation of the fetal gene encoding alpha-fetoprotein (AFP) along with the upregulation of genes associated with adult hepatocyte function. Numerous different protocols have been described in the literature that claim to promote the development of mature hepatocytes, but the resulting cells still express relatively high levels of AFP, suggesting that fetal characteristics are retained. Given that T3 thyroid hormone levels increase dramatically after birth and that it is known to play a pivotal role in the development, growth, and function of many tissues, we added T3 to hPSC-derived hepatocyte cultures to determine whether T3 promotes the maturation of hPSC-derived hepatocytes. For these studies, T3 was added to the cultures during the maturation step from day 38 to day 56. During this stage, cells are cultured as aggregates in the presence of 40 ng / ml dexamethasone, as previously described. Because the addition of cAMP has previously been shown to promote the maturation of hPSC-derived hepatocytes, we compared the effects of T3 with those of cAMP. As shown in Figure 4, the addition of T3 resulted in a dramatic decrease in AFP expression levels, as demonstrated by flow cytometry and qRT-PCR analysis. In many cases, the observed levels were equivalent to those found in adult liver and significantly lower than those observed in cAMP-treated cells. This is the first demonstration that it is possible to generate hPSC-derived mature hepatocytes expressing such low levels of AFP.

[0064] Figure 5 shows that the Wnt signaling pathway regulates the zonation of hPSC-derived hepatocytes. The adult liver contains distinct populations of hepatocytes that are localized in different regions (or zones) and perform distinct functions. To model the development and function of human hepatocytes from hPSCs, it is essential to generate these distinct subtypes of cells. Previous studies in mice have shown that Wnt signaling plays a role in the development of distinct zoned hepatocyte populations. Therefore, we manipulated this pathway in culture by adding a small molecule Wnt agonist, CHIR, or an antagonist, XAV, to cultures from days 38 to 56 (the maturation step). T3 was included in these cultures to promote maturation. As shown in Figure 5, Wnt inhibition promotes the development of cells that express genes associated with zone 1 hepatocytes, which are localized in the portal vein region. These cells upregulate genes related to fatty acid oxidation, ureogenesis, gluconeogenesis, and cholesterol synthesis, including ASS, CPS1, ARG1, OTC, PCK1, G6P, and HMGCS2. hPSC-derived hepatocytes generated in the presence of Wnt signaling expressed genes associated with zone 3 hepatocytes, found near the central vein. These cells express genes encoding P450 enzymes, including CYP3A4 and 2D6.

[0065] Figure 6A is a schematic diagram showing that modulation of Notch inhibitors, TGFβ inhibitors, T3, and Wnt signaling pathways was manipulated in the maturation protocol described herein to promote hepatic maturation in monolayer culture conditions, which differentiated cells into zone 1- and zone 3-like cells. Notch signaling was inhibited by the addition of 0.5 μM to 1.0 μM GSI or 25 μM DAPT, and TGFβ signaling was inhibited by the addition of 6 μM SB43152.

[0066] Figure 6B shows that after 24 days of culture of day 27 hepatoblasts under monolayer conditions, the cells expressed albumin and suppressed AFP, as confirmed by confocal microscopy and flow cytometry. The upper panel shows the characteristics of cells cultured under zone 3 conditions (T3 / Wnt agonist / TGFβ inhibitor / Notch inhibition), while the lower panel shows cells cultured under zone 1 conditions (T3 / Wnt agonist / TGFβ inhibitor / Notch inhibition).

[0067] Figure 6C is a graph of qPCR analysis showing that the gluconeogenic gene G6P is upregulated in zone 1-like cells cultured with a Wnt pathway inhibitor, while CYP3A4, involved in drug metabolism, is upregulated in zone 3-like cells in the presence of a Wnt pathway agonist. These findings demonstrate that liver maturation and zone engineering were achieved in monolayer culture conditions as well as in 3D aggregates.

[0068] Figure 6D is a schematic diagram showing the estimated number of differentiated zone 1- and zone 3-like hepatocytes from a single ES cell after the expansion protocol described herein. The method described herein can produce six hepatoblasts from a single ES cell after 27 days (top). After aggregate formation and promotion of maturation by adding a Wnt agonist / antagonist and thyroid hormone, 0.6 zone 3-like cells and 0.3 zone 1-like cells can be differentiated from a single ES cell (top). Maturation in monolayer culture with inhibition of Notch signaling and TGFβ signaling resulted in a more than 10-fold increase in the number of zone 1 / 3-like hepatocytes generated compared to the number of cells generated in 3D culture. After the third passage expansion of hepatoblasts, more than 1,000 zone 1 / 3-like cells can be generated from a single human ES cell.

[0069] cholangiocytes Figure 7 shows that retinoic acid signaling promotes the generation of CFTR-expressing cholangiocytes (bile duct cells) in monolayer culture. It has previously been reported that it is possible to generate cholangiocytes expressing multiple markers indicative of mature cells, including the cystic fibrosis transmembrane conductance regulator (CFTR) gene, a mutation of which causes cystic fibrosis. The development of mature cholangiocytes relied on the growth of cells as cysts in 3D semi-solid cultures composed of Matrigel and collagen. While this approach yielded relatively mature cholangiocytes, the culture system was not amenable to cell expansion or high-throughput screening (e.g., for drugs for cystic fibrosis and other biliary diseases). To improve differentiation efficiency in monolayer culture, a panel of cytokines and small molecules known to play a role in bile duct development was screened to identify those that would promote the upregulation of CFTR expression as a sign of maturation. Because Notch signaling is required for the generation of cholangiocytes, hepatoblasts were cultured for 6 days on OP9-Jagl cells or Matrigel as previously described. This screen revealed that retinoic acid (RA) signaling significantly induced CFTR expression in the population cocultured with OP9-Jagl. To further investigate the role of RA signaling, we also tested the effects of specific RA receptor agonists as well as pan-antagonists. BMS493, an RA receptor antagonist, inhibited the induction of CFTR expression, whereas the addition of RA receptor α agonist (AM580), RA receptor β agonist (AC55649), and RA receptor γ agonist (CD437) all induced CFTR expression. These findings indicate that RA signaling is important for the generation of CFTR-expressing cholangiocytes in monolayer culture, and RA-treated cells exhibit functional CFTR responses in FLIPR assays.

[0070] Figure 8 shows the identification of signaling pathways that promote the development of functional cholangiocytes, ciliated cholangiocytes, in monolayer culture. One of the primary determinants of cholangiocyte maturation and function is the development of primary cilia. These cilia extend from the apical plasma membrane into the lumen of the bile duct and function as mechanosensors that transmit signals initiated by fluid flow within the bile duct to cholangiocytes. At the molecular level, ciliogenesis correlates with upregulation of the expression of genes including PDK1, PDK2, and TRPV4. RA signaling induced CFTR expression but did not promote ciliogenesis in cholangiocytes. To identify pathways that promote further maturation of hPSC-derived cholangiocytes, we used a screening approach based on flow cytometry identification of cells expressing an epitope recognized by the antibody DHCS-4D9, which stains mature bile ductal cells (cholangiocytes) in the adult liver. We hypothesized that maturation to the DHCS-4D9-positive stage correlates with ciliogenesis. For this screen, different combinations of agonists and antagonists for the following signaling pathways were added to the cultures for 6 days: cAMP, Wnt, Hedgehog, EGF, BMP, HGF, TGFβ, FGF10, IL6, VEGF, and Extendin 4. After this maturation step, cells were harvested and analyzed by flow cytometry for reactivity with DHC5-4D9. As shown in Figure 8, inhibition of the ROCK inhibitor (R), cAMP signaling (forskolin, F), and BMP pathway (N) all promoted the development of DHC5-4D9 cells. The triple-action combination (NFR) consistently gave rise to the greatest proportion of DHC5-4D9+ cholangiocytes, up to 80% of the population.

[0071] Figure 9 shows the characteristics of NFR-induced cholangiocytes. QRT-PCR-based expression analysis revealed that cholangiocytes induced with NFR in a monolayer format expressed many genes associated with mature cholangiocyte function, including those involved in ciliogenesis, such as TRPV4, PDK1, and PDK2 (Figure 9). Furthermore, the majority of cells contained primary cilia (H9: 77.3 ± 11.0%; two iPS-derived F508del CF patient cell lines: 76.1 ± 10.9%, 77.5 ± 4.9%). Cells generated with this protocol exhibited robust CFTR responses in high-throughput FLIPR assays, indicating that they are suitable for screening for novel CF drugs.

[0072] Figure 10 shows that hPSC-derived mesothelial cells support hepatoblast function in vitro. The adult liver is surrounded by a population of mesothelial cells (MCs) that form an epithelium around the organ. While the function of this cell population is not fully understood, studies in model organisms suggest that they interact with hepatocytes, undergo epithelial-mesenchymal transition (EMT), and contribute to the stellate cell population within the liver. To model this interaction in vitro, we modified a published protocol designed for the generation of cardiac epicardial cells to generate a mesothelial population from hPSCs. The cardiac epicardium and the mesothelium surrounding the liver share many characteristics, including expression of WT1, RALDH2, and TBX18. To enable tracking of MCs, we generated them from an hPSC line constitutively expressing RFP. Single-cell suspensions of day 20–25 mesothelial cells were mixed with day 27 hepatoblasts generated from an hPSC line constitutively expressing GFP. Cells were mixed at a 4:1 hepatoblast / MC ratio, and developing aggregates were cultured under the expansion conditions described herein. Within 4 days of culture, the cells formed aggregates called organoids, with RFP+ mesothelial cells forming distinct layers around GFP+ hepatoblasts and appearing to segregate into distinct regions within the structures. This segregation appears to recapitulate the positioning of these cell types in the developing liver. Analysis of the aggregates on day 6 revealed that those cultured in the presence of MC secreted significantly more albumin than those cultured without these cells. After 6 days of culture, the total number of hepatoblasts was not significantly different between organoids cultured with and without MC. Culture in the presence of MC over a 3-4 week period during maturation to zone 1 and zone 3 fates promoted hepatoblast survival within the aggregates; those with MC contained 2-3 times more cells than those without MC. These observations suggest that coculture with mesothelial cells may provide a novel approach to maintaining hepatoblast function in vitro.

[0073] Example 3A - Experimental Materials and Methods for Therapeutic Applications Liver injury was induced in mice by administering GSV to 6-8 week-old TK NOG mice. Differentiated cholangiocytes from 50-56 days old in monolayer conditions were dissociated using TrypLE to prepare single-cell suspensions. Under appropriate anesthesia, a skin incision was made in the left abdominal region below the ribs. The abdomen was entered through the same incision. The spleen was gently removed from the incision. One million cholangiocytes in 50 μl of PBS were injected into the inferior pole of the removed spleen. After confirming hemostasis at the injection site, the skin incision was closed. Six weeks after transplantation, the animals were euthanized, and the livers were removed and fixed for immunohistochemical studies. For immunostaining of human CK19 and mitochondria, paraffin-embedded sections were delipidated and subjected to heat-induced epitope retrieval. Transplanted iPSC-derived cholangiocytes were confirmed by the presence of human mitochondria and CK19-positive cells according to standard immunostaining methods.

[0074] Liver organoids were self-organized using GFP-positive hepatoblasts and RFP-positive mesothelial cells differentiated from hPSCs. The aggregates were maintained in culture medium for 6 days, and liver organoids, consisting of 6 million hepatoblasts, were embedded in 2.4 mg / ml type 1 collagen gel with 1 to 2 million human umbilical cord endothelial cells (HUVECs). After the collagen gel solidified, the gel containing liver organoids, with or without HUVECs, was removed from the culture plate and transplanted under the skin on the back of NOG mice. Six weeks after transplantation, the transplanted mice were euthanized, and the transplanted tissues were harvested for immunohistochemical analysis. Prior to euthanasia, blood samples were collected to measure human serum albumin.

[0075] Liver organoids containing 6 million hepatoblasts and 1.5 million mesothelial-like cells were embedded in 2.4 mg / ml type 1 collagen gel in the presence of 1 to 2 million HUVECs. Under appropriate anesthesia, a midline abdominal incision was performed. After ligation and removal of the central and lateral segments of the mouse liver, one or two solidified collagen gels containing liver organoids and HUVECs were embedded on the surface of the proximal mesentery near the liver. The embedded gels on the mesentery were covered with SURGICEL to prevent migration. In a control experiment, collagen gels containing liver organoids and HUVECs were embedded in the same site without partial hepatectomy. Four weeks after transplantation, blood samples were collected for measurement of human serum albumin.

[0076] Example 3B - Experimental Results for Therapeutic Applications Figure 11 shows the engraftment of hPSC-derived cholangiocytes. To determine whether NFR-induced cholangiocytes could function in vivo, 1 x 10E6 mature day 62 cells were transplanted into ganciclovir-treated TK-NOG mice. Treatment of these engineered mice with ganciclovir killed host mouse hepatocytes, allowing engraftment of human cells. Six weeks after transplantation, the mice were sacrificed, and their livers were analyzed for the presence of human cholangiocytes. In two independent experiments, ductular structures composed of human cytokeratin 19 (CK19)-positive cells were detected in the livers of all recipients. These findings are the first to demonstrate the engraftment of hPSC-derived cholangiocytes into the mouse liver.

[0077] Figure 12 shows the subcutaneous (ectopic) transplantation and engraftment of liver organoids. To determine whether liver organoids can function in vivo, 27-day-old organoids generated using mesothelial cells and hepatoblasts were encapsulated in collagen gel with or without HUVEC endothelial cells. This gel was then implanted subcutaneously into NSG recipients. Six weeks after transplantation, mice exhibited measurable levels of human serum albumin (HSA). Grafts were detected in all transplanted mice, and those with HUVEC tended to be larger than those without. Histological analysis showed that the grafts contained albumin-positive hepatocyte clusters (arrowheads) surrounded by small capillaries containing red blood cells (arrows). Together, these preliminary findings indicate that liver organoids can engraft in ectopic sites and function to produce HSA over a 6-week period. Hepatoblast aggregates without MCs generated grafts composed of fibrous tissue with few albumin-positive cells, suggesting that mesothelial cells support the development of functional hepatocytes in vivo.

[0078] Figure 13 shows the intraperitoneal (heterotopic) transplantation and engraftment of liver organoids. In this experimental set, recipient NSG mice underwent partial hepatectomy before transplantation of collagen gel containing organoids and HUVEC feeder cells. After surgery, the gel was placed in the hepatic porta covering the portal vein and bile duct. Four weeks after transplantation, HSA was detected in the serum of all animals (n=3). The level in those that underwent partial hepatectomy was significantly higher than that in those that did not undergo surgery, suggesting that the increased demand on liver function provides a stimulus for improving the engraftment and / or function of ectopic tissue.

[0079] Example 4 - Cryopreservation and expansion of hPSC-derived hepatoblasts Protocol for cryopreservation of hepatoblasts: Day 27 hepatoblasts were expanded for 6-8 days with treatment with FGF19 / SB43152 / CHIR99021 ("expansion cocktail"). The medium was changed every 2 days. Expanded hepatoblasts were dissociated with TrypLE for 5 minutes and harvested as single-cell hepatoblasts. Hepatoblasts were cryopreserved using conventional cryopreservation methods in the presence of 10% DMSO, 40% FSC, and 50% DMEM / F12 at a density of 0.5-1 million cells per cryovial.

[0080] Thawing of cryopreserved hepatoblasts: After thawing the cryopreserved hepatoblasts in a water bath for 5 minutes, the cells were washed once with DMEM / F12 and resuspended in fresh DMEM / F12 containing the expansion cocktail. The recovered hepatoblasts were plated at a density of 1.0 x 10e5 cells per well in DMEM / F12 containing the expansion cocktail and 10 μM Rho kinase (Rock) inhibitor in a 12-well culture plate. After the first medium change at 48 hours, the Rock inhibitor was not included, and the medium was then changed every 48 hours until the hepatoblast population reached confluency.

[0081] 1.0 x 10e5 hepatoblasts were plated in each well of a 12-well culture dish (3.5 cm2) and cultured in Expansion Medium. The medium was changed every 2 days until the hepatoblast population reached confluency on day 8 of culture. At this stage, the cell number increased an average of 6.65 ± 2.35-fold, and more than 98% of the cells in the population expressed both ALB and AFP (Figures 14A, 14B, 14C). Populations that were not expanded in Expansion Cocktail before cryopreservation did not expand when cultured in Expansion Cocktail after thawing. The population remaining after 8 days of culture contained a significant proportion of ALB- cells. These findings indicate that expanding the hepatoblast population before cryopreservation allows for improved recovery of a functional cell population that can be further expanded and differentiated into mature, zoned hepatocytes.

[0082] When thawed and cultured (8 days) hepatoblasts were subjected to zone 1 (T3, XAV, GSI, and SB) or zone 3 (T3, CHIR, GSI, and SB) maturation stimuli, they differentiated, giving rise to distinct populations containing few AFP+ cells (Figure 14D), and exhibited the expected zone 1 and zone 3 gene expression patterns. In addition to mature zoned hepatocytes, cryopreserved hepatoblasts were also able to generate functional CFTR+ ciliated cholangiocytes when cultured under cholangiocyte induction / maturation conditions (data not shown).

[0083] Example 5 - Ectopic kidney subcapsular transplantation of hPSC-derived zoned hepatic aggregates into NSG mice To examine the functional capacity of differentiated hepatocytes in vivo, day 21 or day 27 hepatoblasts or zone 1 / zone 3 mature hepatocyte aggregates were ectopically transplanted into the subcapsular space of NSG mice. Zone 1 and zone 3-like hepatocytes were differentiated in monolayer culture from expanded, non-cryopreserved hepatoblasts. The expanded hepatoblasts were subjected to zone 1 maturation stimuli (T3, XAV, GSI, and SB) or zone 3 maturation stimuli (T3, CHIR, GSI, and SB) in monolayer conditions. After 15–18 days of monolayer culture, 3D aggregates were generated from the monolayer cells and maintained in culture for an additional 4–6 days. Aggregates were also generated from day 21 and day 27 hepatoblasts. Aggregates from different populations were transplanted into the subcapsular space of NSG mice. Each mouse received 8–10 x 10e6 aggregates generated from the monolayer cells. For mixed populations, zone 1 and zone 3 aggregates were mixed in equal proportions (equivalent to 4-5 x 10e6 monolayer cells each) prior to transplantation (Figure 15A).

[0084] Human serum albumin (HSA) was measured in serum by ELISA 4 weeks after transplantation. Mice transplanted with either zone 1 or zone 3 liver aggregates had higher levels of HSA than mice transplanted with aggregates generated from progenitor cells on days 21 and 27. Notably, mice receiving a mixture of zone 1 and zone 3 aggregates exhibited the highest levels of HSA (Figure 15B). These data suggest that both zone 1 and zone 3 hepatocytes cooperate to maintain liver function in vivo.

[0085] While the methods and compositions have been described herein in connection with a number of different aspects, it will be understood that the foregoing description of the various aspects is intended to illustrate, but not limit, the scope of the methods and compositions. Other aspects, advantages, and modifications are within the scope of the following claims.

[0086] Disclosed are methods and compositions that can be used for, can be used with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, although specific reference to various individual and collective combinations and permutations of each of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition or a particular method is disclosed and discussed, and multiple compositions or methods are discussed, each and every combination and permutation of the compositions and methods is specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

Claims

1. 1. A method for expanding hepatoblasts, comprising: Culturing hepatoblasts in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or an equivalent thereof. A method comprising:

2. 2. The method of claim 1, wherein the activator of the Wnt pathway is CHIR99021, CHIR98014, BIO, a GSK-3β inhibitor, or a natural Wnt agonist, such as Wnt3.

3. The method of claim 1 or claim 2, wherein the TGF-β receptor inhibitor is SB431542, A83-01, or an ALK4 and / or ALK7 inhibitor.

4. The method of any one of claims 1 to 3, wherein the FGF19 or its equivalent is NGM282.

5. 5. The method of any one of claims 1 to 4, wherein the method is carried out under hypoxic conditions.

6. 1. A method for expanding hepatoblasts, comprising: Culturing hepatoblasts under hypoxic conditions A method comprising:

7. Culturing hepatoblasts in the presence of a Wnt pathway activator, a TGF-β receptor inhibitor, and FGF19 or an equivalent thereof.

7. The method of claim 6, further comprising:

8. 1. A method for expanding hepatoblasts, comprising: Culturing hepatoblasts under hypoxic conditions in the presence of a Wnt pathway activator, a TGFβ inhibitor, FGF19 or an equivalent thereof. A method comprising:

9. 5. The method of any one of claims 1 to 4, wherein the number of hepatocytes is expanded from about 100-fold to about 400-fold within 3 to 5 passages when cultured under ambient O2 conditions.

10. 9. The method of any one of claims 5 to 8, wherein the number of hepatocytes is increased by about 75 to about 1000 fold within 3 to 5 passages when cultured under hypoxic conditions.

11. 1. A method for obtaining mature hepatocytes, comprising: Culturing hepatoblasts in the presence of thyroid hormone or a thyroid hormone receptor agonist A method comprising:

12. 12. The method of claim 11, wherein the thyroid hormone is triiodothyronine or thyroxine.

13. 12. The method of claim 11, wherein the thyroid hormone receptor agonist is GC-1.

14. The method of any one of claims 11 to 13, wherein the hepatoblasts are cultured as a monolayer.

15. The method of any one of claims 11 to 14, wherein the hepatoblasts are cultured as aggregates.

16. The method of any one of claims 11 to 15, wherein the hepatoblasts are cultured in the absence of cAMP.

17. 17. The method of any one of claims 11 to 16, wherein the mature hepatocytes express little or no alpha-fetoprotein (AFP).

18. 18. The method of any one of claims 11 to 17, wherein the mature hepatocytes express albumin.

19. 1. A method for producing zone 1 hepatocytes, comprising: Culturing hepatoblasts in the presence of an inhibitor of the Wnt pathway A method comprising:

20. 20. The method of claim 19, wherein the inhibitor of the Wnt pathway is XAV939, IWP2, IWP4, or ICRT14.

21. 21. The method of claim 19 or claim 20, wherein the hepatoblasts are cultured in monolayer or in aggregates.

22. 1. A method for producing zone 3 hepatocytes, comprising: Culturing hepatoblasts in the presence of an activator of the Wnt pathway. A method comprising:

23. 23. The method of claim 22, wherein the hepatoblasts are cultured in monolayer or aggregates.

24. The method of any one of claims 19 to 23, wherein the hepatoblasts are cultured in the presence of a NOTCH inhibitor.

25. 1. A method for producing cholangiocytes, comprising: Culturing hepatoblasts in the presence of retinoic acid, retinol, or an RA receptor agonist A method comprising:

26. 26. The method of claim 25, wherein the cholangiocytes are identified based on the presence of cystic fibrosis transmembrane conductance regulator (CFTR) protein.

27. 27. The method of claim 25 or claim 26, wherein the bile duct cells are identified based on binding to the DHC5-4D9 antibody.

28. 1. A method for producing liver organoids, comprising: Combining mesothelial cells and hepatoblasts under conditions that promote their self-organization into liver organoids A method comprising:

29. Expanding hepatoblasts in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or an equivalent thereof.

29. The method of claim 28, further comprising:

30. 1. A method for producing stellate cells, comprising: Culturing the liver organoids produced using the method of claim 28 under conditions that produce stellate cells. A method comprising:

31. 1. A method of treating liver disease in a subject, comprising: Transplanting a composition comprising cholangiocytes into a subject, wherein the cholangiocytes are produced by the method of claim 25. A method comprising:

32. 32. The method of claim 31, wherein the liver disease is cholangiopathy, specifically bile duct disease or cholangiocytosis.

33. 1. A method of treating a subject having liver disease, comprising implanting a composition into the subject, The composition comprises: Hepatoblasts expanded using the method of any one of claims 1 to 10; Hepatocytes matured using the method of any one of claims 11 to 18; Zone 1 hepatocytes produced using the method of any one of claims 19-21 or 24; Zone 3 hepatocytes produced using the method of any one of claims 22 to 24; Bile duct cells produced using the method of any one of claims 25 to 27; A liver organoid produced using the method of claim 28 or 29; and / or Astrocytes produced using the method of any one of claims 30 to 32. A method comprising:

34. The method of any one of claims 31 to 33, wherein the composition further comprises epithelial cells.

35. Monitoring the subject for albumin levels 35. The method of any one of claims 31 to 34, further comprising:

36. monitoring the subject for levels of one or more liver enzymes 36. The method of any one of claims 31 to 35, further comprising:

37. 37. The method of any one of claims 31 to 36, wherein the transplantation is performed directly into the liver or heterotopically into the abdomen.

38. 1. A method for culturing liver cells, comprising: culturing the liver cells on the substrate under conditions in which the liver cells grow as a monolayer; A method comprising:

39. Culturing liver cells as a monolayer and then as aggregates 39. The method of claim 38, further comprising:

40. 40. The method of claim 38 or 39, wherein the number of liver cells resulting from the monolayer is at least 10 times greater than the number of liver cells resulting from a culture of aggregated cells.

41. 1. A method for cryopreserving liver cells, comprising: Culturing liver cells in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or an equivalent thereof for at least 3 days; Cryopreserving the cultured liver cells A method comprising:

42. thawing the cryopreserved liver cells; and Culturing the thawed liver cells in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or an equivalent thereof.

42. The method of claim 41, further comprising:

43. 1. A method for recovering cryopreserved liver cells, comprising: thawing the cryopreserved liver cells; and Culturing the thawed liver cells in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or an equivalent thereof. A method comprising:

44. Culturing the liver cells in the presence of a Wnt pathway activator, a TGFβ inhibitor, and FGF19 or an equivalent thereof for at least 3 days prior to cryopreserving the liver cells.

44. The method of claim 43, further comprising:

45. 45. The method of any one of claims 41 to 44, wherein the cryopreservation comprises freezing the liver cells at -80°C in a medium comprising DMSO, FSC, and DMEM / F12.

46. 46. ​​The method of any one of claims 41 to 45, wherein the thawing step comprises heating the liver cells to 37°C for about 5 minutes.

47. 48. The method of claim 47, wherein the liver cells are hepatoblasts.

48. 1. A method of screening for therapeutic compounds for cystic fibrosis and / or ciliopathies, comprising: contacting the cholangiocytes produced using the method of any one of claims 25 to 27 with a test compound; and Determining the presence or absence of CFTR function Including, The method wherein the presence or absence of CFTR function is indicative of a test compound that is therapeutic for cystic fibrosis and / or a ciliary-associated disorder.

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