Method for generating hepatocytes and cholangiocytes from pluripotent stem cells

Abstract

The present invention provides a method for generating hepatocyte and / or cholangiocyte lineage cells from pluripotent stem cells, the method comprising (a) specializing a prolonged nodal agonist-treated induced endoderm cell population by contacting the prolonged nodal agonist-treated induced endoderm cell population with a specialized medium to obtain a cell population including hepatic progenitor cells and / or cholangiocyte progenitor cells, the specialized medium comprising an FGF agonist and a BMP4 agonist and / or their active conjugates and / or fragments; (b) inducing maturation of the hepatic progenitor cells and / or cholangiocyte progenitor cells of the cell population and optionally further lineage specialization and / or expansion to obtain a population including hepatocyte lineage cells such as hepatoblasts, hepatocytes and / or cholangiocytes, the inducing maturation step comprising generating aggregates of the cell population. Optionally, the method also comprises activating the cAMP pathway within the aggregates and forming co-aggregates.

Description

[0001] This application is a divisional application. The application number of the original application is 201480021570.7 (international application number is PCT / CA2014 / 000122), the application date is February 18, 2014, the name of the invention is “Method for generating hepatocytes and bile duct cells from pluripotent stem cells”, and the application date “February 18, 2014” is the international application date. Technical Field

[0002] The present invention relates to a method for generating functional hepatocytes from human pluripotent stem cells. Background Art

[0003] The ability to generate functional hepatocytes from human pluripotent stem cells (hPSCs; including embryonic stem cells; hESCs and induced pluripotent stem cells; hiPSs) will provide a source of hepatocytes for drug metabolism studies and cell-based therapies for treating liver diseases. Hepatocytes are particularly important because they are the cells responsible for drug metabolism and therefore for the elimination of xenobiotics by the body. 1- 3. Given this effect and the fact that individuals may differ in their ability to metabolize specific drugs 4 The fact that functional hepatocytes can be obtained from a representative sample of the population has enormous implications for drug discovery and testing within the pharmaceutical industry. In addition to providing a new platform for drug testing, hPSC-derived hepatocytes could provide potential new therapies for patients with liver disease. While liver transplantation offers an effective treatment for end-stage liver disease, the shortage of viable donor organs limits the patient population that can be treated with this approach. 5-7 Hepatocyte transplantation and bioartificial liver devices developed with hPSC-derived hepatocytes represent life-saving therapies for patients with certain types of liver disease. However, these applications rely on the ability to generate mature, metabolically functional cells from hPSCs. Reproducible and efficient generation of such cells has so far been challenging due to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood.

[0004] Given the potential processing and commercial importance of functional human hepatocytes, significant efforts have been directed toward optimizing the generation of these cells from hPSCs. 8-16 Almost all approaches attempt to recapitulate the key stages of liver development in differentiation cultures, including the induction of definitive endoderm, specification of the endoderm to a hepatic fate, the generation of liver progenitor cells termed hepatocytes, and the differentiation of hepatoblasts to mature hepatocytes. 17In most studies, differentiation was induced in a monolayer format with the sequential addition of known pathway agonists and antagonists to modulate early stages of development, including induction of endoderm and hepatic specification. Through this strategy, it has been possible to optimize these early stages of differentiation and generate populations highly enriched in definitive endoderm, hepatoblasts, and immature hepatocytes as defined by the expression of markers such as Hex, alpha-fetoprotein, and albumin. 17 Although these early differentiation stages are reasonably well established, conditions are not described that, for example, promote maturation of hepatocytes to functional cells as defined by the activity of phase I and phase II drug metabolizing enzymes. Populations generated with different protocols vary widely in their maturation status and in most cases represent immature hepatocytes. Summary of the invention

[0005] One aspect of the invention includes a method for generating hepatocyte lineage cells from a population of induced endoderm cells treated with an extended nodal agonist, the method comprising:

[0006] (a) specializing the induced endoderm cell population treated with a prolonged nodal agonist to obtain a cell population including hepatic progenitor cells by contacting the induced endoderm cell population treated with a prolonged nodal agonist with a specialized medium comprising an FGF agonist and a BMP4 agonist and / or active conjugates and / or fragments thereof;

[0007] (b) inducing maturation, optional further lineage specification and / or expansion of the hepatic progenitor cells of the cell population to obtain a population comprising hepatocyte lineage cells, such as hepatoblasts, hepatocytes and / or cholangiocytes, the maturation inducing step comprising generating aggregates of the cell population.

[0008] In an embodiment, the hepatocyte and / or cholangiocyte lineage cells are hepatoblasts. In an embodiment, the method produces an expanded population of hepatoblasts. In another embodiment, the hepatocyte lineage cells are mature hepatocytes, or the cholangiocyte lineage cells are mature cholangiocytes.

[0009] In some embodiments, the induced endoderm cell population of prolonged nodal agonist treatment is induced from pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). The pluripotent stem cells are optionally human ESCs (hESCs) or human iPSCs (hiPSCs).

[0010] In an embodiment, the induced endoderm population of the nodal agonist treatment is obtained by inducing endoderm cells in embryoid bodies (EBs). In another embodiment, the induced endoderm population of the nodal agonist treatment is obtained by inducing the endoderm cells of a monolayer. In each case, the induced endoderm population is cultivated under the presence of a nodal agonist (e.g., activin) and the time of extension is continued to produce the induced endoderm population of the nodal agonist treatment.

[0011] In embodiments, the induced endoderm population of prolonged nodal agonist treatment comprises at least 80%, 85%, 90%, 95% CXCR4+ and CKIT+ positive cells and / or at least 70%, 75%, 80% SOX17+ cells.

[0012] In an embodiment, the specialization step includes contacting the extended nodal agonist-treated (e.g., activin-treated) induced endoderm population with a specialization medium, the specialization medium includes FGF and BMP4. For example, the FGF can be bFGF, FGF10, FGF2 or FGF4 or a combination thereof. For example, the combination can be added sequentially.

[0013] In an embodiment, the specification step comprises: first contacting the induced endoderm population treated with a prolonged nodal agonist with a specification medium comprising FGF10 and BMP4 for about 40 hours to 60 hours, optionally about 40 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours or 60 hours, and then contacting the induced endoderm population treated with a prolonged nodal agonist with a specification medium comprising bFGF and BMP4 for about 4 days to 7 days, optionally about 4 days, 5 days, 6 days or 7 days.

[0014] In another embodiment, aggregates are generated from a cell population comprising at least 70%, 80%, 85% or 90% albumin positive cells. In another embodiment, aggregates are generated after 24, 25, 26, 27 or 28 days in culture.

[0015] In some embodiments, aggregates are generated from a monolayer of a population of cells comprising hepatic progenitor cells and / or biliary progenitor cells by enzymatic treatment and / or manual dissociation.

[0016] Inducing maturation and optionally further lineage specification and / or expansion may include one or more additional steps. In a further embodiment, the cell population comprising hepatic progenitor cells and / or bile duct progenitor cells and / or aggregates is cultured in the presence of hepatocyte growth factor (HGF), dexamethasone (DEX) and / or oncostatin M (OSM) and / or their active conjugates and / or fragments.

[0017] In one embodiment, inducing maturation and optionally further lineage specification and / or expansion also includes activating the cAMP pathway within the cells of the aggregate to induce the maturation of hepatic progenitor cells and biliary progenitor cells into hepatocytes and / or biliary cells. In another embodiment, activating the cAMP pathway includes exposing the aggregate to cAMP and / or cAMP analogs (e.g., such as 8-bromoadenosine-3'5"-cyclic monophosphate, butyryl-cAMP, adenosine 3', 5'-cyclic monothiophosphate, Sp-isomer (SP-cAMP) and / or 8-bromoadenosine-3', 5'-cyclic monothiophosphate, Sp-isomer (Sp-8-Br-cAMP)) and / or any other cAMP agonist.

[0018] For example, in embodiments, after culturing the pre-aggregate population in a maturation medium comprising HGF, DEX and OSM for, e.g., about 10 days, 11 days, 12 days, 13 days or 14 days, a maturation medium comprising a cAMP agonist and DEX and optionally HGF is added to the aggregates.

[0019] In embodiments, the population of hepatocytes produced is a population comprising functional hepatocytes.

[0020] In embodiments, the hepatocytes, optionally functional hepatocytes, comprise increased expression of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes or proteins selected from the group consisting of ALB, CPS1, G6P, TDO, CYP2C9, CYP2D6, CYP7A1, CYP3A7, CYP1A2, CYP3A4, CYP2B6, NAT2, and UGT1A1, compared to a cell population comprising hepatic progenitor cells and / or bile duct progenitor cells and / or hepatocytes generated from an induced endoderm cell population that is not treated with a nodal agonist (e.g., an induced endoderm population that has not been treated with a nodal agonist (such as activin) for an extended period of time, without aggregation and / or cAMP signaling induction. In other embodiments, at least 40%, 50%, 60%, 70%, 80% or 90% of the hepatocytes, optionally functional hepatocytes, are ASGPR-1+ cells.

[0021] In an embodiment, cholangiocyte fate is specified by treating aggregates of a population of cells with a nodal agonist.

[0022] In an embodiment, the population of the cholangiocytes produced is a population of functional cholangiocytes.Compared with cells of a cell population including liver progenitor cells and cholangiocyte progenitor cells and / or compared with a cell population produced from an aggregate never treated with a nodal agonist, functional cholangiocytes include, for example, at least 1, at least 2 or 3 genes or protein selected from Sox9, CK19 and CFTR (cystic fibrosis transmembrane transduction regulator).In other embodiments, at least 40%, 50%, 60%, 70%, 80% or 90% of the population of cholangiocytes are CK19+ cholangiocytes.In other embodiments, at least 40%, 50%, 60%, 70%, 80% or 90% of the functional cholangiocytes are CFTR+ cholangiocytes.

[0023] As mentioned, this method can be applied to populations of endoderm cells grown in a monolayer.

[0024] Thus, another aspect includes a method of generating hepatocytes and / or cholangiocytes from a population of pluripotent stem cells, the method comprising:

[0025] a) contacting pluripotent stem cells cultured as a monolayer with an induction medium comprising a nodal agonist (such as ActA) and optionally a Wnt / β-catenin agonist (such as i) Wnt3a and / or ii) a selective inhibitor of GSK-3, such as CHIR-99021) to provide an induced endoderm cell population;

[0026] b) contacting the induced endoderm cell population with a nodal agonist to provide a prolonged nodal agonist-treated induced endoderm cell population;

[0027] c) specializing the prolonged nodal agonist-treated induced endoderm cell population to obtain a cell population comprising hepatic progenitor cells and / or bile duct progenitor cells by contacting the prolonged nodal agonist-treated induced endoderm cell population with a specialized medium comprising an FGF agonist and a BMP4 agonist and / or active conjugates and / or fragments thereof;

[0028] d) optionally contacting the cell population comprising hepatic progenitor cells and / or bile duct progenitor cells with a maturation medium comprising HGF, dexamethasone and / or oncostatin M and / or active conjugates and / or fragments thereof; and

[0029] e) inducing maturation, optionally further lineage specification and / or expansion of the hepatic progenitor cells and biliary progenitor cells of the cell population into expanded hepatoblasts, hepatocytes and / or biliary cells, the inducing maturation comprising generating aggregates of the cell population.

[0030] Additionally, endoderm populations may also be included in embryoid bodies.

[0031] Therefore, another aspect of the present invention provides a method for generating hepatocytes and / or bile duct cells from a pluripotent stem cell population, the method comprising:

[0032] a) forming embryoid bodies (EBs) of pluripotent stem cells, optionally by contacting the pluripotent stem cells with a BMP4 agonist;

[0033] b) contacting the EBs with an induction medium comprising a nodal agonist (such as ActA) and optionally a Wnt / β-catenin agonist (such as i) Wnt3a and / or ii) a selective inhibitor of GSK-3, such as CHIR-99021) to provide an induced endoderm cell population;

[0034] c) dissociating the induced endoderm cell population to provide a dissociated induced endoderm cell population;

[0035] d) contacting the dissociated induced endoderm cell population with a nodal agonist to provide a prolonged nodal agonist-treated induced endoderm cell population;

[0036] e) specializing the prolonged nodal agonist treated induced endoderm cell population to obtain a cell population comprising hepatic progenitor cells and / or bile duct progenitor cells by contacting the prolonged nodal agonist treated induced endoderm cell population with a specialized medium comprising an FGF agonist and a BMP4 agonist and / or active conjugates and / or fragments thereof,

[0037] f) optionally contacting the cell population comprising hepatic progenitor cells and / or bile duct progenitor cells with a maturation medium comprising HGF, dexamethasone and / or oncostatin M and / or active conjugates and / or fragments thereof; and

[0038] g) inducing maturation, further lineage specification and / or expansion of hepatic progenitor cells and / or biliary progenitor cells of the cell population into hepatocytes and biliary cells, the inducing maturation, further lineage specification and / or expansion comprising generating aggregates of the cell population.

[0039] In some embodiments, the inducing maturation, further lineage specification and / or expansion step further comprises activating the cAMP pathway within the aggregate to induce the maturation of the hepatic progenitor cells and / or bile duct progenitor cells of the cell population into a population comprising hepatocytes and / or bile duct cells. In embodiments, the method comprises contacting the aggregate with a cAMP analog and / or a cAMP agonist.

[0040] In embodiments, the monolayer or EBs are contacted with a nodal agonist in an induction medium for at least about 1 day, 2 days, 3 days, or about 4 days.

[0041] In embodiments, the EBs are cultured with a nodal agonist for at least 36 hours, 38 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours, 50 hours, 52 hours, 56 hours, 58 hours, or 60 hours, or for at least about 1 day, 2 days, 3 days, or about 4 days, in a step prior to dissociation of the endoderm population (e.g., embryoid body (EB) stage).

[0042] Therefore, another aspect of the present invention relates to a method for generating hepatocytes and / or cholangiocytes from pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), the method comprising:

[0043] a) contacting pluripotent stem cells cultured as a monolayer or formed into embryoid bodies with an induction medium comprising a nodal agonist (such as ActA) and optionally a Wnt / β-catenin agonist (such as i) Wnt3a and / or ii) a selective inhibitor of GSK-3, such as CHIR-99021) to provide an induced endoderm cell population;

[0044] b) contacting the induced endoderm cell population with a nodal agonist to provide a prolonged nodal agonist-treated induced endoderm cell population; and

[0045] c) specializing the prolonged nodal agonist treated induced endoderm cell population to obtain a cell population comprising hepatic progenitor cells and / or bile duct progenitor cells by contacting the prolonged nodal agonist treated induced endoderm cell population with a specialized medium comprising at least one FGF agonist and one BMP4 agonist and / or active conjugates and / or fragments thereof, and

[0046] d) inducing maturation, further lineage specification and / or expansion of hepatic progenitor cells and / or bile duct progenitor cells into hepatocytes and / or bile duct cells, wherein the inducing maturation, further lineage specification and / or expansion comprises:

[0047] (i) culturing a cell population comprising hepatic progenitor cells and / or bile duct progenitor cells using a maturation medium comprising HGF, OSM and DEX;

[0048] (ii) optionally, generating aggregates of the cell population when the cell population comprises at least 70%, 80%, 85% or 90% albumin-positive cells, or after about 20 to about 40 days of culturing, for example, after about 24 to about 28 days of culturing;

[0049] (iii) culturing the aggregated cells in a maturation medium for the aggregated cells: and

[0050] (iv) optionally, activating a cAMP pathway in the aggregated cells within about 1 day to about 10 days of aggregation, for example, within 6 days of aggregation, optionally after about 27 days to about 36 days of culture.

[0051] In embodiments, the maturation medium of the aggregated cells may include factors that promote hepatocyte maturation or factors that promote cholangiocyte development, or both.

[0052] In another embodiment, the aggregated cells are based on aggregation treated with a Wnt agonist such as CIHR99021, optionally in the presence of a TGFβ antagonist such as SB431542. As demonstrated herein, activation of the Wnt pathway and the SMAD pathway, for example, at day 26 (or optionally one or two days thereafter, for example, day 27, in embodiments using EBs), promotes expression of an expanded albumin+ / HNF4+ progenitor cell population. For example, it was demonstrated that when a Wnt agonist was added, up to a 10-fold expansion of the population could be obtained.

[0053] In embodiments, aggregated cells are treated with a Wnt agonist and optionally a TGFβ antagonist (such as SB431542) for about 6 days to about 12 days, preferably, about 8 days to about 10 days, optionally, about 9 days.

[0054] In yet further embodiments, a Wnt antagonist such as XAV939 (also referred to as XAV) and / or a Mek / Erk antagonist such as PD0325901 (also referred to as PD) is added during the cAMP activation step. Adding a Wnt antagonist and / or a MEK / Erk antagonist during the activation of cAMP signaling enhances the expression of CYP enzymes, for example, up to or greater than the level seen in adult hepatocytes. For example, adding an inhibitor of MEK / Erk added in the presence of cAMP to a culture from about day 28 to about day 32 produces hepatocytes with increased CYP3A4 levels. Adding a combination of a MEK / Erk antagonist and a Wnt antagonist is shown to also increase the level of CYP1A2. In an embodiment, the Wnt antagonist is XAV939. In another embodiment, the MEK / Erk antagonist is PD0325901.

[0055] In an embodiment, the cells are treated with a nodal agonist about 1 day to about 4 days after aggregation. Adding a nodal agonist at this stage promotes maturation of cholangiocytes. In some embodiments, such as where cholangiocyte maturation is preferred, induction of cAMP signaling is omitted.

[0056] Further inhibition of Notch signaling, for example with a Notch antagonist such as γ-secretase inhibitor (GSI) L695,458, is shown herein to inhibit cholangiocyte development, and the resulting cells retain the characteristics of hepatocytes. In embodiments, for example, embodiments in which hepatocyte differentiation is desired, the method comprises treating cells with a nodal antagonist about 1 day to about 4 days after aggregation.

[0057] In another embodiment, a method of generating hepatocytes and / or cholangiocytes from pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), comprises:

[0058] a) contacting pluripotent stem cells cultured as a monolayer or formed into embryoid bodies with an induction medium, optionally for about 4 days to about 8 days, to provide an induced endoderm cell population, the induction medium comprising a nodal agonist such as ActA and optionally a Wnt / β-catenin agonist (such as i) Wnt3a and / or ii) a selective inhibitor of GSK-3, such as CHIR-99021);

[0059] b) contacting the induced endoderm cell population with a nodal agonist, optionally for about 1 day, 2 days, 3 days, or about 4 days, to provide a prolonged nodal agonist-treated induced endoderm cell population;

[0060] c) specializing the prolonged nodal agonist treated induced endoderm cell population to obtain a cell population comprising hepatic progenitor cells and / or bile duct progenitor cells by contacting the prolonged nodal agonist treated induced endoderm cell population with a specialized medium, optionally for about 4 days to about 10 days, the specialized medium comprising at least one FGF agonist and at least one BMP4 agonist and / or active conjugates and / or fragments thereof, and

[0061] d) inducing maturation, further lineage specification and / or expansion of hepatic progenitor cells and / or bile duct progenitor cells into hepatocytes or bile duct cells, wherein the inducing maturation, further lineage specification and / or expansion comprises:

[0062] (i) culturing a cell population comprising hepatic progenitor cells and / or bile duct progenitor cells with a maturation medium comprising HGF, Dex and / or OSM, optionally for about 10 to 14 days;

[0063] (ii) optionally, generating aggregates of the cell population when the cell population comprises at least 70%, 80%, 85% or 90% albumin-positive cells, or after about 20 days to about 40 days of culturing, for example, after about 24 days to about 28 days of culturing;

[0064] (iii) culturing the aggregates in a maturation medium comprising Dex for about 1 day to 10 days;

[0065] iv) a) culturing the aggregates in a maturation medium comprising Dex and a cAMP analog and / or a cAMP agonist for about 6 days to about 10 days, optionally adding a cAMP analog and / or a cAMP agonist within about 1 day to about 10 days of the step of generating the aggregates, for example, within 6 days of the step of generating the aggregates, optionally, after about 27 days to about 36 days of culturing; or

[0066] b) culturing the aggregates in a maturation medium comprising a nodal agonist and optionally a cAMP agonist, HGF and / or EGF for about 6 days to about 20 days, optionally adding the nodal agonist within about 1 day to about 10 days of the step of generating the aggregates, for example, within 6 days of the step of generating the aggregates, optionally after about 20 days to 40 days of culturing.

[0067] In embodiments, the method comprises aggregation, e.g., after about 20 days of culturing and / or before 40 days of culturing.

[0068] The present invention also provides a method for inducing bile duct progenitor cells to mature, further lineage-specificate and / or expand into bile duct cells, wherein the inducing maturation, further lineage-specificate and / or expansion comprises:

[0069] (i) culturing a cell population including bile duct progenitor cells with a Notch agonist to induce at least one bile duct progenitor cell to mature into a bile duct cell, optionally, a functional bile duct cell.

[0070] The notch agonist can be, for example, any notch ligand that is bound to a surface such as a cell, plastic, ECM or bead. In one embodiment, the notch ligand is notch ligand delta. In one embodiment, inducing maturation, further lineage specialization and / or amplification includes contacting a cell population including bile duct progenitor cells with a notch signal donor (e.g., notch agonist) such as OP9, OP9delta and / or OP9 Jagged1 cells and optionally in the presence of EGF, TGFβ1, HGF and EGF and / or HGF, TGFβ1 and EGF for at least or about 5 days to about 90 days to induce bile duct progenitor cells to mature into functional bile duct cells.

[0071] Optionally, contacting a cell population comprising bile duct progenitor cells with a notch agonist (e.g., a notch signal donor) comprises co-culturing a cell population comprising bile duct progenitor cells with a notch signal donor such as OP9, OP9delta and / or OP9 Jaggedl cells and optionally in a maturation medium comprising EGF, TGFβ1, HGF and EGF and / or HGF, TGFβ1 and EGF for at least or about 5 days to at least or about 90 days, optionally, at least or about 5 days to at least or about 60 days, at least or about 30 days, at least or about 25 days, at least or about 21 days, and / or at least or about 14 days to induce maturation of bile duct progenitor cells into bile duct cells, optionally functional bile duct cells, optionally, wherein the functional bile duct cells form branched, encapsulated, tubular or spherical structures.

[0072] In another embodiment, the present application provides a method, comprising:

[0073] (a) producing a population of cells comprising hepatocytes and / or cholangiocytes according to any of the methods described herein; and

[0074] (b) introducing the population of cells, or optionally the hepatocyte and / or cholangiocyte enriched or isolated population, into a subject.

[0075] In certain embodiments, the method also includes enriching or separating the hepatocyte and / or cholangiocyte populations of cells. Optionally, the hepatocyte and / or cholangiocyte populations of cells include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or up to about 95% of hepatocytes and / or cholangiocytes (e.g., optionally, functional hepatocytes and / or cholangiocytes).

[0076] The invention also provides for the use of populations of hepatocytes and / or cholangiocytes for drug discovery, drug metabolism analysis, development of bioartificial liver devices and / or as cell replacement therapy for the treatment of liver conditions and diseases.

[0077] Other features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples are given by way of illustration only, and while the detailed description and specific examples indicate preferred embodiments of the present invention, various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Embodiments of the present invention will now be described with reference to the accompanying drawings, in which:

[0079] Figure 1Shown is the induction of endoderm in hESC-derived embryoid bodies. Figure 1 (a) is a schematic diagram of a differentiation protocol. EBs were digested with trypsin on the sixth day and were plated as a monolayer for two days in the presence of activin to generate the definitive endoderm of appropriate stages. In the presence of BMP4 and FGF, the endoderm population was specialized into a hepatocyte line by culture. Liver maturation was induced by a stepwise process: first by adding HGF, dexamethasone (Dex) and oncostatin M (OSM) and continuing for 12 days, then 3D aggregates were generated, and 3D aggregates were cultured for 8 days in a hepatocyte culture medium supplemented with Dex, and then in the culture medium adding cAMP analogs, 8-br-cAMP, 12 days (32-44 days) were cultivated again. Figure 1 (b) shows flow cytometric analysis showing the proportion of CXCR4+, CKIT+ (CD117), and EPCAM+ cells in the activin- and activin / Wnt3a-induced populations at day 6. Figure 1 (c) shows intracellular flow cytometry analysis showing the proportion of SOX17+ and FOXA2+ cells in activin- and activin / Wnt3a-induced populations at day 6. The size of the SOX17+ and FOXA2+ populations in EBs induced with activin / Wnt3a was significantly larger (SOX17: 96.2±1.1%, FOXA2: 88.5±2.9%) compared to EBs induced with activin alone (SOX17: 91+ / -1.8%, FOXA2: 80.3±2.5%), *P<0.05 (P=0.002), **P<0.01 (P=0.005); n=4 in Scholar's t-test. Figure 1 (d) shows RT-qPCR-based analysis of the expression of T, SOX17, GSC, and FOXA2 in EBs induced by activin and activin / Wnt3a. EBs were analyzed at the indicated time points. Columns represent the SD of the mean of three independent experiments. Figure 1 (e) is a flow cytometric analysis showing the dynamics of the development of CXCR4+, CKIT+, SOX17+, and FOXA2+ populations in Activin / Wnt3a-induced EBs. Figure 1 (f) is a flow cytometric analysis showing the proportions of CXCR4+, CKIT+, EPCAM+, Sox17+, and FOXA2+ cells in EBs induced with Activin in neurobasal medium on day 6.

[0080] Figure 2(a) is RT-qPCR analysis of albumin expression in monolayer cultures specified by the indicated cytokines. Cells were treated with different factors (10 ng / ml bFGF; 50 ng / ml BMP4; 20 ng / ml HGF; or 20 ng / ml bFGF plus 50 ng / ml BMP4) from day 6 to day 12, then cultured with DEX, HGF and OSM and analyzed on day 24. The columns represent the standard deviation (SD) of the mean of three independent experiments. Values ​​are determined relative to TBP and are presented relative to expression in bFGF (20 ng / ml) cultures (values ​​set to 1). ***P<0.001 compared to bFGF-treated cultures. n=3 in scholar's t-test. (b) RT-qPCR analysis of albumin expression in specific populations in the presence and absence of FGF10. Cultures were treated (or not treated) with FGF10 (50 ng / ml) plus BMP4 (50 ng / ml) between days 6 and 8. At this stage, FGF10 was removed and cells were cultured in bFGF / BMP between days 8 and 12. The columns represent the standard deviation (SD) of the mean values ​​of three independent experiments. Values ​​are determined relative to TBP and are presented relative to expression in FGF10 (-) cultures (values ​​set to 1). *P < 0.05, n = 3 in scholar's t-test.

[0081] Figure 3 It was shown that the duration of activin signaling affects liver development. Figure 3 (a) is intracellular flow cytometry analysis, and intracellular flow cytometry analysis shows the ratio of SOX17+ and FOXA2+ cells in the EB induced by activin / Wnt3A on the 6th day and the monolayer population obtained by EB. The monolayer population is directly cultured in the specified culture medium (-activin), or cultured in activin (50ng / ml) for two days and then cultured in the specified culture medium (+activin). After culturing in the specified culture medium for two days or four days (-activin group for 8 days and 10 days and + activation group for 10 days and 12 days), the population is analyzed. The column represents the standard deviation (SD) of the mean value of three independent experiments. Compared with untreated population (73.3+ / -7.5% relative to 45.9±3.7%), the ratio of SOX17+ on the 10th / 12th day of activin treatment is significantly higher. Similarly, the proportion of FOXA2+ cells was significantly higher on days 8 / 10 and 10 / 12 of activin treatment compared to untreated populations (day 8: 96.1+ / -0.9% vs. 76.5+ / -10.1%, day 12: 92.7±2.5% vs. 50.2+ / -6.3%). Figure 3(b) Total number of cells in activin-treated and non-treated monolayer cultures are shown. Day 6 EB-derived cells were cultured directly in hepatic differentiation medium or in the presence of activin for two days and then cultured in hepatic differentiation medium. Figure 3 (c) is a flow cytometric analysis showing the proportion of CXCR4- and CKIT-positive cells in populations cultured on days 8, 10, and 12 generated from non-treated cells and Activin-treated endoderm. Figure 3 (d) shows the expression analysis based on RT-qPCR of the liver monolayer population generated from the (black column) and non-processed (grey column) endoderm of activin treatment. The population was analyzed for the expression of the endoderm (HEX, AFP, ALB and HNF4a) and mesoderm (MEOX1, MESP1, CD31 and CD90) genes specified. The population (grey column) treated with activin was analyzed at the 12th, 18th and 26th day of total culture, while the untreated population (black column) was analyzed at the 10th, 16th and 24th day of culture. The expression level indicated is relative to TBP. The column represents the standard deviation (SD) of the mean value of three independent experiments. Figure 3 (e) is a flow cytometric analysis showing the proportion of CD31+, CD90+, and EPCAM+ cells in monolayer populations derived from activin-treated (day 26) and non-treated (day 24) endoderm. Untreated CD31+ and CD90+ populations were significantly larger compared to treated cultures (CD31: 13.6±2.3% vs. 0.49±0.11%, P<0.001; CD90: 41.2+ / -4.7% vs. 8.5± / -1.19%, P<0.001, Scholar's t-test, n=3). In contrast, a higher fraction of EPCAM+ cells was detected in populations derived from activin-treated endoderm compared to populations generated from non-treated cells (EPCAM: 90.7±2.7% vs. 56.8+ / -7.3%, P<0.01, n=3). Figure 3 (f) shows an immunostaining analysis showing the proportion of albumin-positive cells in cultures generated from activin-treated (day 26) and non-treated (day 24) endoderm. Albumin was visualized with Alexa 488. Scale bar: 200 μm. (g) Intracellular flow cytometry analysis showing the proportion of albumin (ALB) and alpha-fetoprotein (AFP) cells in monolayer cultures generated from activin-treated (grey columns; day 26) and non-treated (black columns; day 24) endoderm. The bars in the figure represent the standard deviation (SD) of the mean of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 (Scholar's t-test; n=3). AL: adult liver, FL: fetal liver.

[0082] Figure 4 It was shown that the aggregates promote liver maturation. Figure 4 (a) is a phase contrast image of hepatic aggregates on culture day 28. Scale bar, 200 μm. Figure 4 (b) shows RT-qPCR-based analysis of ALB, CPS1, TAT, G6P and TDO expression in monolayer (black bars) and 3D aggregate cultures (grey bars) at day 32 of differentiation. Values ​​are determined relative to TBP and are presented relative to expression in adult liver (values ​​set to 1). Figure 4 (c) RT-qPCR-based analysis of CYP7A1, CYP3A7, and CYP3A4 expression in monolayer (black bars) and 3D aggregate cultures (grey bars) at day 32 of differentiation. Expression levels are relative to TBP. Figure 4 (d) is a flow cytometric analysis showing the proportion of asialoglycoprotein receptor-1+ (ASGPR-1) cells in monolayer (2D) and aggregate (3D) cultures on day 36. The frequency of ASGPR-1+ cells was significantly higher in 3D aggregate cultures (2D: 28.8±3.1%, 3D: 64.7+ / -4.26%, P<0.001, n=3). The bars in all figures represent the standard deviation (SD) of the mean of samples in three independent experiments, *P<0.05, **P<0.01, ***P<0.001, scholar's t-test, AL: adult liver, FL: fetal liver, pH; primary hepatocytes cultured for 2 days.

[0083] Figure 5 It is shown that cAMP signaling induces maturation of hESC-derived hepatocyte-like cells. Figure 5 (a) is RT-qPCR analysis of PGC1-a, HNF4a, AFP, ALB, G6P and TAT expression in liver aggregates cultured in the presence and absence of 8-Br-cAMP. Expression levels are relative to TBP. Figure 5 (b) is an intracellular flow cytometric analysis showing the proportion of alpha-fetoprotein (AFP)+ and albumin (ALB)+ cells (day 44) in liver aggregates cultured in the presence and absence of 8-Br-cAMP. The frequency of AFP+ cells was significantly lower in the population induced with cAMP compared to the non-induced population (34.5+ / -12.4% vs. 56.9+ / -3.6%, P<0.05, mean±SD, n=3). On the other hand, the proportion of ALB-positive cells was higher in the cAMP-treated population (89.5+ / -5.6% vs. 82.3+ / -3.0%, P<0.05 (mean±SD, n=3). Figure 5(c) is RT-qPCR analysis of PGC1-α expression in cAMP-treated pancreatic aggregates and hepatic aggregates generated from HES2, H9 and 38-2 cells. Values ​​were determined relative to TBP and are presented as fold changes relative to expression in non-treated cells (values ​​set to 1). Figure 5 (d) shows ICG uptake on day 44 in untreated and cAMP-treated aggregates. Bars in all graphs represent the standard deviation (SD) of the mean of values ​​from three independent experiments. *P<0.05, **P<0.01, ***P<0.001, Scholar's t-test, AL: adult liver, FL: fetal liver.

[0084] Figure 6 It was shown that cAMP signaling increases metabolic enzyme activities in hESC-derived hepatocytes. Figure 6 (a) shows the expression of CYP3A7, CYP3A4, CYP1A2, CYP2B6 and UGT1A1 in liver aggregates cultured in the presence and absence of 8-Br-cAMP (day 44). Levels in primary hepatocytes (PH) are shown as controls. Values ​​are determined relative to TBP and presented as fold changes relative to expression in non-treated cells (values ​​set to 1). Figure 6 (b) shows RT-qPCR analysis showing the expression of PGC1a, TAT, HNF4a, CYP1A2 and CYP3A4 in untreated (-) and cAMP-treated (+) monolayer populations (day 44). Values ​​were determined relative to TBP and presented as fold changes relative to expression in non-treated cells (values ​​set to 1). Figure 6 (c) shows RT-qPCR analysis of CYP1A2 and ALB expression in cAMP-treated aggregates generated from non-treated (-Act) or expanded activin-treated (+Act) endoderm. Figure 6 (d) shows RT-qPCR analysis of CYP1A2 expression in aggregates cultured for 6 days (cAMP+ / -) or 12 days in 8-Br-cAMP (cAMP+). Figure 6 (e) shows that hESC-derived hepatocytes exhibit CYP1A2 activity in vitro. Untreated and cAMP-treated aggregates and primary hepatocytes were incubated with phenacetin (200 μM) for 24 hours. The generation of acetaminophen from the o-deethylated metabolite of phenacetin was monitored by HPLC. Activity was presented per 10,000 cells. (*P<0.05, n=5). Figure 6(f) shows that hESC-derived hepatocytes exhibit CYP2B6 activity in vitro. cAMP-treated aggregates and primary hepatocytes were incubated with bupropion (1 μM) for 48 hours. The formation of the metabolite o-hydroxybupropion from bupropion was measured by HPLC. Activity was presented per 50,000 cells, (n=3). Figure 6 (g) shows that the metabolism of sulfadimethazine (SMZ) to N-acetylated SMZ indicates the presence of the second stage enzymes NAT1 and / or NAT2. Aggregates and primary hepatocytes treated with cAMP were cultured with SMZ (500 μM) for 48 hours, and N-acetylated SMZ was measured by HPLC. Activity was presented per 10,000 cells (n=3). Figure 6 (h) is HPLC analysis showing the generation of 4-MU glucuronide (4-MUG) from 4-methylumbelliferone (4-MU) by cAMP-treated aggregates of total UGT activity. cAMP-treated aggregates and primary hepatocytes were cultured with 4-MU for 48 hours. The formation of 4MUG was measured by HPLC. Activity was present per 10,000 cells, (n=3). The bars in all figures represent the standard deviation (SD) of the mean of samples in three independent experiments, *P<0.05, **P<0.01, ***P<0.001, Scholar's t-test, PH; primary hepatocytes.

[0085] Figure 7 Hepatic differentiation from different pluripotent stem cell lines is shown. Figure 7 (a) is a flow cytometric analysis showing the proportions of CXCR4+, CKIT+, EPCAM+, SOX17+, and FOXA2+ cells in Activin / Wnt3a-induced day 6 EBs generated from H9 hESCs, H1 hESCs, and 38-2 iPSCs. Figure 7 (b) shows RT-qPCR analysis of albumin expression in monolayer cultures generated from H9, H1 and 38-2 derived endoderm treated with activin for different times. The different populations were analyzed at the following times: no activin: day 24, 2 days activin: day 26, 4 days activin: day 28 of differentiation. Figure 7 (c) shows intracellular flow cytometry analysis showing the frequency of ALB and AFP positive cells generated from different HPSC lines (no Activin (-): day 24, 2 days Activin: day 26, 4 days Activin: day 28 of differentiation). Figure 7 (d) is a phase contrast image showing the morphology of H9-derived hepatocytes on culture day 26. Scale bar: 200 μm. Figure 7(e) shows RT-qPCR analysis, RT-qPCR analysis shows CYP3A7, CYP3A4, CYP1A2, CYP2B6 and UGT1A1 in H9- and iPSC (38-2) derived liver aggregates (day 44) cultured in the presence and absence of 8-Br-cAMP. Values ​​were determined relative to TBP and presented as fold changes relative to expression in non-treated cells (values ​​set to 1). The bars in all figures represent the standard deviation (SD) of the mean values ​​from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001, scholar's t test, AL: adult liver, FL: fetal liver, PH: primary hepatocytes.

[0086] Figure 8 (a)(b)(c) show that CHIR99021 can induce definitive endoderm cells. Flow cytometric analysis shows the proportion of CXCR4+, CKIT+, and EPCAM+ cells on day 6 of endoderm induced with (a) activin / wnt3a or (b) activin / CHIR99021 or on day 7 of monolayers induced with (c) activin / wnt3a or (d) activin / CHIR99021. (e), (f), and (g) Ectopic liver tissue in NSG mice. Micrographs of H&E-stained sections of the mesentery show clusters of hESC-derived hepatocytes (arrows) two months after transplantation. Magnification is 5X. Intestine (arrow), transplanted cells (arrow head). (f) from Figure 8 (c) High magnification (10X) micrograph of the H&E stained section. (g) (h) Immunohistochemical staining shows the presence of hESC-derived cells in the mesenteric region two months after transplantation. Double staining of human albumin (Alexa488: green shown as arrow and CK19 (Cy3 labeled: red) shown as arrow head) shows that the transplanted cells have the potential to differentiate into hepatocyte and cholangiocyte lineages. hESC-derived hepatocyte-like cells are observed as albumin-positive cells (arrows), while bile duct epithelial-like cells express CK19 and are found in tubular structures (arrow heads).

[0087] Fig. 9 Factors that influence the proliferation and maturation of hepatic progenitor cells are shown. Fig. 9 (a) shows the expansion of hepatic progenitor populations by Wnt signaling and Smad signaling. Shown is the fold increase in the number of ALB+AFP+ cells after 9 days of culture of H9-derived day 27 hepatic progenitor cells in different concentrations of CHIR99021 (0.3 μM, 1 μM and 3 μM) and TGFB inhibitor SB431542 (6 μM). Fig. 9 (b) and Fig. 9(c) shows immunofluorescence staining showing the presence of ALB-positive cells (b) and double-positive ALB and HNF4α (c) after 9 days of culture (day 36) of H9-derived day 27 hepatic progenitor cells. Fig. 9 (d) and Fig. 9 (e) shows that inhibition of Wnt / p-catenin and MEK / ErkK signaling increases expression of CYP3A4 (erkinhib + sufficient cAMP) and CYP1A2 in day 44 aggregates (all three). Wnt inhibitor XAV939 (1 μM) and MEK / Erk inhibitor PD0325901 (1 μM) were added to aggregate cultures at day 30 of differentiation either alone or together with 8-Br-cAMP. Expression of CYP3A4 (d) and CYP1A2 (e) relative to levels found in adult liver is shown. Addition of MEK / Erk inhibitors together with cAMP induced levels of CYP3A4 expression compared to those found in adult liver, while addition of Wnt and MEK / Erk inhibitors together with cAMP induced the highest levels of CYP1A2 expression. Fig. 9 (f) shows the expression of ALB in hepatocyte-like cells cultured on different extracellular matrices (ECM) at day 26. The values ​​shown are relative to cells cultured on gelatin (values ​​set to 1).

[0088] Fig.10 It is shown that the Notch signaling pathway in hepatic progenitor cells affects the differentiation of the bile duct lineage. (a) High magnification (20X) micrograph of H&E stained sections of three-dimensional (3D) tissues generated in collagen / matrigel. H9-derived day 25 hepatic progenitor cells were mixed (aggregated) with OP9-delta1 stromal cells at a ratio of 5:1 in low-cluster culture dishes for 48 hours. Chimeric aggregates were embedded in a mixture of type 1 collagen (80%) and matrigel (20%) to establish 3D co-cultures. The cultures were maintained in culture medium containing HGF 20ng / ml and EGF 50ng / ml and continued for 9 days in the presence or absence of GSI. Aggregate morphology was maintained in cultures treated with GSI. These aggregates containing hepatocyte-like cells expressed albumin. In the absence of GSI, the aggregates developed extensive branching structures. The cells within the branches showed epithelial morphology and were organized to surround the lumen. These cells expressed CK19, suggesting that they were cholangiocytes and that the branching structures could represent developing bile ducts. Fig.10 (b) shows that activation of Notch signaling upregulates the expression of CK19 and cystic fibrosis transmembrane conductance regulator (CFTR) in ductal structures. The values ​​shown are relative to cells cultured in the presence of GSI. Cells in Notch (-) co-culture (i.e., treated with GSI) retain the characteristics of hepatocytes, as indicated by the expression of albumin. Fig.10(c) shows that the induction of CFTR expression in 3D co-culture is higher than those found in monolayer culture. The values ​​shown are relative to cells cultured in monolayer state.

[0089] Fig.11 Schematic diagram of the hepatocyte / cholangiocyte differentiation protocol. (a) Monolayer cultures were also generated in the presence of activin and wnt3a or CHIR99021 to generate definitive endoderm. Endoderm cells in the monolayer induction on day 5 were equivalent to cells on day 6 in EBs. Additional activin treatment beyond 5 days was also necessary in the monolayer and the culture was used for the generation of hepatic progenitors and mature hepatocytes. (b) EB cultures were used to generate definitive endoderm. (c) Schematic diagram of the protocol was used to generate cholangiocytes. The definitive endoderm generated in monolayer cultures was directed to the hepatic fate leading to the generation of hepatic progenitors (hepatocytes) that were cultured for 25 days. Hepatic progenitors were dissociated and aggregated with OP9 / OP9 delta cells in low cluster dishes for two days. Chimeric aggregates were embedded in collagen / Matrigel gels and cultured in medium supplemented with HGF (20 ng / ml) and EGF (50 ng / ml) in the presence or absence of an inhibitor of panNotch signaling (e.g., notch antagonist) γ-secretase inhibitor (GSI) L-685, 458.

[0090] Fig.12Show that hESC-derived endothelial cells enhance liver maturation. (a) The scheme for generating chimeric aggregates is composed of hESC-derived endothelial cells (RFP positive) and hepatoblasts. RFP(+) / CD34(+) endothelial cells are generated by inducing with BMP4 for 4 days and then inducing with VEGF and bFGF for another 2 days. The RFP(+) / CD34(+) endothelial cells separated by FACS are seeded on the wells coated with type I collagen, and are cultivated for 6 days with EGM-2 culture medium in the presence of VEGF and bFGF. In order to generate chimeric aggregates, the cultured RFP(+) / CD34(+) cells are trypsinized, dissociated and placed in Aggrewell plates at a cell density of 100 cells per well. After culturing for 2 days, the hepatocytes of the 25th day are placed on the RFP(+) / CD34(+) endothelial aggregates at a cell density of 1000 cells per well. Scale bar 100 μm. (b) Phase contrast and fluorescence images show RFP-positive cells within endothelial / liver aggregates at day 33. RFP was not detected in liver aggregates generated without endothelial cells. Scale bar 100 bar. (c) Flow cytometry analysis shows the proportion of RFP-positive cells in endothelial / liver cell aggregates at day 33. (d) RT-qPCR analysis shows the expression of CYP3A4 in aggregates with and without endothelial cells at day 44. Values ​​are determined relative to TBP and are presented as fold changes relative to expression in adult liver samples (values ​​set to 1).

[0091] Fig.13 The effect of 3D gel culture on the maturation of hPSC-derived hepatocytes is shown. Aggregates composed of hepatoblasts or hepatocytes and endothelial cells (ends) are generated on the 25th day of culture, and then cultured in a hepatocyte liquid culture medium supplemented with VEGF and bFGF for an additional 7 days, followed by 12 days of culture in the same culture medium supplemented with cAMP, PD0325901 (PD) and XAV939. In order to test the effect of collagen on maturation, chimeric aggregates on the 32nd day were embedded in collagen type 1 gel and cultured for 12 days in the presence of cAMP, PD0325901 and XAV939. All cultures were collected on the 44th day, and the expression of the specified genes was analyzed by qRT-PCR. Values ​​were determined relative to TBP. The expression of ALB and CYP3A4 is presented as a multiple change relative to their levels in adult liver. The expression of AFP and CYP3A7 is presented as a multiple change relative to their levels in fetal liver. AL: adult liver, FL: fetal liver.

[0092] Fig.14Characterization of hepatocyte development stages in hPSC differentiation culture. (a) Schematic diagram of the differentiation protocol. (b) Flow cytometry analysis showing the development of CXCR4+, CKIT+, and EPCAM+ populations at day 7 of the monolayer induction format. (c) RT-qPCR showing the maintenance of Fig.14 a Expression of the indicated genes in H9-derived hepatoblasts under the indicated culture conditions. The expression of the indicated genes was analyzed on days 7, 13, 19 and 25 of culture. Values ​​were determined relative to TBP and presented as fold changes relative to expression in fetal liver (values ​​set to 1). AL: adult liver, FL: fetal liver.

[0093] Fig.15 .Notch signaling promotes cholangiocyte development from hPSC-derived hepatocyte-like populations. RT-qPCR-based expression analysis of ALB and CK19 in hepatocyte-derived cells after co-culture with OP9 in a medium supplemented with HGF (20 ng / ml), EGF (50 ng / ml) and TGFbl (5 ng / ml) in the presence or absence of γ-secretase inhibitors (GSI), antagonists of the Notch pathway. Cells were collected and analyzed on days 30, 33 and 36 of culture. Values ​​were determined relative to TBP and values ​​were presented as fold changes relative to expression levels in hepatocyte aggregates on day 27 (values ​​set to 1). (b) RT-qPCR-based expression analysis of Notch target genes HES1, HES5 and HEY1 in hepatocyte-derived cells after culture with or without OP9. Cells were measured on day 36 of culture. For this analysis, the 27th day hepatocyte aggregates were plated on OP9 (OP9+) stromal cells or on matrigel (OP9-) in the presence or absence of Notch signaling antagonist γ-secretase inhibitor (GSI) containing HGF, EGF and TGFb1 (5ng / ml) in the culture medium. Values ​​were determined relative to TBP and values ​​were presented as fold changes relative to the expression levels in cells cultured on the 36th day on matrigel (values ​​set to 1). The bars in all figures represent the standard deviation (SD) of the mean values ​​of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 (scholar's t test; n=3).

[0094] Fig.16.Three-dimensional culture promotes cholangiocyte maturation: morphology of chimeric aggregates composed of day 25 hESC-derived cells and OP9 stromal cells (GFP+). H9-derived day 25 hepatoblasts were mixed (aggregated) with OP9 stromal cells at a ratio of 4:1 in low-cluster culture dishes for 48 hours. Chimeric aggregates were embedded in a mixture of type 1 collagen (1.2 mg / ml) and matrigel (40%) to establish 3D gel cultures. Cultures were maintained in medium containing HGF, EGF, and TGFb1 in the presence or absence of GSI for 2 weeks. (b) The proportion of structures showing the morphology of tubes, cysts, or spheres in 3D cultures. Values ​​are presented as the proportion of total structures developed in the presence or absence of GSI. Values ​​represent 3 independent experiments. (c) RT-qPCR-based expression analysis of merged structures developed in 3D gels. Cultures were harvested on day 44 and cells analyzed for gene expression indicative of hepatocytes (ALB, AFP, and CYP3A7) and cholangiocytes (CK19, S o x9 and CFTR) lineages. Values ​​were determined relative to TBP and are presented as fold changes relative to expression levels in populations treated with GSI (values ​​set to 1).

[0095] Fig.17 hPSC-derived cholangiocytes form duct-like structures in vivo. (ab) Histological analysis of bile duct grafts in Matrigel plugs 8 weeks after transplantation of hepatocyte-derived cells on day 25 of co-culture with OP9 stromal cells in medium containing HGF, EGF, and TGFb1 for 9 days. After co-culture, cells were dissociated and transplanted (10 per recipient). e ) was implanted into the mammary fat pad of immunodeficient NOD / SCID / IL2rg- / -(NSG) mice. Multi-tube structures were observed in mammary fat pads with low (a) and high magnification images (b) (H&E staining). (cd) Immunostaining, to detect RFP positive cells in the hESC-derived ductal structures developed in mammary fat pads after transplantation. For these studies, bile duct cells were generated from HES2-RFP hESC, which express RFP from ROSA locus. The bile duct cells generated after 9 days of co-cultivation with OP9 stromal cells were transplanted into the mammary fat pad of NSG mice. Due to the presence of RFP+ cells, the grafts developed in 8 weeks after transplantation were analyzed by immunohistochemistry. RFP-positive cells were detected in all ductal structures, confirming that the cells were human-derived, and derived from HES2-RFP cells. RFP+ structures are visible in images at low (c) and high (d) magnification.

[0096] Fig.18hPSC-derived cyst structures generated in 3D gels contain functional CFTR protein (a) Representative confocal microscopy images of calcitonin-labeled and forskolin / IBMX (F / I)-stimulated cyst structures generated from H9 (hESC)- and Y2-1 (IPSC)-derived cholangiocytes. Images were taken 24 hours after F / I stimulation. Scale bar, 500 μm. (b) Quantification of the extent of cyst swelling 24 hours after F / I stimulation in the presence or absence of CFTR inhibitors. F / I-stimulated cyst swelling was quantified using speed imaging software. The total size of cysts was normalized to before F / I stimulation. Values ​​are from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 (scholar's t-test; n=3).

[0097] Fig.19 Generation of cholangiocytes from iPSCs of cystic fibrosis patients. (a) Phase contrast images of cholangiocyte-like cells derived from CFTR-deleted F508 iPS cells (CF-iPS) at day 44 of 3D gel culture in the presence or absence of forskolin. Fig.14 The scheme shown in A generates CF-iPS cell-derived hepatocytes and chimeric hepatocyte / OP9 aggregates. After embedding collagen / matrigel culture, cyst formation is induced from aggregates by adding forskolin in the first week of a two-week culture period (left). In the absence of forskolin stimulation, CF-iPSC-derived cholangiocytes form branched duct structures, rather than hollow cysts (right). (b) Quantification of the number of cyst structures developed from CF-iPSC and normal iPSC (Y2-1)-derived cholangiocytes cultured for 7 and 14 days. CF-iPSC-derived cholangiocytes are maintained under 3D gel conditions in the presence or absence of forskolin in the first week of a two-week culture cycle (left figure). Normal iPS cell-derived cholangiocytes are maintained in the first week of a two-week culture cycle in the presence or absence of CFTR inhibitors (right figure). Adding forskolin increases the number of cyst structures, which develop from CF-iPSC-derived cholangiocytes in culture for 7 and 14 days (left figure). Adding CFTR inhibitor to normal iPSC-derived cholangiocytes delayed the formation of cysts (right). (c) Histological analysis of cysts from normal iPSC- (upper panel) and CF-iPSC- (lower panel) cholangiocytes at day 44. Cholangiocyte populations were cultured in the presence of forskolin in the first week of two-week culture. Forskolin was added to normal iPSC-derived cholangiocytes to induce the formation of large hollow cysts (upper panel). CF-iiPSC-derived cysts are smaller and usually contain an internal septum (lower panel).

[0098] Fig. 20.CFTR function in CF-iPSC-derived cholangiocytes was restored by treatment with small molecule correctors VX-809 and C4. Immunoblot analysis shows the accumulation of mature complex glycosylated forms of CFTR (band C) in CF-iPSC-derived cholangiocytes treated with VX-809 and C4. The mutant form of the protein (band B) is mainly in uncorrected cells. Human bronchial epithelial cells (HBE) are used as positive controls. (b) Representative confocal microscopy images of calcium green-labeled and forskolin / IBMX (F / I)-stimulated cyst structures generated by CF-iPSCs from two individual patients (997CFTRdel and C1CFTRdel-both of which carry delta F508 mutations). Images were taken 24 hours after F / I stimulation. Scale bar is 500 μm. (c) Quantification of the extent of swelling observed in hPSC-cysts 24 hours after F / I stimulation in the presence or absence of CFTR inhibitors. F / I stimulated cyst swelling was quantified using the Velocity Imaging software. The total size of cysts was normalized to before F / I stimulation in each of three separate experiments. *P<0.05, **P<0.01, ***P<0.001 (Scholar's t-test; n=3).

[0099] Fig.21 Intracellular flow cytometry analysis shows the proportion of ALB+ and CK19+ cells in the hepatocyte-derived population after 9 days of co-culture with OP9. Cells were cultured in medium containing HGF, EGF, and TGFb1 in the presence or absence of GSI. Ctrl shows isotype control.

[0100] Fig. 22 Hepatic specification and differentiation of hepatoblasts from other hPSCs. RT-qPCR analysis showed that Fig.14 a Expression of the indicated genes in HES2 and Y2-1 iPS cell-derived hepatoblasts as indicated. The expression of the indicated genes was analyzed on days 7, 13, 19 and 25 of culture. Values ​​were determined relative to TBP and presented as fold changes relative to expression in fetal liver (values ​​set to 1). AL: adult liver, FL: fetal liver.

[0101] Fig.23.3D gels were used to generate cystic structures from hPSC-derived cholangiocytes. (a) Schematic representation of the differentiation protocol used to generate chimeric aggregates consisting of day 25 hPSC-derived hepatoblasts and OP9 cells (GFP+). In low-cluster culture dishes, day 25 hepatoblasts were dissociated and co-cultured with OP9 cells at a ratio of 4:1. Chimeric aggregates were embedded in a gel consisting of a mixture of type I collagen (1.2 mg / ml) and matrigel (20%). (b) RT-qPCR-based expression analysis of structures developed in gels in the presence or absence of OP9 at day 44 of culture in medium containing HGF, EGF, and TGFb1. In the presence of OP9, the expression of Notch target genes was significantly upregulated. Values ​​were determined relative to TBP and are presented as fold changes relative to expression in cells cultured in the absence of OP9 (values ​​set to 1). (c) Histological analysis (H&E staining) of cystic structures developed from H9-derived cholangiocytes cultured with OP9 cells in the presence (right panel) or absence (left) of GSI at day 44 of culture. (d) Immunoblot analysis showing the presence of mature complex glycosylated forms of CFTR protein (band C) in structures generated from normal iPSC-derived cholangiocytes cultured in the presence or absence of OP9. Undifferentiated normal iPSCs were used as negative controls. (e) RT-qPCR-based expression analysis of CFTR in structures generated from normal iPSC-derived cholangiocytes cultured in the presence or absence of OP9. Cells were analyzed at day 44 of culture. Values ​​were determined relative to TBP and are presented as fold changes relative to expression values ​​detected in Caco-2 cells (intestinal colon cancer cell line) (values ​​set to 1). *P<0.05, **P<0.01, ***P<0.001 (scholar's t-test; n=3).

[0102] Fig.24 . Generation of definitive endoderm and hepatoblasts from iPSCs of patients with cystic fibrosis. Flow cytometry analysis shows the development of CXCR4+, CKIT+, and EPCAM+ populations in CF-iPS cells (C1 del CFTR) from day 7 of monolayer culture. (b) RT-qPCR analysis shows that the CXCR4+, CKIT+, and EPCAM+ populations are maintained at day 7 of monolayer culture. Fig.14 a Expression of the indicated genes in CF-iPSC-derived hepatoblast populations under the culture conditions outlined. The expression of the indicated genes was analyzed on days 7, 13, 19, and 25 of culture. Values ​​were determined relative to TBP and presented as fold changes relative to expression in fetal liver (values ​​set to 1). AL: adult liver, FL: fetal liver. DETAILED DESCRIPTION

[0103] This article describes a robust and reliable platform for efficiently generating hepatocytes and cholangiocytes from pluripotent stem cells (PSCs) and for generating metabolically functional hepatocytes and / or cholangiocytes through a series of steps described herein. For example, it is demonstrated that one or more extended nodal (e.g., activin) signaling treatments induce aggregation and activate cAMP signaling, for example in combination with FGF agonist induction and BMP4 agonist induction, optionally, combined with one or more steps to increase the expansion of specific cell populations and / or specific fate, thereby allowing reproducible generation of hepatocyte and cholangiocyte lineage cells, including, for example, expanded hepatocytes and / or using additional manipulations, from embryoid body induced definitive endoderm or from a single layer of functional and mature hepatocytes and cholangiocytes.

[0104] One aspect of the invention includes a method for preparing hepatocyte or cholangiocyte lineage cells, such as hepatoblasts, hepatocytes and / or cholangiocytes from a population of induced endoderm cells treated with a prolonged nodal agonist, the method comprising: (a) specifying a population of induced endoderm cells treated with a prolonged nodal agonist to obtain a cell population including hepatocytes and / or cholangiocyte progenitor cells by contacting the population of induced endoderm cells treated with a prolonged nodal agonist with a specialized culture medium, the specialized culture medium comprising a combination of an FGF agonist and a BMP4 agonist and / or their active conjugates and / or fragments; and (b) inducing maturation, further lineage specification and / or expansion of the hepatocytes and / or cholangiocyte progenitor cells of the cell population to obtain an expanded population of hepatocytes and / or a population including hepatocytes and / or cholangiocytes, the inducing maturation step comprising generating aggregates of the cell population.

[0105] Aggregation is demonstrated herein to be important for promoting maturation.

[0106] In embodiments, the hepatocytes and / or bile duct progenitor cells include hepatoblasts and / or immature hepatocytes and / or immature bile duct cells.

[0107] The term "contacting" (e.g., contacting a population of endoderm cells with one or more components) is intended to include incubating a component (or multiple components) and cells together in vitro (e.g., adding a compound to cultured cells) and the step of contacting can be performed in any suitable manner. For example, cells can be treated in adherent culture or in suspension culture, and the components can be added substantially simultaneously in time (e.g., together in a mixture) or sequentially (e.g., within 1 hour, 1 day or more from the addition of the first component). The cells can also be contacted with another agent or environment such as a growth factor or other differentiation agent to stabilize the cells, or further differentiate the cells, as well as culturing the cells under conditions known in the art, such as for culturing pluripotent (and / or differentiated) populations such as further described in the Examples.

[0108] As used herein, the terms "endoderm" and "definitive endoderm" refer to one of the three major germ cell layers in the very early embryo (the other two germ cell layers are mesoderm and ectoderm). The endoderm is the innermost layer of the three layers. Endoderm cells differentiate to first produce the embryonic intestine and then to produce derived tissues, including the esophagus, stomach, intestines, rectum, colon, pharyngeal pouch-derived tonsils, thyroid, thymus, parathyroid, lungs, liver, gall bladder, and pancreas.

[0109] As used herein, "induced endoderm cell population" refers to a cell population corresponding to Figure 1 A population of endoderm cells at the "definitive endoderm induction" stage as shown in a. This population can be prepared, for example, from embryoid bodies (EBs) exposed to a nodal agonist such as activin, or optionally from embryoid bodies (EBs) that have been exposed to a nodal agonist and a wnt / β-catenin agonist (such as Wnt3a) or a GSK-3 selective inhibitor (such as CHIR-99021 (Stemolecule TM CHIR99021 Stemgent), 6-bromoindirubin-3'-oxime (BIO) (Cayman Chemical (cat: 13123)) or Stemolecule from Stemgent TMBIO (cat: 04003)). Alternatively, the induced endoderm cell population can be prepared from cells grown in a monolayer. The induced endoderm cell population can be identified, for example, by flow cytometry and molecular analysis for one or more markers, such as surface markers CXCR4, CKIT and EPCAM and transcription factors SOX17 and FOXA2. The induced endoderm cell population can also be identified, for example, by at least or more than 70%, 80%, 90% or 95% of the population co-expressing CXCR4 and CKIT or CXCR4 and EPCAM. The induced endoderm cell population can also be identified, for example, by being greater than 70%, 80%, 90% or 95% of the population expressing SOX17 and / or FOXA2. The induced endoderm cell population can, for example, be in a 2D (monolayer) or 3D (embryoid or other forms of aggregates) format. The induced endoderm population can be derived from, for example, hESC and the induced pluripotent stem cells (iPSC) shown in Example 1.

[0110] The induced endoderm cell population is treated, for example, with a nodal agonist for an extended period of time to provide an extended nodal agonist-treated induced definitive endoderm cell population.

[0111] As described in Example 1 and Figure 3 As shown in a, culturing day 6 (day 5 when the method includes monolayer induction) cells for an additional 2 days in activin prior to specification with FGF / BMP4 resulted in a higher proportion of SOX17+FOXA2+ cells measured on day 12, compared to cells that were not cultured in activin for an additional 2 days (e.g., an example of a nodal agonist). This step is also referred to herein as "extended activin" treatment and is an example of a "extended nodal agonist" treatment.

[0112] As used herein, "prolonged nodal agonist treated induced endoderm cell population" refers to an induced endoderm cell population that has been treated with a nodal agonist such as activin for an extended period of time (e.g., from about 1 to about 4 days or an additional about 1, 2, 3 or 4 days (e.g., "extended period" may include, in addition to the endoderm induction period treated with a nodal agonist)). Prolonged nodal agonist treatment as shown herein results in higher levels of expression of genes specified for hepatoblast (hepatoblast) development, including HEX, AFP, ALB and HNF4a (e.g., at day 26 of culture) Figure 3 d). The extended nodal agonist-treated induced endoderm population is obtained by inducing endoderm cells in embryoid bodies (EBs) or by inducing endoderm cells in a monolayer, and wherein the induced endoderm population is cultured for an extended period of time in the presence of a nodal agonist (e.g., activin) to produce the extended nodal agonist-treated induced endoderm population.

[0113] In an embodiment, the induced endodermal cell population of the nodal agonist treatment is obtained by inducing the endodermal cells in embryoid bodies (EB). In another embodiment, the induced endodermal population of the nodal agonist treatment is obtained by inducing the endodermal cells of a monolayer. In each case, the time of the induced endodermal population extension is cultivated under the presence of a nodal agonist (e.g., activin).

[0114] Optionally, for example, in embodiments where the induced endoderm cell population is derived from EBs, the induced endoderm population is subsequently dissociated. As used herein, "dissociated cells" or "dissociated cell populations" refer to cells that are not in a 3D aggregate, e.g., physically separated from each other. Dissociated cells are different from "cell aggregates," which refer to clusters or clumps of cells.

[0115] In embodiments, the induced endoderm population comprises at least 80%, 85%, 90% CXCR4+ and CKIT+ positive cells and / or at least 70%, 75%, 80% SOX17+ cells.

[0116] In some embodiments, the induced endoderm cell population (and / or the induced endoderm cell population treated with a prolonged nodal agonist) is generated from a pluripotent stem cell (PSC) such as an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC). The pluripotent stem cell is optionally a human ESC (hESC) or a human iPSC (hiPSC).

[0117] As used herein, the term "pluripotent" refers to cells that have the ability to differentiate into more than one differentiated cell type under different conditions, and, for example, the ability to differentiate into cell types characteristic of the three germ cell layers. Pluripotent cells are characterized by their ability to differentiate into more than one cell type, for example, using nude mouse teratoma formation assays. Pluripotency is also reflected in the expression of embryonic stem (ES) cell markers.

[0118] The term "progenitor cell" refers to a cell having a cellular phenotype that is at an earlier stage along a developmental pathway or progression than a fully differentiated cell relative to a cell that can be generated by differentiation. A progenitor cell can give rise to multiple different differentiated cell types or a single differentiated cell type, depending on the developmental pathway and the environment in which the cell develops and differentiates.

[0119] The term "stem cell" as used herein refers to an undifferentiated cell that is capable of proliferation, self-renewal, and production of more progenitor cells that are capable of generating a large number of mother cells that in turn can produce differentiated or differentiable daughter cells. Daughter cells can, for example, be induced to proliferate and produce offspring that subsequently differentiate into one or more mature cell types while also retaining one or more cells with the developmental potential of their parents.

[0120] The term "embryonic stem cell" is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see, e.g., U.S. Patent Nos. 5843780, 6200806). Such cells can also be obtained from the inner cell mass of the blastocyst derived from somatic cell nuclear transfer (see, e.g., U.S. Patent Nos. 5945577, 5994619, 6235970). The distinguishing features of embryonic stem cells define the embryonic stem cell phenotype. Therefore, if the cell has one or more unique features of embryonic stem cells so that the cell can be distinguished from other cells, the cell has the phenotype of embryonic stem cells. Exemplary distinguishing features of embryonic stem cells include but are not limited to gene expression profiles, proliferation capacity, differentiation capacity, karyotype, responses to specific culture conditions, etc.

[0121] In one embodiment, a method for generating hepatocytes and / or cholangiocytes from an induced endoderm cell population treated with a prolonged nodal agonist comprises: (a) specializing the induced endoderm cell population treated with a prolonged nodal agonist into a cell population comprising hepatocytes and / or cholangiocytes by contacting the induced endoderm cell population with a specialized medium comprising an FGF agonist and a BMP4 agonist and / or active conjugates and / or fragments thereof.

[0122] In an embodiment, the specialization step includes contacting the induced endoderm population of the extended nodal agonist treatment with a specialized culture medium comprising an FGF agonist and BMP4. For example, the FGF agonist can be bFGF, FGF10, FGF2 or FGF4, their active fragments and / or their combinations. For example, the combination can be added to the cell in sequence.

[0123] In an embodiment, the specification step comprises: first contacting the induced endoderm population treated with a prolonged nodal agonist with a specification medium comprising FGF10 and BMP4 for about 40 hours to about 60 hours, for example, about 40 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours, 50 hours, 52 hours, 54 hours, hours, 56 hours, 58 hours or about 60 hours, and then contacting the induced endoderm population treated with a prolonged nodal agonist with a specification medium comprising bFGF and BMP4 for about 4 days to about 7 days, for example, about 4 days, 5 days, 6 days or about 7 days.

[0124] In one embodiment, the specialized culture medium includes Iscove's modified Dulbecco's medium (IMDM), IMDM supplemented with 1% volume / volume B27 supplement (Invitrogen: A11576SA), ascorbic acid, MTG, FGF10 (50 ng / ml) (e.g., from day 8 to day 10), bFGF (20 ng / ml) (e.g., from day 10 to day 14), and BMP4 (50 ng / ml).

[0125] Optionally, the endoderm cell population is contacted with FGF10 and BMP4 for 1 to 3 days, optionally, 2 days, and subsequently with bFGF and BMP4 for 2 to 6 days, optionally, 3 to 5 days, optionally, 4 days. In some embodiments, the endoderm cell population is incubated in a cell culture medium comprising BMP4 and FGF10 or bFGF. Optionally, the endoderm cell population is incubated in a cell culture medium comprising Iscove's modified Dulbecco's medium (IMDM), and IMDM is supplemented with 1% volume / volume B27, ascorbic acid, thioglycerol, BMP4 and FGF10 or bFGF.

[0126] In some embodiments, a population of endoderm cells is dissociated and the monolayer of cells is then contacted with FGF and a BMP4 agonist.

[0127] In other embodiments, the monolayer of cells is contacted with activin for 1 to 4 days, optionally 1 day, 2 days, 3 days or 4 days prior to contact with FGF and a BMP4 agonist such as BMP4. Optionally, the monolayer of cells is incubated in a cell culture medium comprising activin A, optionally the culture medium comprises StemPRO-34 supplemented with bFGF, activin A and BMP4.

[0128] In further embodiments, the specification step comprises contacting the cells with a specialized medium comprising one or more factors that promote maturation, further lineage specification, and / or expansion.

[0129] As used herein, the term "specialized culture medium" refers to a culture medium for promoting or facilitating the specialization of cells or cell groups. An example of a liver-specialized culture medium includes Iscove's modified Dulbecco's medium (IMDM), which is supplemented with 1% volume / volume B27 (Invitrogen: A11576SA) and ascorbic acid, MTG, BMP4 agonist, and at least one FGF agonist selected from FGF10, bFGF, FGF4, and FGF2. For some stages and in some embodiments, for example, the same specialized culture medium can be used for specialized hepatocytes and cholangiocyte lineages. At other stages and in other embodiments, the specialized culture medium includes one or more factors that promote the specialization of hepatocytes and / or the development of cholangiocytes, for example, notch antagonists or notch agonists. The term "specialization" used herein refers to the process by which cells evolve toward a specific cell fate, before which the cell type has not yet been determined, and any bias toward a cell of a specific fate can be reversed or converted to another fate. Specialization causes the fate state of the cell to be unable to change under typical conditions.

[0130] For example, the specification of induced endoderm along the hepatic fate can be confirmed by measuring genes expressed by hepatic and / or cholangiocytes, for example, genes expressed by hepatic and / or cholangiocytes include Tbx3 ALB, AFP, CK19, Sox9, NHF6β and Notch2 as shown in Example 9. For example, it was demonstrated that the expression of Notch2 was upregulated in HGF / DEX / OSM treated hepatic blasts. Detection and / or expression of Notch2 protein can be used to confirm that the cell population is specialized to cholangiocytes, for example, Notch2 can be detected as described in Example 9.

[0131] In the context of cells, the terms "differentiated" or "differentiation" are relative terms, and a "differentiated cell" is a cell that has progressed further down the developmental pathway than the cell to which it is being compared. Thus, a stem cell can differentiate into a lineage-restricted precursor cell (such as an induced endoderm progenitor cell), which in turn can differentiate into other types of precursor cells, further down the pathway, and then mature into a final stage functional cell that plays a key role in a specific tissue type and may or may not retain the ability to proliferate further. The term "differentiation" as used herein includes steps for generating induced germ layer populations and specialized cell populations (e.g., hepatocyte or cholangiocyte specialized cell populations).

[0132] In another embodiment, aggregates are generated from a cell population comprising at least 70%, 80%, 85%, or at least 90% albumin-positive cells. In another embodiment, aggregates are generated after 24, 25, 26, 27, or 28 days of culture (e.g., the day on which PSCs are obtained is considered day 0).

[0133] Optionally, aggregates are generated by enzyme treatment and / or artificial dissociation. In certain embodiments, aggregates are generated by dissociating cells with collagenase and / or TrypleLE. In certain embodiments, cells are subsequently cultured in ultra-low cluster dishes. In other embodiments, monolayer cultures can be mechanically separated by a pipette, or enzymatically dissociated and aggregated in low attachment plates for incubation, or mechanically separated by shaking cell clusters. Aggrewells can be optionally used.

[0134] In an embodiment, cells (e.g., cells before monolayer and aggregation) are grown on a matrix-coated plate, optionally on a matrigel-coated plate. Other matrix-coated plates that support the attachment of hepatoblasts, hepatocytes, and / or cholangiocytes, such as laminin, fibronectin, and collagen-coated plates, may also be used. For example, Fig. 9 f shows ALB expression at day 26 of induction using several different matrix-coated substrates.

[0135] Inducing maturation, further lineage specification and / or expansion may comprise one or more sub-steps.

[0136] In a further embodiment, a cell population comprising hepatocytes and / or bile duct progenitor cells and / or aggregates is cultured in the presence of hepatocyte growth factor (HGF), dexamethasone (DEX) and / or oncostatin M (OSM) and / or their active conjugates and / or fragments. For example, before aggregation, a cell population comprising hepatocytes and / or bile duct progenitor cells can be cultured in a maturation medium comprising HGF, DEX and / or OSM for about 10 days, 11 days, 12 days, 13 days or 14 days, and / or after aggregation, the aggregates can be cultured in a maturation medium comprising HGF, DEX and / or OSM for about 6 days, 7 days, 8 days, 9 days or 10 days. For example, the addition of about 10 ng / mL of HGF promotes the survival of the aggregates.

[0137] In one embodiment, inducing maturation and optionally inducing further lineage specification and / or expansion further comprises activating a cAMP pathway within cells of the aggregate to induce differentiation and / or maturation of hepatocytes and cholangiocytes progenitor cells into hepatocytes and / or cholangiocytes.

[0138] Prolonged nodal agonist treatment and aggregation of cells (e.g., monolayer induced cells at day 25 and EB induced cells at day 26) produce populations that, for example, are able to respond to cAMP signaling. As shown herein, activation of cAMP increases CYP expression and hepatocyte maturation.

[0139] In another embodiment, activating the cAMP pathway comprises contacting the aggregate with a cAMP agonist analog, such as 8-bromoadenosine-3'5"-cyclic monophosphate (8-Br-cAMP), dibutyryl-cAMP, adenosine-3',5'-cyclic monothiophosphate, Sp-isomer (SP-cAMPS) and / or 8-bromoadenosine-3',5'-cyclic monothiophosphate, Sp-isomer (Sp-8-Br-cAMPS)) and / or any other cAMP agonist, such as cholera toxin, forskolin, caffeine, theophylline, and pertussis toxin. For example, SP-8-Br-cAMP (Biolog: Cat. No.: B002 CAS No.: [127634-20-2]), 8-Br-cAMP and forskolin (FSK) (Sigma: 66575-29-9) were used for experiments. For example, the combination of forskolin (FSK) (Sigma: 66575-29-9) + XAV939 + PD0325901 effectively increases CYP expression and induces liver progenitor cells to mature into hepatocytes. "CAMP agonists" as used herein include cAMP, cAMP analogs that activate cAMP, and molecules that activate cAMP, such as cholera toxin, forskolin, caffeine, theophylline, and pertussis toxin. In some embodiments, IBMX, which is a phosphodiesterase inhibitor (phosphodiesterase is an inhibitor of cAMP), can also be used, for example, in combination with forskolin.

[0140] It has been found, also for example, that the addition of 10 ng / ml of HGF (reduced from 20 ng / ml) promotes the survival of aggregates, while the maintenance of OSM has an inhibitory effect on the induction of expression of phase 1 CYP enzymes, particularly CYP 3A4. Thus, in some embodiments, aggregates are incubated with cAMP analogs and / or agonists in the absence of OSM.

[0141] The term "maturation" as used herein refers to the process by which a cell (e.g., a hepatoblast) becomes more specialized and / or achieves a fully functional state (e.g., its functional state in vivo). In one embodiment, the process by which immature hepatocytes or hepatic progenitor cells become mature, functional hepatocytes are referred to as mature.

[0142] Figure 1 a refers to "liver maturity A" and Fig.14a refers to the differentiation of hepatoblasts. The cell populations mentioned in both cases are cell populations of hepatoblasts, which can generate hepatocytes if cultured, for example, in the presence of DEX (optionally in combination with HGF and OSM and / or cAMP), or bile duct cells if cultured in combination with Notch agonists (e.g., Notch signal donors) (such as OP9, OP9delta and / or OP9 Jagged1 cells) in the presence of EGF, TGB1, HGF and EGF.

[0143] The term "maturation medium" as used herein refers to a medium for promoting or facilitating the maturation of a cell or cell population, and it may include maturation factors as well as cell expansion inducers and lineage inducers. An example of a maturation medium for inducing hepatocyte maturation includes Iscove's modified Dulbecco's medium (IMDM), IMDM supplemented with 1% volume / volume B27 (Invitrogen: A11576SA) and ascorbic acid, glutamine, MTG and optionally hepatocyte growth factor (HGF), dexamethasone (Dex) and / or oncostatin M. Another example of a maturation medium for inducing hepatocytes includes hepatocyte medium (HCM) without EGF (Lonza: CC-4182). The maturation medium optionally also includes cAMP analogs and / or cAMP agonists, which, for example, induce the expansion of the hepatocyte lineage and the maturation of the hepatocyte lineage. The maturation medium may include factors that promote hepatocyte and / or cholangiocyte development, further lineage specialization and / or expansion and / or further lineage selection. For example, Wnt antagonists alone or in combination with TGFβ antagonists and / or MEK / Erk antagonists promote hepatocyte maturation. For example, Notch agonists are shown herein to induce cholangiocyte lineage development and are added when cholangiocytes are desired. Similarly, Notch antagonists promote hepatocyte lineages and can be added when hepatocytes are desired, for example, to inhibit cholangiocyte development.

[0144] Different maturation media can be used sequentially (e.g., monolayer maturation medium (e.g., used before aggregation), aggregate maturation medium (used after aggregation); for example, hepatocyte maturation medium includes factors that promote hepatocyte development, while cholangiocyte maturation medium, for example, promotes cholangiocyte development.

[0145] For example, in an embodiment, the maturation medium comprises a cAMP analog and / or a cAMP agonist and DEX, and HGF is optionally added to the aggregates, and the pre-aggregation population is subsequently cultured in the maturation medium comprising HGF, DEX and OSM for, e.g., about 10 days, 11 days, 12 days, 13 days or 14 days.

[0146] As used herein, the term "hepatocyte" refers to parenchymal liver cells. Hepatocytes make up most of the cytoplasmic mass of the liver and are involved in protein synthesis and storage, carbohydrate metabolism, synthesis of cholesterol, bile salts and phospholipids, and detoxification, modification and excretion of exogenous and endogenous substances.

[0147] The term "primary hepatocytes" as used herein are hepatocytes that are taken directly from living tissue (eg, biopsy material) and established for growth in vitro.

[0148] As used herein, the term "hepatoblast" refers to a progenitor cell with the ability to differentiate into a cell of a hepatocyte and cholangiocyte lineage (e.g., hepatocyte or cholangiocyte). Hepatoblasts are subsets of progenitor cells such as hepatocytes and cholangiocytes, and hepatoblasts may include immature hepatocytes and immature cholangiocytes (e.g., cells with a specific cell fate and that can only mature into hepatocytes or only mature into cholangiocytes). In certain embodiments, hepatoblasts are defined by the expression of markers, such as Hex, HNF4, alpha-fetoprotein (AFP) and albumin (ALB). For example, when Notch signaling is activated in a culture comprising hepatoblasts on the 28th day, hepatoblasts can produce cholangiocytes (e.g., CK19+ cells). As used herein, the term "liver progenitor cell" means a cell with the ability to differentiate into a functional hepatocyte, and a functional hepatocyte is, for example, albumin-positive and / or expresses a CYP enzyme.

[0149] As used herein, the term "biliary progenitor cell" means a cell that has the ability to differentiate into a functional bile duct cell, for example, a cell that is CK19 positive and / or expresses CFTR.

[0150] As used herein, the term "immature hepatocytes" refers to cells of the hepatocyte lineage that express albumin but do not express significant levels of functional CYP3A4 and / or CYP1A2 enzymes. In some embodiments, immature hepatocytes must be matured to obtain the function of mature hepatocytes. In some embodiments, immature hepatocytes are defined by the expression of markers such as Hex, alpha-fetoprotein, and albumin.

[0151] As used herein, "mature hepatocytes" refer to hepatocyte lineage cells that express CYP enzymes (e.g., CYP3A4 and CYP1A2) and albumin. Optionally, mature hepatocytes include functional or measurable levels of metabolic enzymes, such as phase I and phase II drug metabolizing enzymes equivalent to adult cells. Examples of phase I drug metabolizing enzymes include, but are not limited to, cytochrome P450 CYP1A2, CYP3A4, and CYP2B6. Examples of phase II drug metabolizing enzymes include, but are not limited to, arylamine N-acetyltransferases NAT1 and NAT2 and UDP glucuronyltransferases UGT1A1. For example, mature hepatocytes can be metabolically active hepatocytes. Cellular uptake of indocyanine green (ICG) is considered to be a characteristic of adult hepatocytes 29 and is used clinically as a test substrate to evaluate liver function. 3 0. For example, mature hepatocytes are ICG positive staining hepatocytes. In a population of hepatocytes, 50%, 60%, 70%, 80%, 90% or more of the hepatocytes are ICG, and the hepatocyte population can be considered mature. Mature hepatocytes express increased albumin compared to "immature hepatocytes", for example, at least 5%, 10%, 25%, 50%, 75%, 100% or 200% more albumin than immature hepatocytes.

[0152] In embodiments, the hepatocytes are functional hepatocytes.

[0153] As used herein, the term "functional hepatocyte" refers to a hepatocyte that displays one or more characteristics of an adult hepatocyte (e.g., a mature hepatocyte) and / or an immature hepatocyte that is committed to a hepatic fate and is more differentiated than a starting cell (e.g., compared to an endoderm cell population, a hepatocyte precursor, or an immature hepatocyte), for example, expressing albumin and / or increased albumin compared to a starting cell. Optionally, a functional hepatocyte is a mature hepatocyte and includes functional or measurable levels of metabolic enzymes equivalent to adult cells, such as phase I and phase II drug metabolizing enzymes. For example, a functional hepatocyte can be a metabolically active hepatocyte. Cellular uptake of indocyanine green (ICG) is considered a characteristic of adult hepatocytes. 29 And it is used clinically as a test substrate to evaluate liver function 30Functional hepatocytes are, for example, ICG-positive stained hepatocytes. In a population of hepatocytes, for example, at least 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or more of the hepatocytes are ICG-positive, then the population of hepatocytes can be considered to be a functional population of hepatocytes. In another example, the functional hepatocytes are albumin-secreting hepatocytes, and if, for example, at least 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or more of the hepatocytes are albumin-secreting, then the population of hepatocytes can be considered to be a functional population of hepatocytes.

[0154] In embodiments, the hepatocytes, optionally functional hepatocytes, comprise increased expression of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes or proteins selected from the group consisting of ALB, CPS1, G6P, TDO, CYP2C9, CYP2D6, CYP7A1, CYP3A7, CYP1A2, CYP3A4, CYP2B6, NAT2, and UGT1A1, compared to a cell population comprising hepatocytes and / or bile duct progenitor cells, and / or hepatocytes generated from an induced endoderm population treated with a non-prolonged nodal agonist, without aggregation and / or cAMP signaling induction. In other embodiments, at least 40%, 50%, 60%, 70%, 80% or 90% of the hepatocytes, optionally functional hepatocytes, are ASGPR-1+ cells.

[0155] In some embodiments, the functional hepatocytes exhibit nucleic acid or protein levels of CYP1A2, CYP2B6, CYP3A4, CYP2C9 and / or CYP2D6 that are equivalent to or greater than those found in primary mature hepatocytes, optionally, at least 1.1-fold, 2-fold, 3-fold, 4-fold or 5-fold or any 0.1 increment between 1.1-fold and 5-fold, optionally, at least 50% to 100%, 75% to 125%, 85% to 115%, 90% to 110% or 95% to 105%. For example, it is an increased expression of 1.15-fold to 6.1-fold found in primary hepatocytes, (e.g., CYP1A2 6.1-fold (610%), CYP3A4 13.2-fold (1320%), CYP 2B6 2-fold (200%), CYP2C9 1.52-fold 152%, UGT1A1 2-fold (200%), CYP2D6 1.15-fold (115%)). In some embodiments, the hepatocytes exhibit nucleic acid or protein levels of ALB, HNF4, AFP, CPS1, G6P, TDO1, NAT1, NAT2 and / or UGT1A1 that are equivalent to or higher than those found in primary hepatocytes of similar stage, optionally, at least 50% to 100%, 75% to 125%, 85% to 115%, 90% to 110% or 95% to 105% of those found in primary hepatocytes.

[0156] In other embodiments, the functional hepatocytes exhibit nucleic acid or protein levels of CYP1A2, CYP2B6, CYP3A4, CYP2C9 and / or CYP2D6 that are higher than those in hepatoblasts and / or immature hepatocytes, optionally, levels of at least 110%, 125%, 150%, 175%, 200%, 300%, 400% or 500% of those found in primary hepatocytes. In some embodiments, the functional hepatocytes exhibit nucleic acid or protein levels of ALB, CPS1, G6P, TDO1, NAT1, NAT2 and / or UGT1A1 that are higher than those in hepatoblasts and / or immature hepatocytes, optionally, levels of at least 110%, 125%, 150%, 175%, 200%, 300%, 400% or 500% of those found in primary hepatocytes.

[0157] In other embodiments, the functional hepatocytes express the receptor asialoglycoprotein receptor 1 (ASGPR1). In other embodiments, at least 40%, 50%, 60%, 70%, 80% or 90% of the hepatocytes, optionally functional hepatocytes, are ASGPR-1+ cells.

[0158] In further embodiments, the functional hepatocytes exhibit CYP1A2 activity in vitro. Optionally, the functional hepatocytes exhibit CYP1A2 activity equivalent to or greater than that found in primary hepatocytes, optionally at levels of at least 50% to 100%, 75% to 125%, 85% to 115%, 90% to 110%, or 95% to 105% of those found in primary hepatocytes. In some embodiments, CYP1A2 activity is measured by incubating cells with phenacetin and monitoring the generation of accumulated ortho-diethylated metabolites in the cells.

[0159] In further embodiments, the functional hepatocytes exhibit CYP2B6 activity in vitro. Optionally, the functional hepatocytes exhibit CYP2B6 activity that is equivalent to or greater than that found in primary hepatocytes, optionally at levels of at least 50% to 100%, 75% to 125%, 85% to 115%, 90% to 110%, or 95% to 105% of those found in primary hepatocytes. In some embodiments, CYP2B6 activity is measured by incubating cells with bupropion and monitoring the formation of the metabolite o-hydroxybupropion in the cells.

[0160] In further embodiments, the hepatocytes exhibit NAT1 and / or NAT2 activity in vitro. Optionally, the hepatocytes exhibit NAT1 and / or NAT2 activity that is equivalent to or greater than that found in primary hepatocytes, optionally at least 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, or about 50% to 100%, 75% to 125%, 85% to 115%, 90% to 110%, or 95% to 105% of that found in primary hepatocytes. In some embodiments, NAT1 and / or NAT2 activity is indicated by the metabolism of sulfadimethazine (SMZ) to N-acetyl SMZ.

[0161] In further embodiments, the hepatocytes exhibit UGT activity in vitro. Optionally, the hepatocytes exhibit UGT activity comparable to or greater than that found in primary hepatocytes, optionally at levels of at least 50% to 100%, 75% to 125%, 85% to 115%, 90% to 110%, or 95% to 105% of those found in primary hepatocytes. In some embodiments, UGT activity is indicated by the generation of 4-MU glucuronide (4-MUG) from 4-methylumbelliferone (4-MU) in the cells.

[0162] In embodiments, the hepatocytes, optionally functional hepatocytes, comprise at least one, two, three, four, five, six, seven, eight, nine, ten or more genes or proteins selected from the group consisting of ALB, CPS1, G6P, TDO, CYP2C9, CYP2D6, CYP7A1, CYP3A7, CYP1A2, CYP3A4, CYP2B6, NAT2 and UGT1A1, compared to a cell population comprising hepatocytes and / or bile duct progenitor cells, and / or hepatocytes generated from an induced endoderm cell population treated with a non-prolonged nodal agonist, without aggregation and / or cAMP signaling induction. In other embodiments, at least 40%, 50%, 60%, 70%, 80% or 90% of the hepatocytes, optionally functional hepatocytes, are ASGPR-1+ cells.

[0163] In yet another embodiment, the functional hepatocytes show a global gene expression profile indicating hepatocyte maturation. Optionally, the functional hepatocytes show a global gene expression profile that is more similar to that of primary hepatocytes than the global gene expression profile of hepatoblasts and / or immature hepatocytes. The global gene expression profile is obtained by any method known in the art, e.g., microarray analysis.

[0164] In an embodiment, the fate of cholangiocytes is specified by treating aggregates of a population of cells with a notch agonist.

[0165] The term "cholangiocyte" as used herein refers to cells that form the bile ducts.

[0166] As used herein, the term "cholangiocyte precursor" refers to cells that have the ability to differentiate into cholangiocytes (e.g., hepatoblasts) and immature cholangiocytes that can mature into functional cholangiocytes. In some embodiments, cells of the cholangiocyte lineage are defined by the expression of markers such as CK19, pancreatic juice receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and bicarbonate chloride anion exchanger 2 (Cl(-) / HCO(3)(-)AEs).

[0167] As used herein, the term "immature cholangiocyte" refers to a cholangiocyte lineage cell that must undergo maturation to obtain the function of a mature cholangiocyte. In some embodiments, the immature cholangiocyte expresses CK19 and / or Sox9, optionally including cells treated with an early Notch agonist, optionally treated for at least 1 day, at least 2 days, at least 3 days, or at least 4 days.

[0168] In embodiments, the cholangiocytes are functional cholangiocytes.

[0169] As used herein, the term "functional cholangiocyte" refers to a cholangiocyte that exhibits one or more characteristics of an adult cholangiocyte (e.g., a mature cholangiocyte) and / or a cholangiocyte lineage cell that expresses CK-19, MDR1, and / or CFTR. For example, a functional cholangiocyte expresses the MDR1 transporter and can transport a tracer dye such as rhodamine 123 into the luminal space of the structure when located in a cystic structure. As another example, Fig.18 As shown, the functional activity of CFTR can be evaluated, for example, using a forskolin-induced swelling assay on cystic structures. In a population of cholangiocytes, a population can be considered functional if, for example, at least 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or more of the cells express the secretin receptor (SR), the cystic fibrosis transmembrane conductance regulator (CFTR), CK-19 and / or the chloride bicarbonate anion exchanger 2 (Cl(-) / HCO(3)(-)AEs).

[0170] The term "mature cholangiocyte" as used herein is a cholangiocyte that expresses specific transporter or cell membrane receptor activity, such as the secretin receptor (SR), the cystic fibrosis transmembrane conductance regulator (CFTR) and optionally the chloride bicarbonate anion exchanger 2 (Cl(-) / HCO(3)(-)AEs).

[0171] In an embodiment, the population of the cholangiocytes produced is a population of functional cholangiocytes.Compared with cells of a cell population including hepatocytes and cholangiocyte progenitor cells and / or compared with population cells produced from aggregates never treated with notch agonists, functional cholangiocytes include, for example, at least 1, at least 2 or 3 genes or protein increases selected from Sox9, CK19 and CFTR (cystic fibrosis transmembrane conductance regulator).In other embodiments, at least 40%, 50%, 60%, 70%, 80% or 90% of the population of cholangiocytes are CK19+ cholangiocytes.In other embodiments, at least 40%, 50%, 60%, 70%, 80% or 90% of functional cholangiocytes are CFTR+ cholangiocytes.

[0172] The term "expression" refers to the cellular processes involved in producing RNA and protein and the appropriate secretion of the protein, including applicable but not limited to, such as transcription, translation, folding, modification and processing."Expression products" include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene.

[0173] The term "cell culture medium" (also referred to herein as "culture medium" or "medium") refers herein to a medium for culturing cells containing nutrients that maintain cell viability and support proliferation and optional differentiation. The cell culture medium may contain any suitable combination of salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum replacements, and other ingredients such as peptide growth factors, vitamins, etc. The cell culture medium typically used for a particular cell type is well known to those skilled in the art.

[0174] Suitable culture media may include a suitable basal medium including, for example, DMEM (Life Technologies), IMDM, RPMI, CMRL and / or any other medium that supports the growth of endoderm cells to provide, for example, basal medium components, to which ingredients and optionally other agents may be added.

[0175] Various days of culture are referred to herein. Those skilled in the art will recognize that the culture period can vary.

[0176] In certain embodiments, " the 5th day " as used herein generally refers to the endodermal cell group of the induction derived from, for example, a PSC monolayer. The endodermal cell group derived from the induction of EB is cultured for about 6 days, to arrive at a similar culture point that needs to be processed for about 24 hours to induce EB to form. Therefore, the 7th day monolayer induction culture is equivalent to the 8th day embryoid body induction culture, etc. At this stage, the induced endodermal population can include cells expressing, for example, Foxa2 and SOX17. Similarly, " the 7th day " generally refers to the induced endodermal population that has been extended nodal agonist treatment for two days (for example, this will be the 8th day of EB method). " the 25th day " generally refers to the stage of cell aggregation (if derived from a monolayer, or if derived from the 26th day of EB). Wherein, using monolayer cells, using EB can use equivalent methods, and the culture period is generally delayed by 1 day. Fig.11 An example of a schedule using monolayer cells is provided ( Fig.11 A) and examples using EB ( Fig.11 B). Fig.11 C and Fig.14 An example timeline for generating cholangiocytes is provided in A.

[0177] As used herein, the term "FGF agonist" refers to molecules such as cytokines, including, for example, FGF or small molecules that activate FGF signaling pathways (e.g., bind and activate FGF receptors). For example, FGF receptor activation can be assessed by measuring the activity of MEK / ERK, AKT and / or PI3K by immunoassay.

[0178] As used herein, the term "FGF" refers to any fibroblast growth factor, for example, human FGF1 (gene ID: 2246), FGF2 (also known as bFGF; gene ID: 2247), FGF3 (gene ID: 2248), FGF4 (gene ID: 2249), FGF5 (gene ID: 2250), FGF6 (gene ID: 2251), FGF7 (gene ID: 2252), FGF8 (gene ID: 2253), FGF9 (gene ID: 2254) and FGF10 (gene ID: 2255), optionally including their active conjugates and fragments, including naturally occurring active conjugates and fragments. In specific embodiments, FGF is FGF10, FGF4 and / or FGF2.

[0179] As used herein, "active conjugates and fragments of FGF" include conjugates and fragments of fibroblast growth factor that bind to and activate FGF receptors and optionally activate FGF signaling.

[0180] The concentration of FGF can be, for example, in the range of from about 1 ng to about 500 ng / ml, for example, from about 1 ng to about 250 ng / ml, from about 10 ng to about 250 ng / ml, from about 10 ng to about 100 ng / ml. In another embodiment, the concentration of FGF is about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 150 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, or about 500 ng / ml.

[0181] The concentration of FGF10 can be, for example, in the range of from about 1 ng to about 500 ng / ml, for example, from about 1 ng to about 250 ng / ml, from about 10 ng to about 250 ng / ml, from about 10 ng to about 100 ng / ml. In another embodiment, the concentration of FGF is about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 150 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml or about 500 ng / ml.

[0182] The concentration of bFGF can be, for example, in the range of from about 1 ng to about 500 ng / ml, for example, from about 1 ng to about 250 ng / ml, from about 10 ng to about 250 ng / ml, from about 10 ng to about 100 ng / ml. In another embodiment, the concentration of FGF is about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 150 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, or about 500 ng / ml.

[0183] In an embodiment, the BMP4 agonist is selected from the group BMP4, BMP2 and BMP7. For example, BMP4, BMP7 and BMP2 share the same receptors in embryonic development.

[0184] The term "BMP4" as used herein (e.g., Gene ID: 652) refers to bone morphogenetic protein 4, e.g., human BMP4, and active conjugates and fragments thereof, optionally including naturally occurring active conjugates and fragments that can activate BMP4 receptor signaling. The concentration of BMP (e.g., BMP4) can be, for example, in the range of from about 1 ng to about 500 ng / ml, e.g., from about 1 ng to about 250 ng / ml, from about 10 ng to about 250 ng / ml, from about 10 ng to about 100 ng / ml. In another embodiment, the concentration of BMP (e.g., the concentration of BMP4) is about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 150 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml or about 500 ng / ml.

[0185] As mentioned, this method can be applied to populations of endoderm cells grown in a monolayer.

[0186] Thus, further aspects include a method of generating hepatocytes and / or cholangiocytes from a population of pluripotent stem cells, the method comprising:

[0187] a) contacting pluripotent stem cells cultured as a monolayer with an induction medium comprising a nodal agonist such as ActA and optionally a Wnt / β-catenin agonist (such as i) Wnt3a and / or ii) a GSK-3 selective inhibitor such as CHIR-99021) to provide an induced endoderm cell population;

[0188] b) contacting the induced endoderm cell population with a nodal agonist to provide a prolonged nodal agonist-treated induced endoderm cell population; and

[0189] c) specializing to obtain a cell population comprising hepatocytes and / or bile duct progenitor cells by contacting the prolonged nodal agonist-treated induced endoderm cell population with a specialization medium comprising an FGF agonist and a BMP4 agonist and / or active conjugates and / or fragments thereof,

[0190] d) optionally contacting the cell population comprising hepatocytes and / or bile duct progenitor cells with a maturation medium comprising HGF, dexamethasone and / or oncostatin M and / or active conjugates and / or fragments thereof;

[0191] e) inducing maturation, and optionally inducing further lineage specification and / or expansion of the hepatocyte and biliary progenitor cells of the cell population to hepatocytes and / or biliary cells, the step of inducing maturation comprising generating aggregates of the cell population.

[0192] In addition, the endoderm population may also be contained in embryoid bodies. Thus, further aspects include a method for generating hepatocytes and / or bile duct cells from a population of pluripotent stem cells, the method comprising:

[0193] a) forming embryoid bodies (EBs) of pluripotent stem cells, optionally by contacting the pluripotent stem cells with a BMP4 agonist;

[0194] b) contacting the EBs with an induction medium comprising a nodal agonist such as ActA and optionally a Wnt / β-catenin agonist (such as i) Wnt3a and / or ii) a GSK-3 selective inhibitor such as CHIR-99021) to provide an induced endoderm cell population;

[0195] c) dissociating the induced endoderm cell population to provide a dissociated induced endoderm cell population;

[0196] d) contacting the dissociated induced endoderm cell population with a nodal agonist to provide a prolonged nodal agonist-treated induced endoderm cell population;

[0197] e) specializing to obtain a cell population comprising hepatocytes and / or bile duct progenitor cells by contacting the prolonged nodal agonist-treated induced endoderm cell population with a specialized medium comprising an FGF agonist and a BMP4 agonist and / or active conjugates and / or fragments thereof,

[0198] f) optionally contacting the cell population comprising hepatocytes and / or bile duct progenitor cells with a maturation medium comprising HGF, dexamethasone and / or oncostatin M and / or active conjugates and / or fragments thereof; and

[0199] g) inducing maturation, further lineage specialization and / or expansion of hepatocytes and / or bile duct progenitor cells of the cell population to hepatocytes and / or bile duct cells, inducing maturation, further lineage specialization and / or expansion including generating aggregates of the cell population. In some embodiments, the inducing maturation and optionally inducing further lineage specialization and / or expansion steps also include activating the cAMP pathway within the aggregate to induce the maturation of hepatocytes and / or bile duct progenitor cells of the cell population into a population including hepatocytes and / or bile duct cells. In an embodiment, the method includes contacting the aggregate with a cAMP analog and / or a cAMP agonist.

[0200] Another aspect includes a method of generating functional hepatocytes and / or cholangiocytes from a pluripotent stem cell (PSC), such as an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC), the method comprising:

[0201] a) contacting pluripotent stem cells cultured as a monolayer for forming embryoid bodies with an induction medium comprising a nodal agonist such as ActA and optionally a Wnt / β-catenin agonist (such as i) Wnt3a and / or ii) a GSK-3 selective inhibitor such as CHIR-99021) to provide an induced endoderm cell population;

[0202] b) contacting the induced endoderm cell population with a nodal agonist to provide a prolonged nodal agonist-treated induced endoderm cell population;

[0203] c) specializing to obtain a cell population comprising hepatocytes and / or bile duct progenitor cells by contacting the prolonged nodal agonist-treated induced endoderm cell population with a specialization medium comprising at least one FGF agonist and one BMP4 agonist and / or active conjugates and / or fragments thereof, and

[0204] d) inducing maturation, further lineage specification and / or expansion of hepatocytes and / or cholangiocytes and / or cholangiocytes, wherein the induction of maturation, further lineage specification and / or expansion comprises:

[0205] (i) culturing a cell population comprising hepatocytes and / or bile duct progenitor cells using a maturation / specialization medium comprising HGF, OSM and DEX;

[0206] (ii) optionally, generating aggregates of the cell population when the cell population comprises at least 70%, 80%, 85% or 90% albumin-positive cells, or after about 20 days to about 40 days of culturing, for example, after about 24 days to about 28 days of culturing;

[0207] (iii) culturing the aggregated cells in a maturation medium of the aggregated cells; and (iv) optionally activating a cAMP pathway in the aggregated cells, optionally within about 1 day to about 10 days of aggregation, for example, within 6 days of aggregation, optionally after about 27 days to about 36 days of culturing.

[0208] In one embodiment, embryoid bodies (EBs) of pluripotent stem cells are formed by culturing pluripotent stem cells in the presence of BMP4 (optionally, 1-5 ng / ml BMP4 or about 5 ng / ml BMP4) for 12 to 36 hours (optionally, about 24 hours).

[0209] After forming EBs, they can be cultured in an induction medium supplemented with a nodal agonist for another 3 to 10 days (optionally, 4 to 8 days or about 5 or 6 days) to induce an endoderm cell population. Optionally, the nodal agonist is activin A. EBs are also optionally cultured with a Wnt / β-catenin agonist (such as Wnt3a or a GSK-3 selective inhibitor, such as CHIR-99021) to induce an endoderm cell population.

[0210] In some embodiments, EBs are cultured in the presence of a nodal agonist such as activin A for an additional 1 to 4 days (eg, extended nodal agonist treatment) (ie, prior to contacting the population of endoderm cells with at least one FGF agonist and one BMP4 agonist).

[0211] As mentioned, the cells are then treated to induce maturation, further lineage specialization and / or amplification, including, for example, aggregation and treatment with various maturation factors. These steps, for example, can generate a cell population that responds to cAMP activation. Extended nodal agonist treatment and aggregation are steps that generate cells that respond to cAMP activation.

[0212] For monolayer-based methods, cells that respond to cAMP activation are, for example, cells after 26 days in culture.

[0213] In embodiments, the maturation / specialization medium for aggregated cells may include factors that promote hepatocyte maturation or factors that promote cholangiocyte development or both and / or factors that increase expansion of precursor populations.

[0214] For example, the aggregates include hepatoblasts, which can specialize into hepatocytes or cholangiocytes as shown.

[0215] Optionally, inducing maturation, further lineage specification and / or expansion further comprises contacting the cell aggregates with i) a cAMP signaling activator (e.g., a cAMP analog and / or agonist) and / or ii) an antagonist of Wnt / β-catenin signaling (e.g., Wnt inhibitor XAV 939) and / or an inhibitor of MEK / Erk signaling (e.g., MEK / Erk inhibitor PD0325901). Adding Wnt antagonists and / or MEK / Erk antagonists during activation of cAMP signaling enhances expression of CYP enzymes, e.g., up to or above the levels in adult hepatocytes. For example, in the presence of cAMP, an inhibitor of MEK / Erk, e.g., is added to cultures from about day 28 to about day 32, resulting in hepatocytes having increased levels of CYP3A4. Adding a combination of a MEK / Erk antagonist and a Wnt antagonist is shown to also increase the levels of CYP1A2. In an embodiment, Wnt antagonists include, for example, XAV939, IWP2, DKK1, XXX (IWP2 (STEMGENT 04-0034), Dkk-1 (R&D, 5439-DK-010), IWR-1endo (Calbiochem 681699-10). Known antagonists of Wnt signaling include Dickkopf (Dkk) protein, Wnt inhibitory factor-1 (WIF-1), and secreted frizzled-related protein (SFRP) and can be used in an embodiment. In another embodiment, the MEK / Erk antagonist is selected from PD0325901, U0126 (Promega V1121), PD098059 (Sigma-Aldrich P215-1MG).

[0216] Optionally, the cell aggregates are contacted with 0.1 μM to 10 μM (optionally, 0.5 μM to 2 μM or about 1 μM) of XAV939 and / or PD0325901.

[0217] Concentrations of other inhibitors / activators are, for example, concentrations that give similar activation / inhibition as the inhibitors / activators described herein.

[0218] In another embodiment, inducing maturation, further lineage specification and / or expansion further comprises contacting the cell aggregates with cAMP analogs and / or cAMP agonists and Wnt agonists, such as selective inhibitors of GSK3 (e.g., CHIR99021) or TGF-β antagonists (e.g., inhibitor SB431542). Optionally, the cell aggregates are contacted with 0.1 μM to 10 μM (optionally, 0.2 μM to 4M) of CHIR99021 and / or about 2 μM to about 10 μM or about 6 μM of SB431542. TGF-β inhibitors include SB431542 (Sigma-Aldrich S4317-5MG), SB525334 (Sigma-Aldrich S8822-5MG), and A83-01 (Tocris, 2929).

[0219] As demonstrated herein, activation of the Wnt pathway and inhibition of the TGF-β / SMAD pathway promotes expansion of the Albumin+ / HNF4+ progenitor population, for example, at day 27. It is demonstrated that, for example, when a Wnt agonist is added, up to a 10-fold expansion of the population can be obtained.

[0220] In an embodiment, the aggregated cells are treated with a wnt agonist and an optional TGFβ antagonist (such as SB431542) for about 6 days to about 12 days, preferably, about 8 days to about 10 days, optionally 9 days. For example, such treatment results in the expansion of hepatoblasts. In an embodiment, the method includes generating an expanded population of hepatoblasts. These cells can be used to generate a more differentiated population of cells including mature hepatocytes and / or bile duct cells.

[0221] In an embodiment, the TGF-β antagonist is selected from SB431542 (Sigma-Aldrich S4317-5MG), SB525334 (Sigma-Aldrich S8822-5MG), A83-01 (Tocris, 2929).

[0222] In embodiments, the maturation medium of the aggregated cells includes one or more factors that promote maturation, further lineage specification and / or expansion, optionally:

[0223] A Wnt agonist such as CIHR99021, optionally in combination with a TGF-β antagonist such as SB431542 that promotes expansion of the Albumin+ / HNF4+ population; or

[0224] Wnt antagonists such as XAV939 and / or MEK / Erk antagonists, e.g., PD0325901, added during the cAMP activation step, which enhances the expression of CYP enzymes and promotes the maturation of hepatocyte precursors;

[0225] In another embodiment, the maturation medium of the aggregated cells includes one or more factors that promote maturation, further lineage specification and / or expansion, optionally:

[0226] Notch agonists that promote cholangiocyte lineage specification.

[0227] Notch antagonists such as gamma-secretase inhibitor (GSI) L695,458 that promote hepatocyte lineage specification.

[0228] As described below, addition of the cAMP analog 8-Br-cAMP induced significant levels of expression of CYP3A4 (16-fold), CYP1A2 (100-fold), and CYP2B6 (10-fold), as well as the phase II enzyme UGT1A1 (16-fold) in H9-derived aggregates ( Figure 7 e).

[0229] In another embodiment, cell aggregates are generated from a monolayer of a cell population comprising hepatocytes and bile duct progenitor cells by enzymatic treatment and / or manual dissociation.

[0230] In an embodiment, the cell population includes hepatocytes and / or bile duct progenitor cells cultured in a maturation / specialized medium comprising HGF, OSM and DEX, and optionally, before cell aggregation, the cell population is co-cultured with endothelial cells (optionally, CD34+ positive endothelial cells). In an embodiment, the endothelial cells are derived from embryonic ESCs, preferably human. In an embodiment, the endothelial cells are mature endothelial cells, optionally human, and / or derived from mature endothelial cells.

[0231] For example, CD34+ endothelial cells can be generated from hESCs as described in Example 8. As described in Example 8, endothelial cells can be generated by induction with a combination of BMP4, bFGF, and VEGF for about 6 days, at which point CD34+ cells (with CD31+ and KDR+) can be isolated by FACS. The selected CD34+ cells can be further cultured in endothelial cell growth medium (optionally, EMG2 medium), for example, for 6 days, and then used for the generation of chimeric aggregates.

[0232] In an embodiment, a cell population includes hepatocytes and / or bile duct progenitor cells that have been cultured in a maturation / specialization medium comprising HGF, OSM, and DEX, and the cell population is co-cultured with CD34+ positive endothelial cells to form chimeric aggregates, optionally using a culture medium such as Aggrewells. TMAggregation containers (e.g., containers that promote aggregation of a single cell type or mixed cell types) are placed in an aggregation container until chimeric aggregates are obtained, for example, about 1 day, about 2 days, or about 3 days when Aggrewells are used. Aggregation can also be performed using methods described herein or known in the art. As described in Example 8, endothelial cells can be added to the container before the hepatocyte population to coat the bottom of the wells. The hepatocyte population can be added as a single cell suspension, for example, day 25 / 26 hepatoblasts can be added on top of the endothelial cells and in a mixture cultured in Aggrewells. After appropriate aggregation, the chimeric liver / endothelial aggregates can then be removed from the Aggrewells and cultured. As Fig.12 As shown in b, aggregates cultured with endothelial cells contained endothelial cells and were larger than those cultured alone. qRT-PCR analysis also showed that chimeric liver / endothelial aggregates cultured for an additional 12 days expressed substantially higher levels of CYP3A4 message than liver aggregates generated without endothelial cells ( Fig.12 d). Since these levels are achieved without the addition of cAMP, endothelial cells can promote the maturation of hPSC-derived hepatocytes. In embodiments, the liver / endothelial chimeric aggregates are cultured for at least or about 6 days, at least or about 8 days, at least or about 10 days, at least or about 12 days, or until a desired or preselected level of CYP3A4 information is obtained.

[0233] In further embodiments, the hepatic endothelial chimeric aggregates are cultured in a gelatinous matrix, optionally in a collagen comprising matrix, optionally in a gel. In embodiments, the collagen is collagen I or IV. In embodiments, the gelatinous matrix comprises matrigel, laminin, fibronectin, extracted ECM (e.g., extracellular matrix extracted from liver tissue), and / or combinations thereof.

[0234] In an example, aggregates cultured in a collagen comprising matrix were cultured in the presence of cAMP, PD0325901, and XAV939.

[0235] The combination of 3D aggregation, cAMP and PD / XAV was shown to promote significant differentiation of human pluripotent stem cell-derived hepatocytes ( Fig. 9 ),( Fig. 9 d and Fig. 9 e) Maintaining some expression of AFP and fetal CYP3A7. In Example 8, it was demonstrated that treating liver endothelial chimeric aggregates with a combination of cAMP, PD, and XAV in a collagen gel to provide a source of extracellular matrix proteins promoted further maturation of the population. Fig.13As shown, when the aggregates were maintained in liquid culture, the addition of endothelial cells to the aggregates (end) did not significantly affect the expression levels of ALB, CYP3A4, AFP or CYP3A7. In contrast, culturing the aggregates in collagen gels had a significant effect on the expression of AFP and CYP3A7, as both were reduced to almost undetectable levels, similar to those found in adult liver.

[0236] In an embodiment, cells are treated with a notch agonist, for example within about 1 day to about 4 days after aggregation. Adding an agonist at such a stage promotes the maturation of cholangiocytes. In some embodiments, for example, where cholangiocyte maturation is preferred, induction of cAMP signaling is omitted.

[0237] Therefore, the present invention also provides a method for inducing bile duct progenitor cells to mature into bile duct cells, wherein the induction of maturation, further lineage specification and / or expansion comprises:

[0238] (i) culturing a cell population including bile duct progenitor cells with a notch agonist to induce maturation of the bile duct progenitor cells into bile duct cells, optionally, functional bile duct cells.

[0239] In an embodiment, a method of generating functional cholangiocytes from pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs):

[0240] a) contacting pluripotent stem cells cultured as a monolayer or forming embryoid bodies with an induction medium to provide an induced endoderm cell population, the induction medium comprising a nodal agonist such as ActA and optionally a Wnt / β-catenin agonist (such as i) Wnt3a and / or ii) a GSK-3 selective inhibitor such as CHIR-99021;

[0241] b) contacting the induced endoderm cell population with a nodal agonist to provide a prolonged nodal agonist-treated induced endoderm cell population;

[0242] c) obtaining a cell population comprising hepatocytes and / or bile duct progenitor cells by contacting the induced endoderm cell population subjected to prolonged nodal agonist treatment with a specialized medium comprising an FGF agonist and a BMP4 agonist and / or active conjugates and / or fragments thereof; and

[0243] d) inducing maturation of bile duct progenitor cells and inducing further lineage specification and / or expansion of bile duct progenitor cells to bile duct cells, wherein the inducing maturation, further lineage specification and / or expansion comprises:

[0244] (i) optionally, generating aggregates of the cell population when the cell population comprises at least 70%, 80%, 85%, 90% or 95% albumin-positive cells, or after about 20 days to about 40 days of culturing, for example, after about 24 days to about 28 days of culturing;

[0245] (ii) culturing the cell population including bile duct progenitor cells with a maturation medium including a Notch agonist,

[0246] Wherein, when the Notch agonist is a Notch signal donor cell (optionally, OP-9, OP-Jagged1 and / or OP-9deltal cells), the Notch signal donor cell is co-polymerized with the cell population.

[0247] In further embodiments, the aggregates are cultured (or co-cultured when Notch signal donor cells are included) in a gelatinous matrix (optionally, collagen comprising the matrix, optionally, a gel). In embodiments, the collagen is collagen I or IV. In embodiments, the gelatinous matrix comprises matrigel, laminin, fibronectin, extracted ECM (e.g., extracellular matrix extracted from liver tissue), and / or combinations thereof.

[0248] In addition, for example, inhibition of Notch signaling with Notch antagonists such as γ-secretase inhibitor (GSI) L695,458 (Tocris #2627), DAPT (Sigma-Aldrich D5942), LY411575 (Stemgent 04-0054), and L-685458 is shown herein to inhibit cholangiocyte development, and the resulting cells retain the properties of hepatocytes.

[0249] In another embodiment, a method of generating a hepatocyte lineage cell such as a hepatoblast, a hepatocyte and / or a cholangiocyte from a pluripotent stem cell (PSC), such as an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC), the method comprising:

[0250] a) contacting pluripotent stem cells for forming embryoid bodies as a monolayer culture with an induction medium, optionally for about 4 to 8 days, to provide an induced endoderm cell population, the induction medium comprising a nodal agonist such as ActA and optionally a Wnt / β-catenin agonist (such as i) Wnt3a and / or ii) a GSK-3 selective inhibitor, such as CHIR-99021);

[0251] b) contacting the induced endoderm cell population with a nodal agonist (optionally, for about 2 days, 3 days, or about 4 days) to provide a prolonged nodal agonist-treated induced endoderm cell population; and

[0252] c) obtaining a cell population comprising hepatocytes and / or bile duct progenitor cells by contacting the prolonged nodal agonist-treated induced endoderm cell population with a specialized medium (optionally, for about 4 days to about 10 days), the specialized medium comprising at least one FGF agonist and one BMP4 agonist and / or active conjugates and / or fragments thereof, and

[0253] d) inducing maturation, further lineage specification and / or expansion of hepatocytes or cholangiocyte progenitor cells to an optionally expanded hepatoblast population and / or hepatocytes and / or cholangiocytes, the induction of maturation and optionally further lineage specification and / or expansion comprising:

[0254] (i) culturing a cell population comprising hepatocytes and / or bile duct progenitor cells with a maturation medium comprising HGF, Dex and OSM, optionally for about 10 to 14 days;

[0255] (ii) optionally generating aggregates of the cell population when the cell population comprises at least 70%, 80%, 85% or 90% albumin-positive cells, or after about 20 days to about 40 days of culturing, for example, after about 24 days to about 28 days of culturing;

[0256] (iii) culturing the aggregates in a maturation medium optionally including Dex for 1 day to 10 days;

[0257] IV)

[0258] a) culturing the aggregates in a maturation medium (e.g., aggregation maturation medium) comprising Dex and optionally a cAMP analog and / or a cAMP agonist for about 6 days to about 10 days, optionally within about 1 day to about 10 days of aggregate generation, for example, within 6 days of aggregation, optionally after about 27 days to about 36 days of culturing; or

[0259] b) culturing the aggregates in a maturation medium comprising a notch agonist and optionally a cAMP agonist, HGF and / or EGF for about 6 days to about 20 days, optionally adding the notch agonist within about 1 day to about 10 days of the step of generating the aggregates, for example, within 6 days of the step of generating the aggregates, optionally after about 20 days to 40 days of culturing.

[0260] For example, the Notch agonist can be any notch ligand that binds to a surface such as a cell, plastic, ECM, or bead. In one embodiment, the notch ligand is notch ligand delta Jagged-1 (EUROGENTEC 188-204), Jagged1 peptide (Abcam, ab94375), recombinant human Pref-1 / DLK-1 / FA1 (R&D 1144-PR). In one embodiment, inducing maturation and further lineage specification and / or expansion comprises contacting a cell population comprising bile duct progenitor cells with a notch signal donor such as OP9, OP9delta and / or OP9 Jagged1 cells, and optionally, in the presence of EGF, TGFβ, HGF and EGF and / or HGF, TGFβ and EGF, for at least or about 5 days to about 10 days, about 14 days or longer, for example, 90 days, optionally at least or about 5 days to at least or about 60 days, at least or about 30 days, at least or about 25 days, at least or about 1 day, and / or at least or about 14 days, to induce maturation of bile duct progenitor cells into functional bile duct cells. It has been demonstrated that the resulting structures can be maintained in culture for more than 60 days. Thus, in an embodiment, a cell population comprising bile duct progenitor cells is contacted with a notch signal donor (notch agonist) such as OP9, OP9delta and / or OP9 Jagged1 cells, and optionally in the presence of EGF, TGFβ, HGF and EGF and / or HGF, TGFβ, and EGF, for at least 5 days and optionally up to any number of days between 5 and 90 days or between 5 and 60 days.

[0261] Optionally, contacting the cell population comprising bile duct progenitor cells with a notch signal donor comprises co-culturing the cell population comprising bile duct progenitor cells with a notch signal donor such as cells of OP9, OP9delta and / or OP9 Jagged1, and optionally in a maturation medium comprising EGF, TGFβ, HGF and EGF and / or HGF, TGFβ and EGF, for at least or about 5 days to at least or about 90 days, optionally at least or about 5 days to at least or about 60 days, at least or about 30 days, at least or about 25 days, at least or about 1 day, and / or at least or about 14 days to induce maturation of bile duct progenitor cells into bile duct cells, optionally, functional bile duct cells.

[0262] In one embodiment, inducing maturation and optionally further lineage specification and / or expansion comprises co-culturing a cell population comprising biliary progenitor cells with cells of OP9, OP9delta and / or OP9 Jagged1, and optionally in the presence of EGF, TGFβ, HGF and EGF and / or HGF, TGFβ and EGF, for at least 5 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or more than 14 days, 90 days, optionally, co-culturing for at least or about 5 days to at least or about 60 days, at least or about 30 days, at least or about 25 days, at least or about 1 day and / or at least or about 14 days.

[0263] As used herein, the term "activator of Notch signaling" or "notch agonist" refers to any molecule or cell as used herein that activates Notch signaling in hepatocytes and / or bile duct cells and induces notch signal donors such as OP9 cells, bone marrow-derived mouse stromal cell lines, and notch ligands. OP-9 cells endogenously express and have been designed to overexpress one or more notch ligands. OP-9-Jagged1 is designed to overexpress recombinant / exogenous Jagged1 notch ligands, and OP9-delta1 is designed to overexpress recombinant deltalnotch ligands. In an embodiment, the OP9 notch signal donor is selected from OP9, OP9-Jagged1 or OP-delta1 cells. OP9 cells express the notch ligand delta. When they express Notch ligands, they can thereby be used as activators of Notch signaling. Also included are molecules and / or cells expressing Jagged-1 (EUROGENTEC 188-204), Jagged1 peptide (Abcam, ab94375), and recombinant human Pref-1 / DLK-1 / FA1 (R&D 1144-PR). For example, a "notch agonist" can be bound to a surface such as a cell, plastic, ECM, or bead.

[0264] In one embodiment, a cell population comprising bile duct progenitor cells is co-cultured with OP9, OP9delta and / or OP9Jagged1 cells in the presence of 10 ng / ml to 20 ng / ml (optionally, about 20 ng / ml) of HGF and / or 25 ng / ml to 75 ng / ml (optionally, about 50 ng / ml) of EGF to induce at least one bile duct progenitor cell to differentiate into a functional bile duct cell.

[0265] As mentioned, hepatoblasts (e.g., Figure 1 a and Fig.14a) can be aggregated as described in step g) (also referred to as 3D aggregation), step g) includes inducing maturation, further lineage specialization and / or expansion of hepatocytes and bile duct progenitor cells of the cell population to hepatocytes and / or bile duct cells, inducing maturation, further lineage specialization and / or expansion including generating aggregates of the cell population. These 3D aggregates include hepatoblasts that can mature / differentiate into hepatocytes or further specialize into bile duct cells. In embodiments where bile duct cells are desired, optionally when the chimeric aggregates include hepatoblasts and Notch signal donor cells, further lineage specialization is achieved by co-culturing the aggregates with Notch signal donors such as OP9, OP9delta and / or OP9 Jagged1 cells.

[0266] In an embodiment, a cell population of hepatoblasts including biliary progenitor cells is co-cultured with OP9, OP9delta and / or OP9Jagged1 cells in a matrix / gel including matrigel and / or collagen, optionally as chimeric aggregates.

[0267] In embodiments, the matrix / gel comprises at least 20%, at least 30%, at least or up to 40%, at least or up to 50%, at least or up to 60%, at least or up to 70%, at least or up to 80%, at least or up to 90% and / or up to 100% matrix glue.

[0268] In embodiments, the collagen comprises collagen I and / or collagen IV. In embodiments, the matrix / gel comprises from about 0 mg / mL to about 5 mg / mL of collagen I, optionally about 1.0 mg / mL, about 2 mg / mL, about 3.0 mg / mL, or about 4.0 mg / mL of collagen I. In embodiments, the matrix / gel comprises about 1.0 mg / mL, about 1.2 mg / mL, about 1.4 mg / mL, about 1.6 mg / mL, about 1.8 mg / mL, about 2.0 mg / mL, about 2.25 mg / mL, about 2.5 mg / mL, about 2.75 mg / mL, or about 3.0 mg / mL of collagen I.

[0269] As demonstrated in Example 9, encapsulated structures are obtainable where the co-culture comprises at least 30% or more of the Matrigel composition. If increased branching structures are desired, the Matrigel concentration can be reduced, for example to about 20%.

[0270] As demonstrated in Example 9, cholangiocyte branches and cystic structures expressing CFTR can be generated using the methods described herein. CFTR is functional as shown using a swelling assay. Thus, in an embodiment, the cholangiocytes generated and / or isolated are cholangiocytes expressing CFTR.

[0271] As used herein, the term "nodal agonist" refers to any molecule that activates nodal signaling such as "nodal" (eg, human nodal such as Gene ID: 4338) or "activin" in hepatocyte lineage cells.

[0272] As used herein, the term "activin" or "ActA" refers to "activin A" (e.g., Gene ID: 3624), e.g., human activin, and active conjugates and fragments thereof, optionally including, for example, naturally occurring active conjugates and fragments that can activate nodal signaling, and active conjugates and fragments thereof, including naturally occurring active conjugates and fragments. The concentration of activin can be, for example, in the range of from about 1 ng to about 500 ng / ml, e.g., from about 1 ng to about 250 ng / ml, from about 10 ng to about 250 ng / ml, from about 10 ng to about 100 ng / ml. In another embodiment, the concentration of activin is about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 150 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml or about 500 ng / ml.

[0273] As used herein, the term "HGF" refers to hepatocyte growth factor (gene ID: 3082), for example, human HGF and their active conjugates and fragments, including naturally occurring active conjugates and fragments. The concentration of HGF can be, for example, in the range of from about 1 ng to about 500 ng / ml, for example, from about 1 ng to about 250 ng / ml, from about 10 ng to about 250 ng / ml, from about 10 ng to about 100 ng / ml. In another embodiment, the concentration of HGF is about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 150 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml or about 500 ng / ml.

[0274] As used herein, the term "TGFβ" refers to any one of TGFb1, TGFb2 and TGFb3, for example, human TGFb1, TGFb2 and TGFb3 and their active conjugates and fragments, including naturally occurring active conjugates and fragments. As described below, when hepatoblasts are co-cultured with OP9, TGFb1 promotes bile duct cell branching. TGFb2 and TGFb3 have also been tested and promote branched structures under similar conditions.

[0275] As used herein, the term "TGFβ1" refers to transforming growth factor β1, for example, human TGFβ1 (gene ID 7040) and their active conjugates and fragments, including naturally occurring active conjugates and fragments. The concentration of TGFβ1 for cholangiocyte specialization can be, for example, in the range of from about 5 ng / ml to about 10 ng / mL.

[0276] As used herein, the term "wnt / β-catenin agonist" refers to any molecule that activates wnt / β-catenin receptor signaling in hepatocytes and includes, for example, Wnt3a and GSK3 selective inhibitors such as CHIR99021 (Stemolecule TM CHIR99021 Stemgent), 6-bromoindirubin-3'-oxime (BIO) (Cayman Chemical (cat: 13123)) or Stemolecule from Stemgent TM BIO (cat: 04003). CHIR99021 is a selective inhibitor of GSK3. Expected selective inhibitors of GSK3 are, for example, selective inhibitors for GSK-30 / β in the Wnt signaling pathway. For example, using the Cignal TCF / LEF reporter gene from Qiagen (CignalTCF / LEF reporter gene (luc) kit: CCS-018L), Wnt / β receptor signaling in hepatocytes can be determined, for example, by measuring an increase in Axin2 gene expression by qPCR and / or measuring β-catenin phosphorylation.

[0277] As used herein, the term "Wnt3a" refers to the wingless MMTV integration site family, member 3A factor (e.g., gene ID: 89780), such as human Wnt3a and their active conjugates and fragments, including naturally occurring active conjugates and fragments. The concentration of Wnt3a can be, for example, in the range of from about 1 ng to about 500 ng / ml, for example, from about 1 ng to about 250 ng / ml, from about 10 ng to about 250 ng / ml, from about 10 ng to about 100 ng / ml. In another embodiment, the concentration of Wnt3a is about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 150 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml or about 500 ng / ml.

[0278] As used herein, the term "agonist" refers to an activator of, for example, a pathway or signaling molecule. For example, a nodal agonist refers to a molecule that selectively activates nodal signaling.

[0279] As used herein, the term "antagonist" refers to, for example, a selective inhibitor of a pathway or signaling molecule. For example, a TGFβ antagonist is a molecule that selectively inhibits TGFβ signaling, for example, by measuring the phosphorylation of Smad. A83-01 is a more potent inhibitor of Smad2 than SB431542.

[0280] As used herein, the term "selective inhibitor" refers to an inhibitor that more effectively inhibits a selective entity or pathway than a related molecule by at least 1.5X, 2X, 3X, 4X or 10X. For example, a selective inhibitor of GSK-3 inhibits GSK-3 in a Wnt pathway more effectively than at least 1.5X, 2X, 3X, 4X or 10X inhibited by, for example, LiCl, or inhibits other kinases, other GSKs and / or GSK3 in other pathways by at least 1.5X, 2X, 3X, 4X or 10X more effectively than a selective inhibitor of GSK-3. For example, CHIR99021 has been shown in an in vitro kinase assay to specifically inhibit GSK3B with an IC50 of about 5nM and specifically inhibit GSK3a with an IC50 of 10nM when other kinases are not greatly affected. Therefore, a selective inhibitor can show an IC50 of at least 1.5X, 2X, 3X, 4X or 10X lower than other unrelated kinases (e.g., other 2, other 3, etc.). Similarly, the term "selective activator" refers to an activator that activates a selective entity or pathway at least 1.5X, 2X, 3X, 4X or 10X more effectively than a related molecule. As used herein, the term "active fragment" is a polypeptide having an amino acid sequence of a smaller size than a full-length polypeptide, but substantially homologous to the full-length polypeptide, being a fragment of the full-length polypeptide, and wherein the active fragment has at least 50%, or at least 60, or at least 70%, or at least 80%, or at least 90%, or at least 100% of the effective biological effect compared to the full-length polypeptide, the active fragment is a fragment of the full-length polypeptide, or optionally, the active fragment has greater than 100% (e.g., 1.5 times, 2 times, 3 times, 4 times or greater than 4 times) of the effective biological effect compared to a polypeptide that is a fragment of the full-length polypeptide.

[0281] As used herein, the term "active conjugate" refers to a polypeptide (or other molecule) linked to a tag or stabilizing entity, such as a fluorescent tag, for example, to improve stability under conditions of extended storage, heat, enzymes, low pH, agitation, etc., the active conjugate does not interfere at all or substantially with the activity of the active portion of the molecule. For example, the conjugate can have about at least 50%, or 60%, or 70%, or 80%, or 90%, or 100% or more than 100% of the effective biological effect, for example, 1.5 times, 2 times, 3 times, 4 times or more than 4 times the effective biological effect (e.g., receptor activation activity) compared to the unlinked polypeptide or other molecule.

[0282] In one embodiment, the term "active fragments and conjugates" refers to fragments and conjugates of molecules that retain the ability to activate the cognate receptor of the molecule. Optionally, the active fragments and conjugates have at least 60%, 70%, 80%, 90% or 95% of the activity of the full-length and / or unlinked molecule.

[0283] Variants such as conservative mutation variants and activating mutation variants of each polypeptide may also be used.

[0284] The term "Dex" as used herein refers to dexamethasone (Dex). The concentration of dexamethasone can be, for example, in the range of from about 1 ng to about 500 ng / ml, for example, from about 1 ng to about 250 ng / ml, from about 10 ng to about 250 ng / ml, from about 10 ng to about 100 ng / ml. In another embodiment, the concentration of Dex is about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 150 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, or about 500 ng / ml.

[0285] As used herein, the term "OSM" refers to Oncostatin M. The concentration of OSM can be, for example, in the range of from about 1 ng to about 500 ng / ml, for example, from about 1 ng to about 250 ng / ml, from about 10 ng to about 250 ng / ml, from about 10 ng to about 100 ng / ml. In another embodiment, the concentration of OSM is about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 150 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, or about 500 ng / ml.

[0286] As mentioned, some embodiments of the invention include activating the cAMP pathway within the aggregate to induce hepatocyte and / or cholangiocyte maturation.

[0287] As used herein, the term "cAMP pathway" refers to the adenylate cyclase pathway, a G protein-coupled receptor-triggered signaling cascade used in cellular communication. Optionally, the cAMP pathway is a human cAMP pathway.

[0288] As used herein, the term "activation of the cAMP pathway" refers to inducing the pathway to convert ATP to cAMP, for example, increasing the level of cAMP. When the cAMP pathway is activated, the activated GPCR causes a conformational change in the attached G protein complex, which causes the Gα subunit to convert GDP to GTP and separate from the β and γ subunits. The Gα subunit in turn activates adenylate cyclase, which converts ATP to cAMP. The cAMP pathway can also be activated by directly activating adenylate cyclase or downstream of PKA. Molecules that activate the cAMP pathway include, but are not limited to, cAMP, such as 8-bromoadenosine-3', 5'-cyclic monophosphate (8-Br-cAMP), butyryl-cAMP, adenosine-3', 5'-cyclic monothiophosphate, Sp-isomer (SP-aAMPS) and / or cAMP analogs of 8-bromoadenosine-3', 5'-cyclic monothiophosphate, Sp-isomer (Sp-8-Br-cAMPS)). 8-Br-cAMP, butyryl-cAMP and Sp-cAMPS are examples of cell permeable analogs of cAMP. Multiple other cAMP analogs that activate cAMP signaling are also known in the art and can be used. Other compounds that activate the cAMP pathway (e.g., cAMP agonists) include, but are not limited to, cholera toxin, forskolin, caffeine, theophylline, and pertussis toxin. For example, experiments have been conducted using Sp-8-Br-cAMP (Biolog: Catalog No.: B002 CAS No.: [127634-20-2]), 8-Br-cAMP and forskolin (FSK) (Sigma: 66575-29-9), which have shown that these compounds can be interchangeable.

[0289] In some embodiments of the present method, the cAMP pathway is optionally activated by contacting the liver aggregates with 0.5 mM to 50 mM of a cell-permeable cAMP analog, such as 8-Br-cAMP (optionally, 1-40 mM, 1-30 mM, 1-20 mM, 5-15 mM, 8-12 mM, or about 10 mM 8-Br-cAMP). The liver aggregates are optionally contacted with a cell-permeable cAMP analog, such as 8-Br-cAMP, for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days.

[0290] Inducing maturation, further lineage specification and / or expansion may involve a series of steps.

[0291] In some embodiments, a cell population comprising hepatocytes and / or bile duct progenitor cells and / or aggregates is cultured in a cell culture medium comprising HGF, dexamethasone and oncostatin M. In other embodiments, aggregates are cultured in a cell culture medium comprising Iscove's modified Dulbecco's medium (IMDM), IMDM supplemented with B27, ascorbic acid, glutamine, MTG, HGF, dexamethasone and oncostatin M. In other embodiments, cells are cultured in a cell culture medium comprising HGF, dexamethasone and oncostatin M, optionally Iscove's modified Dulbecco's medium (IMDM) supplemented with B27, ascorbic acid, glutamine, MTG, HGF, dexamethasone and oncostatin M, prior to and / or during aggregation. The cell culture medium is optionally also supplemented with a Rho kinase inhibitor and BSA. For example, a cell population comprising hepatocytes and / or bile duct progenitor cells can be cultured in a maturation medium comprising HGF, DEX and OSM for 10 days, 11 days, 12 days, 13 days or 14 days, and / or aggregates can be cultured in a maturation medium comprising HGF, DEX and OSM for 6 days, 7 days, 8 days, 9 days, 10 days.

[0292] In another embodiment, the step of inducing maturation, further lineage specification and / or expansion further comprises activating a cAMP pathway within the aggregate to induce maturation of at least one hepatocyte or biliary progenitor cell into a functional hepatocyte and / or biliary cell. In another embodiment, activating the cAMP pathway comprises contacting the aggregate with a cAMP analog and / or a cAMP agonist (e.g., a cAMP analog or cAMP agonist described above).

[0293] For example, in embodiments, after the aggregates are cultured in a maturation medium comprising HGF, DEX and OSM for, e.g., about 10 days, 11 days, 12 days, 13 days or 14 days, a maturation medium comprising a cAMP analog and / or a cAMP agonist and DEX and optionally HGF is added to the aggregates.

[0294] In one embodiment, the aggregates are cultured in a cell culture medium comprising HGF, Dex, and OSM until the cAMP pathway is activated. In one embodiment, the aggregates are cultured in a culture medium comprising a cAMP analog and / or a cAMP agonist and Dex. When the cAMP analog and / or cAMP agonist is added, OSM is removed from the culture medium. In some embodiments, when the cAMP analog and / or cAMP agonist is added, HGF is also removed from the culture medium. In another embodiment, after the addition of the cAMP analog and / or cAMP agonist, the amount of HGF in the culture medium is reduced (e.g., from 20 ng / ml of HGF to 10 ng / ml of HGF).

[0295] In another embodiment, the aggregates are cultured in HGF, Dex and OSM for about 6 days, 7 days, 8 days, 9 days, 10 days, 11 days or 12 days, at which time, cAMP analogs and / or cAMP agonists are added. In one embodiment, OSM and optionally HGF are removed when cAMP analogs and / or cAMP agonists are added. In other embodiments, the concentration of HGF in the culture medium is reduced (e.g., from about 20 ng / ml to about 10 ng / ml) when cAMP analogs and / or cAMP agonists are added.

[0296] In some embodiments, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, or at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the induced endoderm cell population differentiate / mature into functional hepatocytes and / or bile duct cells.

[0297] Thus, in embodiments, the method induces the production of greater than about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or about 95% functional hepatocytes and / or cholangiocytes from a population of induced endoderm cells treated with a nodal agonist.

[0298] For example, maturity can be detected by determining the level of mature hepatocyte markers. For example, CYP1A2, CYP2B6, CYP2D6, CYP3A4, CYP7A1, CYP2C9, ALB, CPS1, G6P, TAT, TDO1, NAT2, UGT1A1 and / or ASGPR1 are markers of mature hepatocytes or functional hepatocytes, and their expression can be detected, for example, by RT-PCR. Differentiation can also be detected using antibodies that recognize mature hepatocytes (e.g., antibodies that detect ASGPR-1).

[0299] In embodiments, the endoderm cell population is differentiated from pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0300] In an embodiment, the pluripotent stem cell is from a mammal such as a human. In an embodiment, the pluripotent stem cell is a human ESC (hESC) or a human iPSC (hiPSC).

[0301] As used herein, the terms "iPSC" and "induced pluripotent stem cells" are used interchangeably and refer to pluripotent stem cells artificially derived from non-pluripotent cells (usually adult somatic cells) (e.g., induced or by complete reversal), for example, by inducing the expression of one or more genes (including POU4F1 / OCT4 (Gene ID; 5460) in combination with, but not limited to, SOX2 (Gene ID; 6657), KLF4 (Gene ID; 9314), cMYC (Gene ID; 4609), NANOG (Gene ID; 79923), LIN28 / LIN28A (Gene ID; 79727)).

[0302] In an embodiment, the method comprises a step for obtaining an endoderm cell population.For example, provided herein is a method for inducing definitive endoderm in pluripotent stem cells such as ESCs or iPSCs.

[0303] In one embodiment, obtaining the endoderm cell population comprises forming embryoid bodies from a pluripotent stem cell culture. EBs are formed by any method known in the art, e.g., Nostro, MC et al. 18 The method described in , where EBs are formed from small aggregates cultured in low levels of BMP4 agonist for 24 h.

[0304] In another embodiment, obtaining a population of endoderm cells comprises obtaining and / or growing a monolayer culture of pluripotent stem cells.

[0305] The EBs and / or monolayers are then exposed to high concentrations of activin A to induce definitive endoderm. Optionally, the EBs and / or monolayers are exposed to 80 ng / ml to 120 ng / ml or 90 ng / ml to 110 ng / ml of activin (optionally, about 100 ng / ml activin A) for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 14 days.

[0306] In another embodiment, EBs and / or monolayers are contacted with a wnt / β-catenin agonist such as Wnt3a or a selective inhibitor of GSK-3 such as CHIR-99021, 6-bromoindirubin-3'-oxime (BIO), or a Stemolecule other than activin A. TM For example, BIO, a specific inhibitor of GSK-3, was shown to maintain the pluripotency of human and mouse ESCs by activating Wnt signaling 53 .

[0307] Optionally, EBs and / or monolayer cells are exposed to from 10 ng / ml to 40 ng / ml of Wnt3a or 20 ng / ml to 30 ng / ml of Wnt3A, optionally, about 25 ng / ml of Wnt3a for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. In another embodiment, EBs and / or monolayer cells are exposed to from about 0.03 μM to about 30 μM of CHIR-99021, or from about 0.1 μM to about 3 μM, optionally about 0.3 μM to about 1 μM of CHIR-99021. In an embodiment, EBs are exposed to from about 0.1 μM to about 2 μM of CHIR-99021. In another embodiment, monolayer cells are exposed to from about 1 μM to about 30 μM of CHIR-99021, for example, from about about 1 μM to about 3 μM of CHIR-99021. One skilled in the art will be able to determine equivalent useful amounts of other GSK-3 inhibitors.

[0308] In some embodiments, EBs and / or monolayer cells are first contacted with 80 ng / ml to 120 ng / ml activin, or 90 ng / ml to 110 ng / ml activin, optionally about 100 ng / ml activin A and 10 ng / ml to 40 ng / ml Wnt3a, or 20 ng / ml to 30 ng / ml Wnt3A, optionally about 25 ng / ml Wnt3a for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or about 10 days prior to contacting with 80 ng / ml to 120 ng / ml activin, or 90 ng / ml to 110 ng / ml activin, optionally about 100 ng / ml activin A and 10 ng / ml to 40 ng / ml Wnt3a, or 20 ng / ml to 30 ng / ml Wnt3A, optionally about 25 ng / ml Wnt3a for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or about 10 days to produce an induced endoderm cell population.

[0309] In the Examples and described elsewhere, a population of induced endoderm cells is cultured with a nodal agonist, such as ActA, for at least 36 hours, 38 hours, hours, 42 hours, 44 hours, 46 hours, 48 ​​hours, 50 hours, 52 hours, 56 hours, 58 hours, or 60 hours, or from about 1 day to about 4 days, to produce an induced endoderm population treated with a prolonged nodal agonist.

[0310] Optionally, the basal medium for inducing definitive endoderm is any medium known in the art for inducing definitive endoderm, optionally a neural basal medium or StemPro34. In some embodiments, the cell culture medium is supplemented with activin A, glutamine, ascorbic acid, MTG, bFGF, and BMP4. In other embodiments, the cell culture medium is also supplemented with a Wnt / β-catenin agonist, such as Wnt3a or a selective inhibitor of GSK-3 (such as CHIR-99021).

[0311] Other methods of differentiating cells can also be used to obtain induced endoderm cell populations.

[0312] The definitive endoderm or induced endoderm cell population is optionally defined by the expression of surface markers CXCR4, CKIT and EPCAM and the transcription factors SOX17 and FOXA2 or any combination thereof. In some embodiments, after activin induction, greater than 50%, 60%, 70%, 80%, 85%, 90% or 95% of the endoderm cell population expresses CXCR4, CKIT and EPCAM. In another embodiment, after activin induction, greater than 50%, 60%, 70%, 80%, 85%, 90% or 95% of the endoderm cell population expresses SOX17 and / or FOXA2.

[0313] In certain embodiments, the method further comprises enriching and / or isolating functional hepatocytes and / or cholangiocytes, optionally to generate an isolated population of functional hepatocytes and / or cholangiocytes.

[0314] In embodiments, the isolating step comprises contacting the cell population with a specific agent that binds to functional hepatocytes and / or cholangiocytes.

[0315] As used herein, the term "isolated population" relative to an isolated population of cells refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, the isolated population is a substantially pure population of cells compared to a heterogeneous population from which cells are isolated or enriched. The cells can be, for example, a single cell suspension, a monolayer and / or an aggregate. In some embodiments, for example, including bile duct cells, the isolated population can also include cells expressing notch ligands such as OP9, OP9delta and / or OP9Jagged1 cells. In some embodiments, for example, including hepatocytes, the isolated population can also include endothelial cells. The isolated population (optionally in a dissociated cell suspension and / or aggregate) can be used for screening applications, disease modeling applications and / or transplantation applications including, for example, stents, etc.

[0316] The term "substantially pure" with respect to a particular cell population refers to a population of cells that is at least about 65%, preferably at least about 75%, at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure relative to the cells that make up the total cell population. Similarly, a "substantially pure" population for functional hepatocytes and / or cholangiocytes refers to a population of cells containing less than about 30%, less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of non-functional hepatocytes and / or cholangiocytes or their descendants as defined by the terms herein. In some embodiments, the present invention includes a method for expanding a population of functional hepatocytes and / or cholangiocytes, wherein the expanded population of functional hepatocytes and / or cholangiocytes is a substantially pure population of functional hepatocytes and / or cholangiocytes.

[0317] The terms "enrich" or "enriched" are used interchangeably herein and refer to an increase in the yield (fraction) of one type of cells relative to the fraction of cells of that type in the prepared starting culture by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60%. Enrich and partially purify can be used interchangeably.

[0318] Populations of cells can be enriched using various methods, such as methods based on markers, such as cell surface markers (eg, FACS sorting, etc.).

[0319] Cells and components

[0320] As discussed above, functional hepatocytes and / or cholangiocytes can be separated using methods described herein. Therefore, further aspects of the present application include populations of cells produced according to methods described herein. In an embodiment, the population of cells is, for example, hepatoblasts, hepatocytes and / or cholangiocytes of markers of, for example, mature and / or functional cells produced according to methods described herein, optionally enriched, purified or separated cell populations of mature and / or functional hepatocytes and / or cholangiocytes. Enriched, purified or separated are optionally single cell suspensions, aggregates, chimeric aggregates and / or structures, including branched structures and / or capsules.

[0321] In embodiments, the mature and / or functional hepatocytes lack expression of AFP and / or fetal CYP3A7.

[0322] In embodiments, mature and / or functional cholangiocytes express MDR transporter genes, aquaporins, CFTR and / or mutants thereof.

[0323] In embodiments, the cell population is a hepatoblastic cell population, optionally expressing Notch2.

[0324] In embodiments, the isolated, purified and / or enriched population is produced in vitro.

[0325] In embodiments, populations of cells are included in the composition with appropriate diluents.

[0326] Suitable diluents include, for example, suitable culture medium or cryopreservation medium containing, for example, serum, serum substitute or serum supplement and / or suitable cryoprotectant (such as dimethyl sulfoxide (DMSO), glyceryl methylcellulose or polyvinyl pyrrolidone). On the other hand, include culture medium supplement components optionally comprising FGF and / or BMP4 agonist, which can be used as supplements for the minimal culture medium of cell culture. Supplements can also include other components discussed herein, such as activin A, Wnt3A, selective inhibitors of GSK-3 (such as CHIR-99021), HGF, dexamethasone, oncostatin M, ascorbic acid, glutamine and B27 supplements.

[0327] In embodiments, functional hepatocytes and / or cholangiocytes are derived from iPS cells of subjects with liver and / or biliary diseases. In embodiments, the disease is a monogenic disease, e.g., cystic fibrosis, Alagille syndrome, progressive familial intrahepatic cholestasis (PFIC types 1, 2, and 3).

[0328] In embodiments, the disease is cystic fibrosis. In embodiments, the subject carries a mutation, e.g., in a cystic fibrosis gene, e.g., deltaF508, 997CFTR del and / or C1 CFTR mutation. Fig. 9 As shown, the described method can generate hepatoblast populations from iPSCs generated / derived from cystic fibrosis patients.

[0329] In embodiments, the disease is a complex biliary disease, optionally primary sclerosing cholangitis or biliary atresia.

[0330] Another aspect includes an implantable construct or in vitro bioartificial liver device (BAL) prepared according to the methods described herein (including the population of cells described herein).

[0331] use

[0332] The functional hepatocytes and / or cholangiocytes and their derivatives described herein can be used in one or more applications. For example, the method can be used to generate a population of hepatocyte lineage cells from iPSCs derived from or obtained from a subject affected by a liver and / or biliary disease.

[0333] Thus, another aspect is a method for generating a cellular model of liver and / or biliary disease comprising:

[0334] i) generating iPSCs from cells derived from or obtained from a subject afflicted with a liver and / or biliary disease; and

[0335] ii) generating hepatocyte lineage cells and / or hepatocyte lineage cells comprising aggregates and / or structures (optionally, branched structures) and / or capsules according to the methods described herein.

[0336] In embodiments, the disease is a monogenic disease, e.g., cystic fibrosis, Alagille syndrome, progressive familial intrahepatic cholestasis (PFIC types 1, 2, and 3). In embodiments, the disease is a complex biliary disease, optionally primary sclerosing cholangitis or biliary atresia.

[0337] Another aspect is a method for generating a cell model of cystic fibrosis comprising:

[0338] i) generating iPSCs from cells derived from or obtained from a subject affected by cystic fibrosis; and

[0339] ii) generating cholangiocyte lineage cells and / or cholangiocyte lineage cells comprising structures (optionally, branching structures) and / or cysts according to the methods described herein.

[0340] For example, functional hepatocytes and / or cholangiocytes can be used for predicting drug toxicology, drug screening, and drug discovery.

[0341] Thus, provided in the embodiments is an assay comprising: contacting a functional hepatocyte and / or cholangiocyte population generated using the methods described herein with a test compound, and measuring: 1) cell expansion, 2) maturation of hepatocytes and / or cholangiocyte specialization, 3) one or more properties of hepatoblasts, hepatocytes and / or cholangiocytes; and / or 4) restoration and / or improvement of one or more defects in a cellular model of liver and / or biliary disease and comparison with a wild-type cell population and / or other controls tested in the absence of the test compound.

[0342] In embodiments, the method further comprises measuring one or more properties of the hepatoblasts, hepatocytes and / or cholangiocytes, including, for example, as measured in Example 9.

[0343] In embodiments, one or more properties of cholangiocytes include:

[0344] a) hepatoblast / cholangiocyte lineage differentiation capacity compared to wild-type iPSCs, optionally evaluating I) the presence and / or number of branching structures and / or cysts; II) cholangiocyte marker expression levels, forms (mature and / or immature forms) and / or expression patterns;

[0345] b) the kinetics of formation of the cholangiocyte lineage compared to wild-type iPSCs; and / or

[0346] c) Transporter activity, optionally CFTR activity.

[0347] For example, using a cAMP agonist such as forskolin to assess CFTR activity can be, for example, by measuring cyst swelling.Example 9 provides an example of a method that can be used to measure CFTR activity.

[0348] For example, it was shown that the chemical corrector VX809 and its Corr-4a were able to restore / enhance mutant CFTR activity in bile duct cysts, as measured by cyst swelling in a forskolin stimulation assay (Example 9). Restoration and / or improvement of this or another property can be assessed when tested with putative or known CFTR treatments, e.g., to provide for evaluation of new drugs / biological products and / or to assess patient-specific responses.

[0349] Another aspect includes functional CFTR assays, including:

[0350] i) contacting cholangiocyte lineage cells, optionally differentiated in capsules from iPSCs derived from patients with CF and / or CF-related diseases, with a cAMP activator, optionally forskolin and IBMX (3-isobutyl-1-methylxanthine)

[0351] ii) measuring swelling, optionally in the presence of a test agent, and

[0352] iii) comparison to wild-type cells or other controls, optionally in the presence or absence of a test agent.

[0353] For example, the test agent can be compared and / or tested in the presence of a CFTR channel potentiator such as VX-770, an FDA-approved drug (also known as Kalydeco) for patients carrying a specific CF gene mutation, G551D.

[0354] The cells described can also be used for cell transplantation. For example, a mixed population of cells, enriched and / or isolated functional hepatocytes and / or cholangiocytes can be introduced into a subject in need thereof, for example, for the treatment of liver disease.

[0355] Thus, one aspect includes obtaining cells and / or preparing isolated hepatocytes and / or cholangiocytes, optionally functional hepatocytes and / or cholangiocytes, according to the methods described herein, and administering the cells to a subject in need thereof, e.g., a subject having a liver and / or biliary disease.

[0356] For example, Yusa et al. (55) described correcting known genetic defects in iPSC-derived hepatocytes and re-transplanting them. The corrected cells were re-transplanted into mice and showed functions that were not previously present in the disease state. Yusa et al. found that the most suitable transposon for their purpose was piggyBac, a moth-derived DNA transposon that can be effectively transposed into mammalian cells including human embryonic stem (ES) cells. The mobile factor is able to remove transgenes on both sides of the piggyBac inverted repeat sequence without leaving any residual sequence. The generated iPSC-3-G5-A7 has a corrected A1AT, compared to the complete genome of the parental fibroblasts and expresses normal A1AT protein when differentiated into hepatocyte-like cells.

[0357] Transposons are optionally used. Other methods of introducing expression constructs include methods based on lentiviruses and adenoviruses. Effective systems for transferring genes into cells in vitro and in vivo are viral-based vectors, including herpes simplex virus, adenovirus, adeno-associated virus (AAV) and lentiviral vectors. Alternative methods for human gene delivery include the use of naked plasmid DNA and liposome-DNA complexes (Ulrich et al., 1996; Gao and Huang, 1995). It should be understood that more than one transgene can be expressed by the vector construct delivered. Alternatively, separate vectors (each expressing one or more different transgenes) can also be delivered to cells.

[0358] Co-transfection (DNA and label on separate molecules) is optionally employed (see, for example, US 5,928,914 and US 5,817,492). Also, labels (such as green fluorescent protein labels or derivatives) are useful within the vector itself (preferably a viral vector).

[0359] Commonly used systems for genome editing in human pluripotent stem cells are the transcription activator-like effector nucleases (TALENS) and the CRISPR-Cas9 system, for example, as described in Joung and Sander 2013 (56) and Ran et al. 2013 (57), respectively.

[0360] Another approach involves ZFNs (zinc finger nucleases), optionally combined with TALENs (transcription activator-like effector nucleases) for gene editing and correction of mutated genes. See, for example, Gaj et al. (58).

[0361] Thus, in an embodiment, the method comprises obtaining cells (optionally, blood cells) from a patient affected by liver and / or biliary disease, genome editing and / or inserting a construct encoding a functional and / or therapeutic protein; and before and / or after inserting the construct, inducing hepatocytes, hepatocytes and / or cholangiocytes according to the methods described herein.

[0362] In embodiments, the population of cells administered is a day 25 / 26 population of cells. In embodiments, the cells are specialized to a hepatocyte or cholangiocyte fate.

[0363] Also included are uses of the cells and compositions comprising the cells for transplantation and / or treatment of a subject in need thereof, e.g., a subject with liver disease.

[0364] In e.g. Figure 8 c) to Figure 8 ESC transplantation-derived hepatocytes engraft and are able to differentiate into cells of the hepatocyte and cholangiocyte lineages, as demonstrated in Example 3. Similarly, Example 9 demonstrates that CFTR-functional cholangiocytes can be generated in vitro and / or in vivo.

[0365] Thus, in an embodiment, a method of transplanting or treating a subject in need using hepatocytes generated according to the methods described herein is provided. In an embodiment, the invention includes the use of cells generated according to the methods described herein for treating a subject in need, e.g., a subject with liver disease and / or biliary disease.

[0366] In embodiments, a therapeutically effective amount is administered.

[0367] Takebe et al. have demonstrated the generation of vascularized and functional human livers from human iPSCs by transplantation of in vitro established liver buds.

[0368] In an embodiment, the obtained cells are derived from autologous cells, for example, iPSCs generated from blood and / or skin cells of a subject. In an embodiment, the PSCs may be iPSCs obtained, for example, from a biopsy, blood cells, skin cells, hair follicles and / or fibroblasts.

[0369] In another embodiment, hepatocytes and / or cholangiocytes generated using the methods described herein are contacted with a test agent in a toxicity screen. CYPs are major enzymes involved in drug metabolism and bioactivation. Various assays can be performed, including drug-drug interaction assays, CYP inhibition assays, and CYP induction assays.

[0370] For example, a drug can increase or decrease the activity of various CYP isozymes by inducing CYP isozymes (CYP induction) or by directly inhibiting the activity of a given CYP (CYP inhibition). Changes in the activity of CYP enzymes can affect the metabolism and / or elimination of various drugs. For example, if one drug inhibits the CYP-mediated metabolism of another drug, the second drug may accumulate in the body to toxic levels.

[0371] In other embodiments, CYP inhibition screening can be performed. Because hepatocytes generated using the methods described herein have been shown to express CYP1A2, CYP2B6, CYP3A4, CYP2B6, CYP2C9, CYP2D6, and / or CYP7A1, one can screen for inhibition of one or more of these isozymes, for example, using LC-MS / MS or fluorometry. CYP IC 50 and / or Ki can be determined.

[0372] In yet other embodiments, the induction of CYP enzymes can be assessed. For example, some compounds induce CYP enzymes, resulting in increased metabolism of co-administered drugs, which are substrates for the induced CYP enzymes. Such co-administered drugs may therefore lose efficacy. CYP enzymes such as CYP1A2, CYP2B6, CYP2C, and CYP3A4 are susceptible to induction. The catalytic activity and mRNA levels of CYP can be measured relative to a control, wherein the results are expressed as multiple inductions.

[0373] Additionally, in other embodiments, drug metabolites can be assessed, for example, a metabolite profile of a drug can be determined.

[0374] In an embodiment, different concentrations of a test agent are added to cells obtained using the methods described herein, and the survival of the cells, CYP 450 isozyme activity, CYP 450 isozyme mRNA levels and / or metabolite profiles are assessed. This method can generally be used, for example, to screen drugs or assess specific toxicity of drugs to patients.

[0375] In an embodiment, functional hepatocytes and / or cholangiocytes are used for tissue engineering. For example, obtaining purified populations of functional hepatocytes and / or cholangiocytes allows the generation of engineered constructs with a defined number of functional hepatocytes and / or cholangiocytes. In other examples, obtaining purified populations of functional hepatocytes and / or cholangiocytes allows the generation of bioartificial liver devices.

[0376] Alternatives to whole-organ liver transplantation investigated include transplantation using isolated cells, tissue engineering of implantable constructs, and in vitro bioartificial liver devices (BAL) (mentioned in 51). As shown on page 451 of this reference, "their future use will depend on the selection and stabilization of cellular components". Although cell lines and non-human cells have been evaluated, there are difficulties in clinical use. The limited sources of human functional hepatocytes and / or cholangiocytes also hinder the development of such devices.

[0377] As mentioned 52 , the liver is the major source of plasma proteins, including albumin, components of the complement system and coagulation, and fibrinolytic factors. Liver failure results in the inability to process low molecular weight substances, some of which are water soluble (ammonia, phenylalanine, tyrosine), but many of which are poorly water soluble and are transported in the blood bound to transport proteins, primarily albumin (medium chain fatty acids, tryptophan and its metabolites, endogenous benzodiazepines and other neuroactive substances, thiols, toxic bile acids, bilirubin, heavy metals, and endogenous vasodilators). This leads to the accumulation of endogenous toxins, which cause multiple auxiliary organ dysfunctions through direct cytotoxicity (e.g., acute tubular necrosis due to jaundice), alterations in functional homeostasis (e.g., hepatorenal syndrome due to hemodynamic disturbances), or a combination of both (e.g., hepatic encephalopathy and coma). Combined dialysis and plasma exchange, selective plasma filtration and adsorption 27 or selective plasma exchange treatment techniques have been developed for liver support therapy. Plasmapheresis techniques utilize, for example, highly selective membranes and albumin dialysis to increase the clearance of albumin-bound toxins versus water-soluble toxins.

[0378] Obtaining functional hepatocytes and / or albumin-producing hepatocytes can be used in the form of BAL. For example, if functional hepatocytes are obtained, it may not be necessary to implement albumin dialysis. ES / iPS-derived hepatocytes are capable of producing albumin protein, which is the main protein secreted from the liver, and ES / iPS-derived hepatocytes can also be used in the form of BAL and albumin dialysis. For example, the production of albumin from human sources is important in BAL. Albumin transports hormones, fatty acids and other compounds including toxic agents. The benefit of albumin dialysis is that toxic compounds bound to albumin can be eliminated from the bloodstream. Human serum albumin, which is used clinically for liver and kidney diseases, is currently only obtained from donated blood. The generation of albumin-secreting cells and / or together with higher liver function activity will be beneficial to the establishment of a BAL system.

[0379] In an embodiment, the method is applied to patient-specific disease hiPSCs and used, for example, to model liver disease. For example, liver or other cells from patients with liver disease can be isolated and processed to obtain hiPSCs, which can then be cultured and induced to differentiate into functional hepatocytes and / or bile duct cells. These cells can be used to assess disease characteristics, such as genes involved in or responsive to the patient's immune cells.

[0380] For example, normal cells and patient-specific disease hiPSCs can be induced into functional hepatocytes and / or bile duct cells and compared. For example, genetic, epigenetic and proteomic analysis of pancreatic progenitor cells and beta cells from normal and patient-specific hiPSCs can be performed. Such detailed analysis can lead to the discovery of signaling pathways, transcriptional regulatory networks and / or cell surface markers that regulate normal human liver development as well as those that play a role in disease.

[0381] The term "subject" as used herein includes all members of the animal kingdom, including mammals, and appropriately refers to humans.

[0382] When applied to isolated cells, the terms "treat," "treating," "treatment," and the like include subjecting the cells to any type of process or condition, or performing any type of manipulation or procedure on the cells. When applied to a subject, the term refers to providing medical or surgical care, health care, or management to an individual.

[0383] When applied to a subject, the term "treatment" as used herein refers to methods (including clinical results) intended to obtain beneficial or desired results and includes medical procedures and applications, including, for example, drug interventions, surgery, radiotherapy, and natural therapy interventions and test treatments. Beneficial or desired clinical results may include, but are not limited to, alleviation or improvement of one or more symptoms or conditions, reduction in the extent of the disease, a stable (i.e., non-deteriorating) state of the disease, prevention of the spread of the disease, delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" may also refer to prolonged survival compared to the expected survival if not receiving treatment.

[0384] As used herein, the terms "administering," "introducing," and "transplanting" are used interchangeably in the context of delivering cells (e.g., functional hepatocytes and / or cholangiocytes) to a subject by a method or route that at least partially localizes the introduced cells at a desired site. The cells may be implanted directly into the liver, or alternatively, the cells may be administered by any appropriate route that enables delivery to a desired location in a subject, wherein at least a portion of the implanted cells or components of the cells remain viable.

[0385] Reagent test kit

[0386] On the other hand, kits are included. Kits can include, for example, one or more of the agonists, antagonists, maturation factors, etc. described above, one or more culture media, containers for growing cells, etc., which can be used for the methods described herein and / or cells amplified and / or prepared according to the methods described herein. In an embodiment, the kit includes instructions for use according to the methods herein. In an embodiment, the kit includes a population of cells produced herein, optionally with instructions, one or more of the agonists, antagonists, maturation factors, etc. described above, including, for example, one or more culture media, containers for growing cells, etc.

[0387] In the scope of understanding the present invention, the term "comprise" and its derivatives as used herein are intended to be open terms, which specify the existence of stated features, elements, components, groups, wholes and / or steps, but do not exclude the existence of other unstated features, elements, components, groups, wholes and / or steps. The foregoing also applies to words with similar meanings, such as the terms "comprising", "having" and their derivatives. Finally, as used herein, degree terms such as "substantially", "about" and "approximately" refer to reasonable deviation amounts of modified terms that do not significantly change the final result. If the deviation does not negate the meaning of the word modified by the modified term, these degree terms should be interpreted as including at least ± 5% deviation of the modified term.

[0388] In understanding the scope of the present invention, the term "consisting of..." and its derivatives as used herein are intended to be open terms, which specify the presence of stated features, elements, components, groups, integers and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0389] Numerical ranges expressed by endpoints herein include all numbers and fractions contained within the range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all numbers and their fractions are assumed to be modified by the term "about". In addition, it should be understood that "a", "an" and "the" include plural objects unless the content clearly indicates otherwise. The term "about" refers to the referenced number plus or minus 0.1% to 50%, 5%-50% or 10%-40%, preferably 10-20%, more preferably 10% or 15%.

[0390] In addition, the definitions and embodiments described in the particular parts are intended to be applicable to other embodiments described herein, and those skilled in the art will appreciate that they are suitable. For example, in the following paragraphs, different aspects of the present invention are defined in more detail. Each aspect defined in this way can be combined with any other one or more aspects, unless there is a clear contrary indication. In particular, any preferred or advantageous feature of the indication can be combined with any other one or more features of the indication that are preferred or advantageous.

[0391] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described only for illustrative purposes and are not intended to limit the scope of the present application. As circumstances may suggest or make expedients, equivalent forms and replacement changes are contemplated. Although specific terms are employed herein, such terms are intended to be descriptive in nature and not for the purpose of limitation.

[0392] The following non-limiting examples serve to illustrate the present invention:

[0393] Examples

[0394] Example 1

[0395] Endoderm induction in EBs

[0396] Figure 1 The strategy for generating hepatocytes from hESCs using embryoid bodies (EBs) is shown in a. Similar to the protocol using monolayer cultures 16 , which involves specialized steps that recapitulate key stages of liver development in the early embryo. 18 EBs are formed from small aggregates cultured in low levels of BMP4 for 24 hours. EBs are then exposed to high concentrations of activin A (hereafter referred to as activin) for five days to induce definitive endoderm, defining populations by the expression of surface markers CXCR4, CKIT, and EPCAM, and transcription factors SOX17 and FOXA2. Figure 1 As shown in b, after five days of activin induction (6 days of co-culture), more than 90% of the induced EB population co-expressed CXCR4 and CKIT or CXCR4 and EPCAM. Intracellular flow cytometric analysis showed that more than 90% of the population expressed SOX17, and more than 80% of the population expressed FOXA2+.

[0397] Studies using a monolayer induction protocol have shown that Wnt signaling enhances Activin-induced endoderm development, likely by enhancing anterior primitive streak formation 10 Addition of Wnt3A to Activin-induced EB cultures resulted in a reproducible increase in the proportion of CXCR4+, SOX17+, and FOXA2+ cells within EBs ( Figure 1 c). Due to the increased expression of CXCR4, the proportion of CKIT+CXCR4+ and CXCR4+EPCAM+ cells increased to greater than 95% of the population. Molecular analysis showed that the elevated levels of SOX17 and FOXA2 expression in Wnt-induced EBs confirmed the flow cytometry data ( Figure 1 d). Addition of Wnt did not accelerate the decrease in Oct3 / 4 expression, but did lead to an increase in T (short tail) expression on day 3 and Goosecoid (GSC) expression on days 4 and 5 ( Figure 1 d) Mouse ESC model 19 and monolayer hESC-derived cultures 10 As shown in Figure 3, Wnt enhances primitive streak formation. Kinetic analysis showed a rapid and dynamic increase in the proportion of CKIT+CXCR4+, SOX17+, and FOXA2+ positive cells between days 3 and 6 of differentiation ( Figure 1 e). Endoderm induction in EBs is affected by the basal medium used. StemPro34 supports more efficient endoderm induction than the neural basal medium we used previously ( Figure 1 f). Induction of highly enriched endoderm is an important first step in the efficient and reproducible generation of hepatocyte-like cells from hPSCs. For example, it was found that induction levels of at least 90% CXCR4+CKIT+ and 80% SOX17+ cells resulted in the development of optimal hepatic lineages.

[0398] Duration of nodal / activin signaling affects liver development

[0399] To specify the CXCR4+CKIT+ population to a hepatic fate, day 6 EBs were dissociated and cells were plated as monolayers on Matrigel-coated plates in the presence of FGF10 and BMP4 for 48 h and then in the presence of bFGF and BMP4 for six days. 20 and human ESC differentiation cultures 16 In , it was demonstrated that FGF and BMP are important for human and mouse specialization. In Si-Tayeb et al., FGF2 and BMP4 were combined and 80%-85% of the generated cells expressed albumin. It was found that the combination of these two factors was necessary to optimize liver induction under the conditions tested ( Figure 2 a). The FGF10 / BMP4 step was included as it was found to increase albumin expression compared to bFGF / BMP4 alone in differentiation cultures ( Figure 2 b) Under these inductive conditions, a significant number of albumin-positive cells continued to develop in culture between days 12 and 24 of differentiation.

[0400] Although BMP4 / FGF-specific steps promote liver development, analysis of day 10 cultures showed that the proportion of SOX17+ and FOXA2+ cells in the culture had dropped significantly, from over 90% to approximately 50% ( Figure 3 a). Without being bound by theory, this reduction suggests that either the day 6 population is contaminated with non-endodermal cells that preferentially expand in the presence of FGF and BMP4, or that the endodermal fate of the cells is not yet fixed and thus some adopt another fate under the conditions used. It was previously demonstrated that prolonged activin / nodal signaling is necessary for the establishment of an endodermal fate by preprimitive streak cells in an in vitro mouse ESC differentiation system 19 Whether prolonged signaling would be useful in human PSCs is unknown. The effect of increasing the duration of this signaling pathway was amplified in the human system by culturing the monolayer for two days prior to the FGF / BMP4 specification step. When induced with activin for an additional two days, the proportion of SOX17+ and FOXA2+ cells measured on day 12 was significantly higher than that of the untreated group ( Figure 3 a). Without being bound by theory, since the total number of cells is lower in the treated population ( Figure 3 b), it is possible that activin signaling at this stage preferentially supports the survival of endoderm cells.

[0401] Prolonged activin treatment maintained the CXCR4+CKIT+ population until day 8 of culture ( Figure 3 c), and resulted in higher levels of expression of genes indicative of liver progenitor cell (hepatoblast) development, including HEX, AFP, ALB, and HNF4a ( Figure 3 d). Cultures generated from untreated CXCR4+CKIT+ endoderm contain contaminating mesoderm as demonstrated by expression of MEOX1, MESP1, CD31, and CD90 and by the presence of CD90+ mesenchymal and CD31+ endothelial cells at day 24 ( Figure 3 d. Figure 3 e). Populations derived from activin-treated endoderm showed reduced expression of mesodermal genes, with a higher fraction of EPCAIVT cells, undetectable CD31+ cells and a much smaller CD90 population ( Figure 3 e). Consistent with these differences, a significantly higher proportion of albumin-positive cells was observed in the treated population compared to the untreated population on day 26 of culture ( Figure 3 f, Figure 3 g). Interestingly, the proportion of AFP positive cells did not differ between the two groups. Without being bound by theory, this suggests that at this stage, the expression of AFP positive cells in the untreated population may not be liver specific.

[0402] Aggregation promotes liver maturation

[0403] Although prolonged activin / nodal signaling promotes liver development, expression levels of genes such as albumin and HNF4α were significantly lower in hESC-derived populations compared with adult liver, indicating that cells in culture at day 26 were still immature. Previous studies have shown that cell aggregation can maintain primary hepatocytes in culture. 21-23 Differentiation phenotypes and in promoting hESC-derived hepatocytes 24 Next, the effect of aggregation on the maturation of a population of day 26 hepatoblasts derived from activin-treated endoderm was investigated. Aggregates were generated from monolayers by a combination of enzyme treatment and artificial dissociation followed by six days of culture in the presence of HGF, dexamethasone (Dex), and oncostatin M (OSM). Figure 4 a). Aggregation affects differentiation and leads to increased expression of multiple genes related to liver function, including ALB (albumin), CPS1 (carbamoylphosphatase synthase 1), TAT (tyrosine aminotransferase), G6P (glucose 6-phosphatase), and TDO (tryptophan 2,3-dioxygenase) ( Figure 4 b). In some cases, the levels are similar to those found in adult liver (ALB) or higher than those found in TDO ( Figure 4 b). Aggregation also increased the expression of several cytochrome P450 genes, including CYP7A1, CYP3A7, and CYP3A4. Levels of CYP3A4 were similar to those found in primary hepatocytes but much lower than those in adult liver ( Figure 4 c). The expression of other P450 genes, including CYP1A2 and CYP2B6 and the phase II enzyme UGT1A1, was not induced to any significant level.

[0404] The cell surface marker asialoglycoprotein receptor-1 (ASGPR-1) is found on mature hepatocytes and has been shown to mark mature cells in hESC differentiation cultures. Aggregation resulted in a significant increase in the proportion of ASGPR-1+ cells detected in the culture, consistently producing populations containing greater than 50% positive cells ( Figure 4 d). Immunostaining showed that ASGPR-1 and E-cadherin were detected on cells in albumin day 32 aggregates. Collectively, these findings suggest that the simple process of aggregation into 3-D structures promotes changes indicative of liver maturation.

[0405] cAMP signaling induces maturation of hESC-derived hepatocyte-like cells.

[0406] To further mature the cells, the role of cAMP signaling was explored. Studies using hepatocyte cell lines have shown that activation of this pathway can induce hepatic gene expression, in part through the induction of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-alpha), a coactivator that works with HNF4a to regulate the expression of many genes involved in hepatocyte function. 25-28 To determine whether cAMP signaling could promote maturation of hESC-derived hepatocyte-like cells, 8-bromoadenosine-3'5"-cyclic monophosphate (8-Br-cAMP), a cell-permeable analog of cAMP, was added to hepatic aggregates from day 32 to day 44 in culture. Treatment with 8-Br-cAMP significantly enhanced the expression of PGC1-a (15-fold), but not HNF4α ( Figure 5 a). 8-Br-cAMP also induced the expression of G6P and TAT, on average 25- and 33-fold, respectively, reaching levels similar to those in adult liver ( Figure 5 a). In contrast, the expression levels of AFP and ALB were downregulated by 8-Br-cAMP. Flow cytometric analysis confirmed the expression analysis of AFP and showed a decrease in the number of AFP-positive cells in 8-Br-cAMP-treated aggregates compared to untreated controls (54% to 26%). The proportion of ALB-positive cells did not decrease, despite the fact that the levels of mRNA decreased ( Figure 5 b). Without being bound by theory, these differences may reflect differences in RNA versus protein expression. Other tissues such as the pancreas also express PGC-1a. However, in contrast to the induction observed in hepatocytes, expression of PGC1-a was not induced by cAMP signaling in hESC-derived insulin-positive pancreatic cells ( Figure 5 c), suggesting that the response may be tissue-specific.

[0407] Cellular uptake of indocyanine green (ICG) is considered a property of adult hepatocytes 29 And used clinically as a test matrix to evaluate liver function 30 cAMP signaling dramatically increased the proportion of cells showing this activity, as demonstrated by the observation that nearly all treated aggregates stained with ICG ( Figure 5 d).

[0408] Confocal microscopy was used to assess the co-expression of ALB and AFP or ALB and HNF4α in day 44 aggregates cultured in the presence or absence of 8-Br-cAMP. Immunostaining analysis was consistent with the flow cytometry data and showed that cAMP-treated aggregates expressed similar levels of ALB but lower levels of alpha-fetoprotein compared to untreated aggregates.

[0409] The levels of albumin (ALB) secreted by hESC-derived monolayer and aggregate populations and by HepG2 cells, Huh7 cells and cryopreserved hepatocytes (PH, lot OSI) were detected using an ELISA assay. The levels of HNF4a protein in the two aggregate populations were comparable, confirming the PCR analysis. Albumin secreted by hESC-derived cells was not affected by 8-Br-cAMP treatment, but was significantly increased by the aggregation step. Low levels of albumin secretion were detected in monolayer cultures at day 20, but increased significantly (approximately 5-fold) in aggregate cultures at day 32. Only low levels were detected in HepG2, Huh7 and PH cells. In contrast, the ability to absorb indocyanine green (ICG) (a characteristic of adult hepatocytes) was enhanced by cAMP signaling (Stieger et al., 2012). The absorption and release of ICG treated and untreated with cAMP was measured in aggregates at day 44.

[0410] cAMP signaling increases metabolic enzyme activities in hESC-derived hepatocytes.

[0411] cAMP signaling also induced changes in the expression patterns of key stage I cytochrome P450 genes, specifically a decrease in the expression level of the fetal gene CYP3A7 and a significant increase in the expression of the adult genes CYP3A4 (2.5-fold), CYP1A2 (18-fold), and CYP2B6 (4.7-fold) ( Figure 6 a). UGT1A1 (an important phase II enzyme) was also significantly induced by 8-Br-cAMP (11-fold) ( Figure 6 a). Induction levels of CYP3A4 and CYP1A2 were significantly higher than those found in primary hepatocytes, whereas levels of CYP2B6 were similar in both populations. UGT1A1 expression in the hESC-derived population did not reach levels found in primary hepatocytes. Without being bound by theory, it is hypothesized that expression of CYP1A2 is restricted to the liver and is only detected after birth. 31 , these findings suggest that cAMP signaling promotes differentiation beyond fetal development.

[0412] The induction of P450 genes by cAMP signaling was observed only in cells in 3D aggregates, whereas no increase in the expression of CYP1A2 and CYP3A4 was detected when cAMP was added to monolayer cultures ( Figure 6b). Expression of PGC1-a and TAT was induced in the monolayer format, likely due to the fact that the promoter regions of these genes contain cAMP response element binding protein (CREB) sites. To further define the variables influencing the cAMP response, aggregates generated from prolonged activin-treated endoderm were compared to those generated from endoderm without activin / nodal treatment for an additional two days. Almost no induction of CYP1A2 and ALB was observed in aggregates from non-treated populations (-Act), suggesting that cAMP signaling only acts on highly enriched, appropriately patterned cells ( Figure 6 c). For the above studies, 8-Br-cAMP was included in the cultures for 12 days (days 32-44). To determine whether changes in gene expression were dependent on continued signaling, cells induced with 8-Br-cAMP for six days and then maintained in the absence of 8-Br-cAMP for the remaining six days were compared to those cultured in 8-Br-cAMP for the full 12 days ( Figure 6 d) CYP1A2 expression is maintained after shorter induction times, suggesting that higher levels of expression are not dependent on continued signaling but instead reflect changes indicative of hepatocyte maturation.

[0413] To explore the functional activity of P450 enzymes, the ability of isozymes to selectively metabolize labeled drugs was measured by high performance liquid chromatography (HPLC). 8-Br-cAMP-treated cells o-deethylated CYP1A2 selective substrate phenacetin ( Figure 6 e) at levels as high as those found in primary cultured hepatocytes. Non-treated cells showed no detectable activity. CYP2B6 activity measured by hydroxylation of bupropion was also detected in 8-Br-cAMP-treated cells at levels comparable to those found in primary hepatocytes ( Figure 6 f). Phase II metabolic enzymes (including arylamine N-acetyltransferases NAT1 and / or NAT2 ( Figure 6 g) and UDP glucuronosyltransferase (UGT) ( Figure 6 H)) analysis showed higher activity than that of primary cultured hepatocytes, indicating that cAMP signaling induces upregulation of expression of a wide range of enzymes, consistent with the maturation of the population. Taken together, these observations suggest that cAMP signaling promotes maturation of hESC-derived hepatocyte-like cells in 3-D aggregates.

[0414] In addition, the inducibility of the metabolic activity of two key enzymes, CYP1A2 and CYP3A4, was also evaluated. Cells treated with 8-Br-cAMP were able to metabolize the CYP1A2 selective substrate phenacetin. Inducing cells with lansoprazole for 72 hours resulted in a 3.4-fold increase in this activity. Untreated (8-Br-cAMP) cells had low levels of activity that were not inducible. Two independent primary hepatocyte samples showed levels lower than or equivalent to basal metabolic activity, but showed higher levels of induction (18-fold and 9-fold). CYP3A4 activity was measured by the ability of cells to metabolize testosterone to 6β-hydroxytestosterone. Cells treated with 8-Br-cAMP showed this activity. The addition of the CYP3A4 inducer rifampicin increased the activity by 2.2 times, indicating that this enzyme can also be induced in hESC-derived cells. When observing CYP1A2, CYP3A4 activity was barely detectable in uninduced cells. Primary hepatocytes showed low but significant levels of CYP3A4 induction.

[0415] Hepatic specialization and maturation from other hPSC lines

[0416] To determine whether the methods detailed above are broadly applicable to different human pluripotent stem cell lines, the protocol was used to differentiate the hESC lines H9 and H1 and the induced pluripotent stem cell (iPSC) line 38-2 towards a hepatic fate. Following the Wnt3a / Activin induction step, day 6 EBs from all three cell lines contained a high proportion of CKIT+CXCR4+ and CKIT+EPCAM+ cells ( Figure 7 a). Although the proportion of EPCAIVT cells was high in all EBs, H9-derived cells expressed substantially higher levels of EPCAM than cells generated from other cell lines. The level of EPCAM expression correlated with the extent of endoderm induction, as greater than 95% of the H9-derived population expressed SOX17+ and FOXA2+, whereas only 65-70% of iPSC-derived cells expressed these transcription factors ( Figure 7 a). These findings suggest that surface marker analysis alone is not sufficient to monitor endoderm development and that quantitative analysis of SOX17 and FOXA2 expression is necessary to measure induction of this germ layer. As observed for HES2 cells, prolonged activin / nodal signaling enhances hepatic development in the CKIT+CXCR4+ population from each cell line ( Figure 7 b). However, the duration of activin treatment required to generate significant levels of ALB expression varied between cell lines. While higher levels of ALB expression were achieved after two days of activin treatment of H9-derived cells, H1 and 38-2 cells required four additional days of activin signaling. As a result of this treatment, it was possible to generate cultures containing 90%, 85%, and 70% ALB+ cells from the H9, H1, and 38-2 cell lines, respectively ( Figure 7c). H9-derived cells at day 26 of differentiation showed a cobblestone morphology very similar to cultured hepatocytes ( Figure 7 d).

[0417] The addition of 8-Br-cAMP did induce significant levels of expression of CYP3A4 (16-fold), CYP1A2 (100-fold), and CYP2B6 (10-fold), as well as the phase II enzyme UGT1A1 (16-fold) in H9-derived aggregates ( Figure 7 e). The magnitude of induction was substantially greater than that in HES2-derived cells, and the expression levels of CYP1A2 and CYP3A4 were significantly higher than those in primary hepatocytes. The reason for the difference in induction between the two hESC cell lines is currently unknown. 8-Br-cAMP also induced the expression of these enzymes in hiPSC-derived aggregates to levels as high as those in primary hepatocytes ( Figure 7 e).

[0418] As observed with the HES2 cell line, H9-derived cells have CYP1A2 activity induced by lansoprazole. H9 and iPSC-derived cells also show CYP3A4 activity induced by rifampicin. Induction of CYP1A2 activity cannot be detected in iPSC-derived cells, which may reflect the suboptimal differentiation of this population.

[0419] Microarray analysis of cAMP-stimulated hepatic populations

[0420] In order to further evaluate the results of cAMP induction and assess the developmental status of hESC-derived liver populations relative to primary hepatocytes, microarray analysis was performed to compare the global expression profiles of different populations. A total of 23,038 filtered transcripts were used for the final analysis. Two-way unsupervised hierarchical cluster analysis showed that the three groups were displayed as different populations. The three cAMP-induced populations were most similar to each other, while the three primary hepatocyte populations showed the most divergent expression patterns. FDR-corrected ANOVA (q < 0.05) was used to identify 784 transcripts, showing the most statistically significant differences in all three sample groups. The hierarchical cluster visualization of these data identified clusters of highly expressed transcripts in each biological group. These clusters include 181 transcripts in primary hepatocytes, 106 transcripts in 8-Br-cAMP-induced cells, and 80 transcripts in non-treated cells. Genes enriched in 8-Br-cAMP-induced cells included ontogenetic categories of most of the key P450 enzymes and those involved in many aspects of liver function, including gluconeogenesis, glucose homeostasis, and lipid metabolism. The highest level of expression in primary hepatocytes included immune system, inflammation-related, and MHC genes. Clusters detected in non-induced hESC-derived cells did not contain any ontogenetic categories of enriched genes.

[0421] For a more detailed comparison of populations, the selected transcript subsets of proteins encoding key aspects of liver function are analyzed. These include subsets of phase I and II drug metabolizing enzymes, transporters, coagulation factors, lipoproteins, nuclear receptors and transcription factors as well as general liver enzymes and other functional molecules. The analysis of these data shows that the expression of many genes is at a level comparable to that of hESC-derived cells and primary hepatocytes processed by 8-Br-cAMP. Compared with untreated cells or primary hepatocytes, the selected genes in each category are expressed at significantly higher levels in cells processed by 8-Br-cAMP. These include phase I enzymes CYP1A2 and CYP3A4, phase II enzyme SULT2A1, transporter SLCO1B1, general liver enzymes TAT, G6P and TDO (responsible for the metabolism of tyrosine metabolism, gluconeogenesis and tryptophan, respectively), surface receptors ASGPR-1 and ALB that confirm qPCR and functional studies. Since the cellular uptake of ICG in hepatocytes is regulated by the organic anion transporters SLCO1B1 and SLCO1B3 and the Na+-independent transporter SLC10A129, the induction of their expression is consistent with the finding that cAMP-treated aggregates show higher levels of ICG uptake. Taken together, these data from microarray analysis indicate that induction of hepatoblast-stage aggregates with cAMP results in global expression changes indicative of hepatocyte maturation. Based on these analyses, the hESC-derived hepatocytes generated by this method appear to represent a developmental stage at least equivalent to primary human hepatocytes.

[0422] discuss

[0423] In order for hPSC-derived hepatocytes to be useful for drug metabolism analysis and for transplantation to treat liver disease, the cells must be relatively mature and display many characteristics of adult hepatocytes, including measurable levels of key stage I and stage II drug metabolizing enzymes. To date, a number of different studies have shown that it is possible to generate immature hepatocyte lineage cells from hESCs and hiPSCs using staged protocols designed to recapitulate key developmental steps in the embryo. The success of these studies reflects the fact that the pathways controlling differentiation at the early stages are reasonably well defined. In contrast, the factors and cellular interactions that control hepatocyte maturation are poorly understood, and therefore only a few studies have reported the development of metabolically functional cells. Duan et al. 12showed that it was possible to derive hepatocytes from H9 hESCs, displaying levels of CYP1A2, CYP3A4, CYP2C9, and CYP2D6 enzyme activities equivalent to those found in primary hepatocytes. The serum used by Duan et al. in their method included a number of factors, some of which varied between serum batches. The factors responsible were not identified. While these findings suggest that relatively mature hESC-derived hepatocytes can be generated, the study does not provide details about the pathways that promote maturation, nor does it suggest that the strategy is broadly applicable to other hPSC cell lines. As Duan et al. Figure 6 In, they measured the metabolic drugs in the hepatocytes from human ES cells (H9). Phenacetin induces and provides the assessment of CYP1A2 activity, midazolam, bufuralol and diclofenac induce and provide the assessment of CYP3A4 and CYP2B6 and CYP2D6 respectively. In the HES2 cell line such as described herein, compared with what is seen in primary hepatocytes, the enzyme activity level (CYP1A2 and CYP2B6) is almost equal. In qPCR analysis, the level of expression of CYP1A2 and CYP2B6 in H9 cells is 5-8 times higher than those found in HES2 cell line. The cells produced by the method described herein are closer to primary hepatocytes in terms of CYP enzymes CYP1A2 and CYP2B6. Similarly, compared with primary hepatocytes, almost the same or comparable level of CYP2D6 expression is seen in the cells generated using the method.

[0424] Several other groups have shown that forced expression of specific transcription factors in hESC-derived populations alone or in conjunction with co-culture with Swiss 3T3 cells can promote maturation that results in the expression of key metabolic enzymes. 32-34 However, the major disadvantage of this approach is the need for viral transduction for each experiment and the variability of these manipulations, including poor infection efficiency and differences in the availability of transcription factors to establish appropriate expression levels. When compared to primary hepatocytes, expression levels in hESC-derived populations generated by this approach are much lower than those found in primary hepatocytes.

[0425] Here, a deep understanding of the pathway regulating maturity is provided. It is also shown that the combination of 3D aggregation and cAMP signal transduction plays a key role in the maturation of hepatoblasts. In addition, the activin / nodal signal transduction after endoderm induction is necessary for the optimal generation of liver progenitor cell groups, and the enriched progenitor cell groups are useful for the separation of mature populations. The combination of these three different steps for example causes the generation of hepatocyte-like cells, which show expression profiles similar to those found in primary adult liver cells and the level of functional metabolic enzymes.

[0426] The observation that sustained activin / nodal signaling within the CXCR4+C-KIT+ population is useful for the generation of mature hepatocytes highlights the importance of proper manipulation of early cells for the efficient generation of mature cells. The effects of prolonged activin / nodal signaling between days 6 and 8 of differentiation (for HES2 cells) affect gene expression patterns and the proportion of albumin-positive cells detected on day 26 of culture. This step also promotes the development of a population of hepatocytes that, in response to cAMP, mature to produce metabolically functional hepatocytes. This additional signaling step does not compensate for poor endoderm induction, as the day 6 EB target population includes greater than 95% CXCR4+CKIT+EPCAM+SOX17+ cells. Instead, without being bound by theory, this appears to reduce contaminating mesodermal derivatives (CD90+ and CD31+ cells), perhaps due to the inability of activin to induce these lineages or promote their survival in the absence of BMP or FGF. In addition to reducing mesoderm contamination, the additional Activin culture step may also play a role in properly patterning endoderm to ventral foregut fates, as this pathway is important in the intestinal tract. 35，36 It has been previously shown that sustained activin / nodal signaling also affects pancreatic development from hESCs. 18 .

[0427] In certain embodiments, the maturation phase of the protocol includes two distinct but interdependent steps. The first is the generation of 3D aggregates. Previous studies have shown that 3D culture can improve hepatocyte survival and primary fetal hepatocytes in mice and humans. 24，37，38 Recently, Miki et al. reported that 3D culture in a perfusion bioreactor can enhance the differentiation of hESC-derived hepatocytes, suggesting that the 3D environment may be important for the maturation of the cells. However, the magnitude of these differences is difficult to interpret as no comparisons were made to fetal and adult liver controls. The present study has extended these findings to show that culture in a static 3D format promotes differentiation, as demonstrated by a significant increase in the expression of key hepatic genes such as ALB, CPS1, G6P, and TDO and by a significant increase in the proportion of cells expressing ASGPR1, a receptor found in mature hepatocytes. Maturation within the aggregates is also important for cAMP responsiveness as genes such as CYP1A2 and CYP3A4 were not induced in 2D culture. The mechanism by which aggregation promotes maturation is currently unclear, but without being bound by theory, it may be related to enhanced cellular interactions and possibly the generation of polarized epithelial cells within the 3D structure, mimicking a certain degree of cellular morphology of hepatocytes within the liver.

[0428] The second step of the maturation strategy is to activate the cAMP pathway within the 3D aggregates, optionally by adding the cell permeable and more slowly hydrolyzed cAMP analog 8-Br-cAMP. Specific genes in the liver, including PGC1-α, TAT, and G6P, contain CREB elements in their promoter regions and are therefore direct targets of cAMP signaling. 25，39，40 . Given this, these target genes were induced in 2D monolayers as well as in 3D aggregates. PCR and microarray analysis clearly demonstrated that the effects of cAMP signaling extend beyond the induction of target genes, as activation of the induced pathways altered the expression patterns of genes associated with different aspects of hepatocyte function, including drug metabolism, mitochondrial biogenesis, lipid synthesis, and glucose metabolism. These global changes support the interpretation that cAMP signaling promotes maturation of hepatoblasts. The fact that sustained cAMP signaling is not required to maintain elevated expression levels further supports the interpretation that the effect is one of maturation and not simply induction and maintenance of expression of specific genes. Some of the most notable changes in expression observed were that levels of major drug-metabolizing enzymes including CYP1A2, CYP3A4, and UGTiA1 were detected at or above those found in primary human hepatocytes. Transcript levels are indicative of function, as HES2-derived cells displayed levels of functional enzymes comparable to those of primary hepatocytes. Similar patterns of induction were observed in hepatocyte-like cells from two hESC lines and one hiPSC line, indicating that this maturation strategy is broadly applicable.

[0429] Endocrine hormones such as insulin and glucagon can affect cAMP levels in the adult liver, which has acute effects on glucose metabolism as well as chronic effects by regulating gene expression. Under fasting conditions, upregulation of cAMP levels leads to rapid induction of PGC1-α and genes involved in gluconeogenesis, thereby ensuring energy supply. 41，42 In addition to fasting conditions, it has been reported that PGC1-α expression is significantly upregulated in mouse liver one day after birth. 43 This upregulation is thought to rapidly promote maturation of neonatal hepatocytes. Without being limited by theory, the effects of cAMP signaling on hESC-derived hepatoblasts through upregulation of PGC-1α expression may recapitulate to some extent the changes observed in the liver during fasting and / or in the hepatocyte lineage at birth, resulting in the generation of cells that display many of the properties of mature cells.

[0430] In summary, the inventors have defined for the first time the steps that promote the maturation of hepatocyte lineage cells from hPSCs, resulting in the generation of cells that display gene expression profiles similar to those of primary human hepatocytes. The development of metabolically functional cells is an important endpoint because it suggests that these advances will enable routine production of hPSC-derived hepatocyte-like cells for drug metabolism analysis in the pharmaceutical industry. cAMP-induced cells also provide an ideal candidate population for the development of bioartificial liver devices and ultimately for transplantation of cell replacement therapies for the treatment of liver disease.

[0431] Materials and methods:

[0432] HPSC culture and differentiation

[0433] HPSCs were maintained on irradiated mouse embryonic feeder cells in hESC medium consisting of DEME / F12 (50:50; Gibco) supplemented with 20% knockout serum replacement (KSR) as previously described. 44 hESCs were passaged on Matrigel before generating embryoid bodies (EBs). TM Coat the plates for 1 day to deplete the feeder cell population. At this stage, hESCs were dissociated by 0.25% trypsin-EDTA to generate clusters as previously described. 44，45 and then cultured in serum-free differentiation (SFD) medium in the presence of BMP4 (3 ng / ml) for 24 hours (day 0 to day 1). On day 1, EBs were collected and cultured in The cells were cultured in induction medium A for another 3 days. Supplemented with glutamine (2 mM), ascorbic acid (50 pg / ml; Sigma), MTG (4.5 × 10 -4 M; Sigma), basic fibroblast growth factor (bFGF; 2.5 ng / ml), activin A (100 ng / ml), Wnt3a (25 ng / ml), and BMP4 (0.25 ng / ml). On day 4, EBs were collected and EBs were cultured again. Supplemented with bFGF (10 ng / ml), Activin A (100 ng / ml), Wnt3a (25 ng / ml) and BMP4 (0.25 ng / ml) (Medium B). EBs were collected on day 6, dissociated into single cells and cultured on Matrigel. TM 4 × 10 5The cells were cultured for 2 days at a concentration of 10 cells / mL, without WNT3A in the culture medium, and with activin A at a concentration of 50 ng / ml. On day 8, culture medium B was replaced with a liver-specific medium comprising Iscove's modified Dulbecco's medium (IMDM), supplemented with 1% volume / volume B27 supplement (Invitrogen: A11576SA), ascorbic acid, MTG, FGF10 (50 ng / ml) (from day 8 to day 10), bFGF (20 ng / ml) (from day 10 to day 14), and BMP4 (50 ng / ml). From day 8 to day 14, the culture medium was changed every 2 days. To promote the maturation of HES2-derived hepatocytes, they were cultured in maturation medium A for 12 days. Maturation medium A consisted of IMDM with 1% vol / vol B27 supplement, ascorbic acid, glutamine, MTG, hepatocyte growth factor (HGF) (20 ng / ml), dexamethasone (40 ng / ml) and oncostatin M (20 ng / ml). Aggregates were generated from the population on day 26 of culture. To generate aggregates, cells were dissociated with collagenase and TrypleLE and then cultured at a concentration of 6×105 cells per well in a six-well ultra-low cluster plate in maturation medium A supplemented with Rho kinase inhibitor and 0.1% BSA. Aggregates were maintained under these conditions until day 32, with medium changes every 3 days. On day 32, the medium was changed to maturation medium B, which consisted of hepatocyte medium (HCM) (Lonza: CC-4182) without EGF. 10 mM 8-Br-cAMP (Biolab: B007) was added at this stage. The medium was changed every 3 days. To generate hepatocyte-like cells from H9, H1, and IPS cells, the following changes were made to the liver-specific medium. The concentration of bFGF was increased to 40 ng / ml, and the basal medium was switched from IMDM to H16 DMEM for culture from day 8 to day 14, and then to H16 DMEM with 25% Ham's F12 from day 14 to day 20. IMDM was replaced with H21 DMEM with 25% Ham's F12 and 0.1% BSA, and maturation medium A was used from day 20 to day 32. All cytokines were human and purchased from R&D Systems unless otherwise stated. EB and monolayer cultures were maintained in a 5% CO2, 5% O2, 90% N2 environment. Polymeric cultures were maintained in an ambient air environment of 5% CO2.

[0434] Flow cytometry

[0435] Flow cytometric analysis was performed as previously described 45For cell surface markers, staining was performed in PBS with 10% FCS. For intracellular proteins, cells fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in PBS were stained. Cells were permeabilized with 90% ice-cold methanol for 20 min for SOX17 and FOXA2 staining as previously described. 45 Albumin and alpha-fetoprotein staining was performed with 10% FCS and 0.5% saponin (Sigma) in PBS. The stained cells were analyzed using a LSRII flow cytometer (BD).

[0436] Immunostaining

[0437] As previously described 45 Immunostaining was performed at 37 ° C. Cells were fixed in culture wells with 4% PFA for 15 minutes at 37 ° C, washed three times with DPBS (with calcium chloride and magnesium chloride) + 0.1% BSA, and then permeabilized in washing buffer with 0.2% Triton-X100 for 20 minutes. After an additional 3 washes in DPBS (with calcium chloride and magnesium chloride) + 0.1% BSA, cells were blocked with protein blocking solution (DAKO; X0909) for 20 minutes at room temperature. To evaluate albumin and alpha-fetoprotein positive cells, cells were stained with goat anti-ALB antibody (Bethyl) or rabbit anti-AFP antibody (DAKO) for 1 hour at room temperature. The concentration of the isotype control matched the primary antibody. To visualize the signal, the cells were then incubated with donkey anti-goat Alexa 488 antibody (Invitrogen) or donkey anti-rabbit Cy3 antibody (Jackson Immuno Research) for 1 hour at room temperature. For SOX17 staining, cells were fixed, permeabilized and blocked as described above. Cells were incubated with goat anti-SOX17 (R&D) overnight at 4°C. Signals were visualized by incubating cells with donkey anti-goat Alexa488 (Invitrogen). For ASGPR-1 staining, cells were plated on Matrigel TM Aggregates were cultured on coated coverslips for 1 day. After attachment and spreading, cells were fixed with 4% PFA for 15 min at 37°C and then permeabilized with cold 100% methanol for 10 min. Cells were washed and blocked as described above. Fixed cells were incubated with goat anti-ASGPR-1 (Santa Cruz) overnight at 4°C and then with rabbit anti-ALB (DAKO) for 1 h at room temperature. Signals were visualized by incubation with donkey anti-goat Alexa488 antibody and donkey anti-rabbit CY3 antibody. Primary and secondary antibodies were diluted in DPBS + 2% BSA + 0.05% Triton-X100. DAPI (Invitrogen) was used for the detection of 100% leukemia cells. Gold anti-fluorescence quencher was used to counterstain the cell nuclei. The stained cells were visualized using a fluorescence microscope (Leica CTR6000) and images captured using Leica Application Suite software.

[0438] Quantitative real-time PCR

[0439] use Total RNA was prepared using the ELISA kit (Ambion) and treated with RNase-free DNase (Ambion). 500 ng to 1 μg of RNA was PCR-PCR-treated using random hexamers and oligo(dT). III reverse transcriptase (Invitrogen) to produce cDNA. The PCR products were reverse transcribed into cDNA using the QuentiFast SYBR Green PCR Kit (Quiagen) as previously described45. Quantitative PCR was performed using ep realplex (Eppendorf). Expression levels were normalized to the housekeeping gene TATA box binding protein (TBP). To measure the expression of UGT1A1, the expression of related genes was assessed using the delta-delta CT method relative to the levels of 8-Br-cAMP(-) treated cells. Total human adult and fetal liver RNA was purchased from Clontech. Two RNA samples of primary hepatocytes were provided by Dr Stephen C. Strom (University of Pittsburgh), and the third sample was purchased from Zenbio (lot number: 2199). Two primary hepatocyte samples were cultured for 2 days and collected. One (HH1892) was isolated from a 1-year-old white male, while the other (HH1901) was isolated from a 14-month-old male, whose liver was transplanted due to cholestasis. The third sample (Zenbio: lot number 2199) was isolated from a 48-year-old male white organ donor.

[0440] Indocyanine green uptake by liver aggregates

[0441] Indocyanine green (ICG, Sigma) solution was added to the cells at a final concentration of 1 mg / mL ICG in HCM (Lonza). The cells were incubated at 37°C for 1 hour, washed 3 times with PBS, and then examined with an inverted microscope (Leica). Drug metabolism analysis by HPLC

[0442] Liver aggregates were incubated for 24 or 48 h in HCM containing the CYP1A2 substrate phenacetin (200 μM), the CYP2B6 substrate bupropion (900 μM), the NAT1 / 2 substrate sulfamethazine (SMZ) (500 μM), or the total UGT substrate 4-methylumbelliferone (4-MU) (200 μM). Following incubation, aliquots of the culture medium were collected and the levels of the metabolites were quantified using individually optimized high-performance liquid chromatography assays. The HPLC method based on Loboz et al. 46 Hydroxybupropion levels were determined by 47 4-MU glucuronide was measured by high performance liquid chromatography coupled with tandem mass spectrometry. Cryopreserved hepatocytes were thawed and plated at 1 × 10 4 The cells were plated on collagen culture dishes at a density of 10 cells / mL for 24 or 48 hours. After 24 or 48 hours of culture, the supernatants were collected and the activities of CYP1A2, CYP2B6, NAT1 / 2 and total UGT were measured.

[0443] Chip processing and data analysis

[0444] Follow the operation criteria of standard Affymetrix company at the University Health Network Genomics Center, run RNA samples on Affymetrix human gene ST V1.0 chip.In brief, for each sample, the starting material of the total RNA of 300ng is used as the input of Ambion company WT expression test kit.2.7 micrograms of amplified cDNA are then fragmented, labeled and hybridized to Affymetrix human gene ST 1.0 chip 18 hours (45 ℃, at 60RPM).Gene chip fluidics station P450 fluid workbench washing array is used, and array is scanned with Affymetrix gene chip scanner 7G.After scanning, each chip is checked, and it is found that the quality control guide is passed through Affymetrix.Original CEL file is imported into Genspring software (Agilent, v11.5.1), and the ExonRMA16 algorithm based on HuGene-1_0-st0v1_na31_hg19_2010-09-03 is used to summarize probe level data. In addition, each gene was normalized to the median value among all considered samples. All statistics were performed on Log2 transformed data. A total of 28869 transcripts were represented on the array.

[0445] As a first step, transcripts were filtered to remove those whose expression percentiles measured in all 3 sample groups were consistently below the 20th. Unsupervised hierarchical cluster analysis using Pearson center distance under the average linkage rule was used to resolve overall similarities and differences between samples and groups. Directed statistical analysis between the 3 sample groups was performed using ANOVA with Benjamini and Hochberg false discovery rate (FDR, q < 0.05) 48 To find sets of biologically significant differentially expressed transcripts, the corrected Benjamini and Yuketieli hypergeometric test was used at a significance level of q < 0.1. 49 Gene Ontology (GO) analysis was performed. More specifically, two a priori defined sets of specific transcripts were examined: transcripts involved in a specific liver-related activity of interest; and transcripts found to be expressed and based on the HOMER database. 55 Public information on liver-specific transcripts.

[0446] Example 2

[0447] CHIR99021 is a selective inhibitor of GSK3 that has been reported to mimic the canonical Wnt signaling pathway. CHIR99021 was tested as a replacement for wnt3a (e.g., added in combination with activin) in the induction of embryoid bodies and for monolayer induction of definitive endoderm from hPSCs. See Figure 8 a) and Figure 8 b), The proportion of CKIT+CXCR4+ and CXCR4+EPCAM+ cells induced with CHIR99021 replacing Wnt3a was greater than 95% of the population and comparable to that seen with activin / wnt3a induction.

[0448] Figure 8 (a) and Figure 8 (b) shows that CHIR99021 can induce definitive endoderm cells. Figure 8 (a) is a flow cytometric analysis showing the proportions of CXCR4+, CKIT+, and EPCAM+ cells in activin / CHIR99021 at day 6 of embryoid body induction using activin / wnt3a. Figure 8 (b) is a flow cytometric analysis showing the proportion of CXCR4+, CKIT+, and EPCAM+ cells in Activin / CHIR99021 at day 6. Figure 8 (c) and Figure 8 (d) shows day 7 of monolayer induction with (c) Activin / wnt3a or (d) Activin / CHIR99021.

[0449] method

[0450] Induction of definitive endoderm using GSK3β inhibitor

[0451] For EB induction, CHIR99021 (0.3 μM) replaced Wnt3a for endoderm induction.

[0452] For monolayer induction, hPSCs were maintained on irradiated mouse embryonic feeder cells in hESC medium consisting of DEME / F12 (50:50: Gibco) supplemented with 20% knockout serum replacement (KSR) as previously described (Kennedy et al., 2007). hESCs were passaged onto Matrigel-coated surfaces (usually 12-well plates) for 1 day prior to induction of endoderm in monolayer culture. On day 0, cells were cultured in RPMI-based medium supplemented with glutamine (2 mM), MTG (4.5 × 10 -4 M: Sigma), activin A (100 ng / ml), CHIR99021 (0.3 μM), or Wnt3a (25 ng / ml). From day 1 to day 3, the medium was replaced every day with RPMI supplemented with glutamine (2 mM:), ascorbic acid (50 pg / ml; Sigma), MTG (4.5 × 10 -4 M; Sigma), basic fibroblast growth factor (bFGF; 5 ng / ml), activin A (100 ng / ml). From day 3 to day 5, cells were cultured in SFD-based medium supplemented with glutamine (2 mM:), ascorbic acid (50 pg / ml; Sigma), MTG (4.5 × 10 -4 M; Sigma), basic fibroblast growth factor (bFGF; 5 ng / ml), activin A (100 ng / ml). The medium was changed every other day. As described above, by treating with FGF and BMP pathway agonists, the definitive endoderm was specified to a hepatic fate on day 7.

[0453] Example 3

[0454] Ectopic liver tissue in NSG mice

[0455] Transplanted hESC-derived hepatocytes engraft and generate cells expressing hepatocyte differentiation markers.

[0456] Figure 8 (e) Figure 8 (f) and Figure 8 (g) shows engraftment of ES-derived hepatocytes prepared using the methods described in Example 1, except that the GSK3 inhibitor CHIR99021 was used to make the transplanted cells described in Example 2.

[0457] Figure 8 (e) Figure 8 (f) and (g), Figure 8 (h) Ectopic liver tissue in NSG mice. Figure 8 (e) is an exemplary photomicrograph of H&E staining of the mesenteric region, showing clusters of hESC-derived hepatocytes (arrows) 2 months after transplantation. Magnification is 5X. Intestine (arrow), implanted cells (arrow head). Figure 8 (f) shows the Figure 8 (e) High magnification (10×) micrograph of an H&E stained section.

[0458] Figure 8 (g) Figure 8 (h) Immunohistochemical staining shows the presence of hESC-derived cells in the mesenteric region 2 months after transplantation. Double staining for human albumin (Alexa488: green) (shown as arrows) and CK19 (Cy3: red) (shown as arrow heads) shows that the transplanted cells have the potential to differentiate into hepatocyte and cholangiocyte lineages. hESC-derived hepatocyte-like cells are observed as albumin-positive cells (arrows), while cholangiocyte-like cells express CK19 and are found in duct-like structures (arrow heads).

[0459] method

[0460] Ectopic liver tissue in NSG mice

[0461] Six-week-old NSG mice were obtained from Jackson Laboratory (Bar Harbor, Maine, USA) and stored in UHN animal facilities. Aggregates (day 27) included hESC-derived hepatic progenitor cells (hepatoblasts), and hESC-derived CD34+ endothelial cells were suspended in 50 μl of matrigel (BD Biosciences) and kept on ice until transplantation. Recipient mice were anesthetized with 1-3% isoflurane and laparotomy. The mesenteric region was exposed, and a cell mixture with matrigel was positioned in the mesenteric region and covered with absorbable hemostatic agent Surgicel (Ethicon360, USA). Two months after transplantation, mice were killed and the presence of hESC-derived cells was assessed by histological analysis.

[0462] Example 4

[0463] Small molecules related to the Wnt / β-catenin pathway can expand hepatic progenitor cells. Hepatic progenitor cells (H9) on day 27 were dissociated and cultured at 1×10 4 The cells were seeded at a density of 100 cells on 96-well matrigel-coated dishes. The cells were treated with different concentrations of CHIR99021 (0.3 μM, 1 μM and 3 μM) and cultured for 9 days. The increase in the proportion of hepatic progenitor cells was examined by counting the number of cells compared to the number of cells on day 27 without treatment ( Fig. 9a).

[0464] Inhibition of Wm and MEK / ERK pathways increased the expression of genes associated with phase I drug-metabolizing enzymes.

[0465] Gene expression of CYP3A4 in 3D liver aggregates cultured at day 44 with small molecules was associated with inhibition of Wnt / β-catenin signaling (XAV939: 1 μM) and MEK / Erk signaling (PD0325901: 1 μM). Together with 8-Br-cAMP, inhibition of Wnt and MEK / Erk signaling had the effect of increasing gene expression of CYP3A4 ( Fig. 9 d).

[0466] Gene expression of CYP1A2 in 3D liver aggregates cultured at day 44 with small molecules was associated with inhibition of Wnt / β-catenin signaling (XAV939: 1 μM) and MEK / Erk signaling (PD032590: 1 μM). Together with 8-Br-cAMP, inhibition of Wnt and MEK / Erk signaling had the effect of increasing gene expression of CYP1A2 ( Fig. 9 d).

[0467] Gene expression of ALB in day 26 hepatocyte-like cells cultured on several different extracellular matrices (ECM). Endoderm cells from embryoid bodies (EBs) at day 26 were dissociated as described above and cultured at 4×10 5 The cells were seeded on ECM at a cell density of 10 cells and cultured in liver specialization and maturation medium. Total RNA was extracted from cells at day 26 and the level of albumin expression was measured. Expression levels were determined by fold difference compared to gelatin-coated culture conditions ( Fig. 9 f).

[0468] method

[0469] Induction of definitive endoderm with GSK3β inhibitor.

[0470] For EB induction, CHIR99021 (0.3 μM) replaced Wnt3a during endoderm induction.

[0471] For monolayer induction, hPSCs were maintained on irradiated mouse embryonic feeder cells in hESC medium consisting of DEME / F12 (50:50: Gibco) supplemented with 20% knockout serum replacement (KSR) as previously described (Kennedy et al., 2007). hESCs were passaged to 12-well Matrigel-coated plates for 1 day prior to induction of endoderm in monolayer culture. On day 0, cells were cultured in RPMI-based medium supplemented with glutamine (2 mM), MTG (4.5 × 10 -4M: Sigma), activin A (100 ng / ml), CHIR99021 (0.3 μM), or Wnt3a (25 ng / ml). From day 1 to day 3, the culture medium was changed every day and consisted of RPMI supplemented with glutamine (2 mM), ascorbic acid (50 μg / ml; Sigma), MTG (4.5×10 -4 M; Sigma), basic fibroblast growth factor (bFGF; 5 ng / ml), activin A (100 ng / ml). On days 3 and 5, cells were cultured in SFD-based medium supplemented with glutamine (2 mM), ascorbic acid (50 μg / ml; Sigma), MTG (4.5×10 -4 M; Sigma), basic fibroblast growth factor (bFGF; 5 ng / ml), activin A (100 ng / ml). The medium was changed every other day. On day 7, definitive endoderm cells began to differentiate using liver-specific medium as described above.

[0472] Example 5

[0473] Treatment with cAMP induces maturation of cell aggregates

[0474] Cell aggregates were generated as in Example 1.

[0475] Cell aggregates were cultured in HGF, Dex, and OSM until day 32, when cAMP was added. OSM was removed when cAMP analogs and / or cAMP agonists were added. In some experiments, HGF was also removed from the culture when cAMP analogs and / or cAMP agonists were added. In other experiments, the addition of 10 ng / ml of HGF (reduced from 20 ng / ml) when cAMP was added was shown to promote survival of the aggregates.

[0476] Without being bound by theory, it is believed that exposure to OSM has an inhibitory effect on the induction of expression of Phase 1 CYP enzymes, particularly CYP3A4.

[0477] Example 6

[0478] Notch signaling pathway in hepatic progenitor cells affects the differentiation of cholangiocyte lineage

[0479] To explore the differentiation of cholangiocyte-like cells, H9-derived day 27 hepatic progenitor cells were co-cultured with OP9 cells (Notch signaling donor) in the presence of 20 ng / ml HGF and 50 ng / ml EGF. H9-derived day 27 hepatic progenitor cells were obtained as in Example 1. When hepatic progenitor cells received Notch signaling activation from OP-9 cells, albumin-positive cells were completely attenuated and became CK19-positive cells with a tissue branching appearance ( Fig.10 a, Fig.10 b). In contrast, when Notch signaling was inhibited in co-cultured cells using the γ-secretase inhibitor (GSI) L-685, 458 (10 μM; Tocris), albumin- and CK-19-positive cells ( Fig.10 b).

[0480] Furthermore, H&E sections of cells co-cultured in the presence or absence of GSI showed that chimeric aggregation was maintained in the presence of GSI. In the absence of GSI treatment, cells were arranged into epithelial duct-like structures containing a lumen ( Fig.10 a).

[0481] like Fig.10 b, increased expression of CK19 and cystic fibrosis transmembrane conductance regulator (CFTR) in OP9 co-culture at day 36 and in the absence of GSI. The values ​​shown are relative to cells cultured in the presence of GSI. When Notch signaling is inactivated by culturing in the presence of GSI, expression of albumin is seen, demonstrating that the cells retain the characteristics of hepatoblasts.

[0482] Finally, 3D co-culture of hepatic progenitor cells with OP9 cells resulted in increased expression of CFTR at day 36 compared to 2D culture ( Fig.10 c).

[0483] The experiments described above show that cholangiocyte-like cells that form bile-like structures can be induced from H9-derived hepatic progenitor cells by activating Notch signaling (e.g., by co-culturing with OP9, OP9delta and / or OP9Jagged1 cells). Expression of CFTR, a marker of functional cholangiocytes, is higher in 3D gel co-cultures than in 2D cultures, indicating that the environment can also affect cholangiocyte maturation.

[0484] Example 7

[0485] Exemplary Maturation Medium Formulations

[0486] For HES2 cell line from day 14 (EB) / day 13 monolayer to day 26 (EB) / day 25 (monolayer)

[0487] Basal medium:

[0488] IMDM, 1% volume / volume B27 supplement, glutamine (2 mM), ascorbic acid (50 μg / ml; Sigma), MTG (4.5×10 -4 M; Sigma)

[0489] Cytokines and Growth Factors:

[0490] Hepatocyte growth factor (HGF) (20 ng / ml), dexamethasone (Dex) (40 ng / ml) and oncostatin M (20 ng / ml).

[0491] For H9 and iPS cell lines from day 14 (EB) / day 13 monolayer to day 20 (EB) / day 19 (monolayer)

[0492] Basal medium:

[0493] H16 / Ham's F12 (75% / 25%), 1% vol / vol B27 supplement, glutamine (2 mM), ascorbic acid (50 pg / ml; Sigma), MTG (4.5×10 -4 M; Sigma)

[0494] Cytokines and Growth Factors:

[0495] Hepatocyte growth factor (HGF) (20 ng / ml), dexamethasone (Dex) (40 ng / ml) and oncostatin M (20 ng / ml).

[0496] From day 20 (EB) / day 19 monolayer to day 26 (EB) / day 25 (monolayer)

[0497] Basal medium:

[0498] H21 / Ham's F12 (75% / 25%), 1% vol / vol B27 supplement, glutamine (2 mM:), ascorbic acid (50 μg / ml; Sigma), MTG (4.5×10 -4 M; Sigma)

[0499] Cytokines and Growth Factors:

[0500] Hepatocyte growth factor (HGF) (20 ng / ml), dexamethasone (Dex) (40 ng / ml) and oncostatin M (20 ng / ml).

[0501] Aggregation phase

[0502] From Day 26 / Day 25 to Day 32 / Day 31

[0503] Basal medium:

[0504] IMDM or H21 / Ham's F12 (75% / 25%), 1% vol / vol B27 supplement, glutamine (2 mM), ascorbic acid (50 μg / ml; Sigma), MTG (4.5×10-4 M; Sigma), Rho kinase inhibitor (10 μM) and 0.1% BSA.

[0505] Cytokines and Growth Factors:

[0506] Hepatocyte growth factor (HGF) (20 ng / ml), dexamethasone (Dex) (40 ng / ml) and oncostatin M (20 ng / ml).

[0507] Aggregation phase with cAMP

[0508] From day 32 / day 31 to day 44 / day 43

[0509] Basal medium:

[0510] Hepatocyte culture medium (HCM) without EGF (Lonza: CC-4182). 10 mM 8-Br-cAMP (Biolab: B007), small molecules associated with inhibition of Wnt / β-catenin signaling (xAV93: 1 μM) and MEK / Erk signaling (PD032590: 1 μM).

[0511] Cholangiocyte Maturation Medium

[0512] Basal medium:

[0513] H21 / Ham's F12 (75% / 25%), 1% vol / vol B27 supplement, glutamine (2 mM), ascorbic acid (50 pg / ml; Sigma), MTG (4.5×10 -4 M; Sigma),

[0514] Cytokines and Growth Factors:

[0515] Hepatocyte growth factor (HGF) (20ng / ml), epidermal growth factor (EGF) (50ng / ml)

[0516] Example 8

[0517] Effects of endothelial cells on hESC-derived liver development.

[0518] Given that endothelial cells play an important role in liver development, the effect of this lineage on the growth and / or maturation of hESC-derived hepatocytes was assessed. For these studies, CD34+ endothelial cells were generated from hESC. For these studies, the HES2 hESC cell line was used, which was designed to express red fluorescent protein (RFP) cDNA from the ROSA locus to enable us to track endothelial cells. Endothelial cells were generated by inducing a combination of BMP4, bFGF and VEGF for 6 days, at which time CD34+ cells (also CD31+ and KDR+) were separated by FACS. The selected CD34+ cells were cultured in EGM2 endothelial cell growth medium for 6 days, and then Aggrewells were used. TM For the generation of chimeric aggregates, endothelial cells were added to Aggrewells 2 days before hepatocytes to allow them to coat the bottom of the wells ( Fig.12 a). At this point, a single cell suspension of day 25 hepatoblasts was added on top of the endothelial cells and the mixture was cultured in Aggrewells for 48 hours. Aggregates were then removed from the Aggrewells and cultured for an additional 6 days, at which point they were harvested and analyzed. Fig.12 As shown in b, aggregates cultured with endothelial cells contained more RFP+ cells than those cultured alone. Flow cytometric analysis showed that RFP+ cells represented more than 30% of the population ( Fig.12 c), indicating that a large number had integrated into the hepatocytes in the aggregates. qRT-PCR analysis showed that chimeric aggregates cultured for an additional 12 days expressed substantially higher levels of CYP3A4 message than hepatic aggregates without endothelial cells ( Fig.12 d). Importantly, these levels were achieved without the addition of cAMP, indicating that endothelial cells can promote the maturation of hPSC-derived hepatocytes. These results suggest that interaction with embryonic endothelial cells influences the survival and maturation of hESC-derived hepatocytes.

[0519] Maturation of hESC-derived hepatocytes in collagen gels.

[0520] The combination of 3D aggregation, cAMP, and PD / XAV indeed promoted significant differentiation of human pluripotent stem cell-derived hepatocytes ( Fig. 9 ),( Fig. 9 d and Fig. 9 e). The cells do retain some expression of AFP and fetal CYP3A7, suggesting that they may not be fully mature. To promote further maturation of the population, chimeric endothelial / liver aggregates were treated with a combination of cAMP, PD, and XAV. These aggregates were maintained in liquid culture or in collagen gels to provide a source of extracellular matrix proteins. Fig.13As shown, when aggregates are maintained in liquid culture, adding endothelial cells to aggregates (ends) does not significantly affect the expression levels of ALB, CYP3A4, AFP or CYP3A7. In contrast, the cultivation of aggregates in collagen gels has a significant effect on AFP and CYP3A7 expression, because both are reduced to almost undetectable levels, similar to those found in adult liver. The findings show that signal transduction pathways, cell interactions and the extracellular environment all play a role in the maturation of hPSC-derived hepatocytes. This shows that it is possible to generate cells expressing very little AFP (if any). This expression pattern shows that these cells have progressed to the stage equivalent to the hepatocytes in the adult liver.

[0521] Example 9

[0522] The development of schemes for effectively generating tissue-specific cell types from human embryonic and induced pluripotent stem cells (pluripotent stem cells; PSC) contributes to the establishment of in vitro models of human development and disease and the design of new platforms for drug discovery and predictive toxicology. The lineage including hepatocytes is particularly important because hepatocytes and bile duct cells that constitute the biliary system of the organ are the main targets of the adverse effects of drugs and the main targets of the scope of genetic and infectious diseases. In view of the central role of hepatocytes in drug metabolism, most efforts to date have been directed to the generation of this cell type from hPSC. It has been possible to develop a staged differentiation scheme that promotes the generation of cells, although inefficient and lacking metabolic function, but showing some characteristics of mature hepatocytes, including the expression of functional P450 enzymes. Recent studies have extended this strategy to patient-specific induced pluripotent stem cells (iPSC), as well as simulating genetic liver diseases that affect hepatocyte function.

[0523] Diseases involving the bile duct are common causes of chronic liver disease, resulting in significant morbidity and often requiring whole organ transplantation for definitive management. The underlying mechanisms of monogenic biliary diseases such as cystic fibrosis liver and Alagille syndrome remain incompletely understood, while more complex biliary diseases such as primary sclerosing cholangitis and biliary atresia lack suitable models for understanding their pathophysiology or for screening new pharmacological agents. The ability to generate functional cholangiocytes from hPSCs will fulfill these unmet needs.

[0524] Successful derivation of cholangiocytes from hPSCs will depend on the ability to accurately mimic the embryonic development of this lineage in differentiation culture. Cholangiocytes develop early in fetal life and are derived from bipotential progenitors called hepatocytes, which also give rise to the hepatocyte lineage. Targeted studies in mice have shown that specification of the cholangiocyte lineage from hepatoblasts is a Notch-dependent event mediated by the interaction of Notch2 expressed by progenitor cells and Jagged-1 present on the developing portal mesenchyme. The discovery that the pediatric biliary disease Alagille syndrome is caused by Notch2 or Jagged1 provides strong evidence that this pathway is also involved in cholangiocyte development in humans. As cholangiocytes mature, they organize to form a polarized epithelium that develops primitive glandular duct structures that give rise to the bile duct.

[0525] As the basis for the exploration of iPSCs from patients with biliary diseases, a powerful scheme for the directed differentiation and maturation of functional cholangiocytes from hPSCs is described. hPSC-derived cholangiocytes can be induced to form epithelial cystic structures expressing markers found in mature bile ducts including the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR function in these structures is demonstrated by the regulation of cyst swelling after stimulating the cAMP pathway with forskolin. Cysts generated from iPSCs of cystic fibrosis patients show defects in forskolin-induced swelling assays, which can be rescued by adding CFTR correctors. Collectively, these findings indicate that it is possible to generate cholangiocytes and bile duct-like structures from hPSCs and use these derived cell types to simulate various aspects of cystic fibrosis biliary diseases in vitro.

[0526] result

[0527] Characterization of the hepatoblast stage of development in hPSC differentiation cultures

[0528] To generate cholangiocytes from hPSCs, it was necessary to first characterize the hepatoblast stage of development in differentiation culture. For these studies, we used a modified version of the protocol ( Fig.14 a) This protocol was developed to generate functional hepatocytes from hPSCs 1 The main difference from our previous approach was that the endoderm induction step was performed in a monolayer rather than in 3D embryoid bodies (EBs). This change resulted in an acceleration of endoderm development in culture, as a population consisting of greater than 90% CXCR4+CKIT+ and EPCAM+ cells was generated by day 3 of differentiation ( Fig.14 b) No equivalent population was detected until day 5 of EB differentiation 1To specify the endoderm to a hepatic fate, cultures were treated with a combination of bFGF and BMP4 on day 7 of differentiation.

[0529] Liver development begins in newly formed hepatoblasts of the embryo, which delaminate from ventral foregut cells and invade the septum to form liver buds. Bud formation depends on the transcription factor TBX3, which is transiently expressed during the early stages of this process. 2，3 As buds expand, progenitors downregulate Tbx3 and maintain and / or upregulate expression of a combination of genes normally expressed in the hepatic and / or cholangiocyte lineages (including albumin (ALB), alpha-fetoprotein (AFP), cytokeratin 19 (CK19), Sox9, NHF6p, and NOTCH2). RT-qPCR analysis of bFGF / BMP4-treated hESC-derived endoderm populations showed a transient upregulation of TBX3 expression at day 13 of differentiation, a time identified as a stage of hepatoblast specification ( Fig.14 c). Immunostaining showed that the majority of cells in the day 13 population were TBX3+, indicating that hepatoblast specification was efficient. The onset of SOX9 and HNF6B expression overlapped with the onset of TBX3 expression ( Fig.14 c). However, unlike TBX3, the expression of these genes remained elevated until day 25, the last day analyzed. The expression of ALB and AFP was upregulated at day 19 and also increased at day 25. CK19 showed a biphasic pattern, with peak levels of expression detected at days 13 and 25. Immunofluorescence staining and flow cytometric analysis showed that the majority of cells at day 25 of differentiation were ALB+, AFP+ and CK19+. Together these findings strongly suggest that the cells in the day 25 population represent an amplifying hepatoblast stage of development, the equivalent of the liver bud in vivo. The expression of Notch2 but not Notch1 was also upregulated at day 25, further supporting the interpretation that this population contains hepatoblasts capable of signaling through this pathway.

[0530] Notch signaling promotes cholangiocyte development from hPsC-derived hepatocyte-like populations.

[0531] To investigate the impact of Notch signaling on cholangiocyte development, we co-cultured hepatocyte populations (day 25) with OP9 stromal cells known to express different Notch ligands including Jagged1. 4，5. hPSC-derived cells did not survive well when cultured as single cell suspensions on a matrix. To overcome this problem, we generated 3D aggregates from day 25 monolayer cells and cultured the 3D aggregates on OP9 stromal cells. Immunostaining and flow cytometric analysis showed that most of the cells in the aggregates before co-culture were ALB+AFP+CD19+NOTCH2+, indicating that they retained hepatoblastic properties. Hepatoblasts appear to be selected in this aggregation step, because after 48 hours of culture, aggregates generated from a day 25 population that included only 80% ALB+AFP+CK19+ cells contained greater than 90% ALB+AFP+CK19+ cells. When co-cultured on an OP9 matrix (9 days), the different clusters of CK19+ cells formed by the aggregates no longer expressed ALB, indicating that they had undergone the initial stage of bile duct cell specialization. As shown in studies in mice, HGF, EGF, and TGFpi signaling play a role in bile duct development 6-8 We next added these factors alone or in combination to the cultures to determine whether activation of these pathways would promote further development of CK19+ clusters. 6-8 Addition of EGF or TGFβ1 or the combination of both resulted in an increase in the size of CK19+ aggregates, in contrast to HGF alone which had little effect. Interestingly, the combination of EGF and HGF or EGF, HGF and TGFpi induced dramatic morphological changes and promoted the formation of branched structures including CK19+ cells. RT-qPCR Fig.15 a and flow cytometry analysis ( Fig.21 ) confirmed the immunostaining findings and demonstrated the complete absence of ALB+ cells after co-culture with OP9.

[0532] Addition of γ-secretase inhibitor (GSI), an antagonist of the Notch pathway, blocked the downregulation of ALB expression, reduced the proportion of CK19+ cells in culture, and inhibited the development of branched structures, suggesting that these effects are mediated by Notch signaling ( Fig.15 a). After 9 days of culture on OP9, Notch targets HES1, HES5 and HEY1 were upregulated. ( Fig.15 b) This increase in expression was prevented by the addition of a γ-secretase inhibitor, indicating that co-culture with OP9 effectively activates the Notch pathway ( Fig.15 b). Analysis of the differentiation potential of two additional hPSC lines (ESC HES2 and iPSC Y2-1) showed similar temporal patterns of expression of TBX3 and hepatoblast markers, suggesting that transition through these stages is characteristic of in vitro liver development. ( Fig. 22) After co-culture with OP9 stromal cells, aggregates derived from both cell lines generated branched structures including CK19+ALB- cells. As observed with H9-derived populations, downregulation of ALB expression and development of these structures are NOTCH-dependent events. Collectively, these findings suggest that activation of Notch signaling in hepatoblast populations induces the initial stages of cholangiocyte development, and that a combination of HGF, EGF, and TGFpi signaling promotes morphological changes that lead to the formation of branched structures, possibly reflecting early stages of duct morphogenesis.

[0533] Three-dimensional culture promotes the maturation of cholangiocytes

[0534] To determine whether the branching observed in OP9 co-cultures indicated the initial stages of bile duct development, we next established a culture system to promote the growth of 3D cell structures. With this approach, chimeric aggregates consisting of day 25 hESC-derived cells and OP9 stromal cells (GFP+) were cultured in a culture medium mixture consisting of 1.2 mg / ml collagen, 40% Matrigel, and HGF, EGF, and TGFpi ( Fig.23 a). Within 2 weeks of culture under these conditions, the aggregates undergo dramatic morphological changes and form tubular structures, hollow cysts, or a mixture of both ( Fig.16 a. Fig.16 b). Cysts are the most abundant structures in these cultures ( Fig.16 b). Expression of Notch target genes Hes1, Hes5, and Heyl were upregulated in populations developed from chimeric aggregates compared to populations derived from aggregates without OP9 cells, indicating that Notch signaling is active in the culture ( Fig.23 b). Histological analysis showed that the tubular and cystic structures had a ductal morphology with an inner lumen and consisted of epithelial-like cells that expressed CK19+ and E-cadherin+ but not ALB-. ZO-1 (zona occludens 1), a marker of tight junctions, was also expressed and was found restricted to the apical surface of the structures, suggesting that the cells had acquired gene expression polarity, characteristic of mature epithelial ducts. The cells in the ducts also expressed the cystic fibrosis transmembrane conductance regulator (CFTR), a transmembrane channel first expressed in the adult biliary locus. Due to the presence of ZO-1, CFTR protein was predominantly detected on the apical surface of the tubular structures. Immunoblot analysis confirmed the presence of the protein in populations generated from the chimeric aggregates ( Fig.23 d). Levels of CFTR message and protein are considerably lower in cells derived from aggregates cultured without OP9, suggesting that its expression is dependent on Notch signaling ( Fig.23 d. Fig.23e). The absence of hepatic markers (including ALB, AFP, and CYP3A7) and upregulation of the expression of cholangiocyte markers CK19, SOX9, and CFTR in these structures were confirmed by RT-qPCR analysis ( Fig.16 c). After culturing under these conditions, similar CK19+CFTR+ tubular and cystic structures developed from iPSC-derived aggregates. In summary, these findings show that when cultured in a mixture of matrigel and collagen, hepatoblast populations can generate tubular structures that express markers found in mature bile ducts.

[0535] The addition of GSI prevented the formation of tubular structures and cysts and promoted the development of dense aggregates ( Fig.16 a, Fig.16 b spheroids), which expressed ALB and low levels of CK19, indicating that, in the absence of Notch signaling, cells with hepatoblastic properties persisted in culture. Addition of GSI also led to an increase in the expression of hepatocyte markers (ALB, AFP, and CYP3A7) and a decrease in the expression of genes associated with cholangiocyte development (CK19, Sox9, and CFTR) ( Fig.16 c).

[0536] hPSC-derived cholangiocytes form tubular structures in vivo.

[0537] To evaluate the developmental potential of hPSC-derived cholangiocytes in vivo, we transplanted hPSC-derived cholangiocytes (106 cells) in Matrigel plugs into the mammary fat pads of immunodeficient NOD / SCID / IL2rg- / - (NSG) mice. For these studies, we used cells dissociated from branching structures generated by co-culture of a 25-day hepatoblast population derived from HES2-RFPhESCs with OP9 stroma. These hESCs were engineered to express RFP from the ROSA locus. 9 At 6 to 8 weeks after transplantation, multiple tubular structures were detected in the Matrigel plugs ( Fig.17 a and Fig.17 b). The cells within the duct are RFP+, indicating that they are of human origin and derived from HES2-RFP cells ( Fig.17 c. Fig.17 d). In addition, cells expressing CK19 and CFTR showed that they displayed characteristics of bile duct cells. As observed with the in vitro generated constructs, CFTR expression was isolated from the apical surface of the duct. No teratomas were observed in any of the transplanted animals.

[0538] hPSC-derived cholangiocytes are functional

[0539] As a first step to assess the functionality of hPSC-derived cholangiocytes, we evaluated their ability to efflux rhodamine 123, a tracer dye used to measure the functional activity of the MDR1 transporter present in normal cholangiocytes. Cystic structures derived from H9 hESCs or iPSCs transported the dye to the luminal space, indicating active transport activity. In the presence of 20 μM verapamil, an inhibitor of the MDR transporter, rhodamine did not accumulate in the lumen of the structures, confirming that movement of the dye, reflecting active transport, is likely through the MDR transporter.

[0540] To demonstrate CFTR functional activity, we next performed a forskolin-induced swelling assay of cystic structures. Using this assay, activation of the cAMP pathway by the addition of forskolin / IBMX increases CFTR function, which results in fluid transport and swelling of the cysts. Swelling can be visualized following staining with calcein green, a cell-permeable fluorescent dye ( Fig.18 a). When measured 24 hours later, addition of forskolin and IBMX to the cultures induced a 2.09+ / -0.21- and 2.65+ / -3.1-fold increase in the size of H9- and iPSC-derived cysts, respectively (Fig. 18b). Addition of the CFTR inhibitor (CFTR inh -172) blocked forskolin / IBMX-induced swelling, indicating that the increase in cyst size is CFTR-dependent. Findings from these assays indicate that cells within hPSC-derived cysts / tubular structures display properties of functional cholangiocytes found in the hepatic bile ducts.

[0541] Generation and functional analysis of cholangiocytes from cystic fibrosis patient iPSCs.

[0542] To demonstrate the usefulness of this system to model disease in vitro, we next analyzed cyst formation from iPSCs generated from two different cystic fibrosis patients carrying a common F508 deletion (e.g., deltaF508). The hiPSC lines generated hepatoblast populations with kinetics similar to those observed with wild-type hPSCs ( Fig.24 a and Fig.24 b). Although hepatoblast development was not altered, cyst formation by patient-derived iPSCs was clearly impaired, as only branching structures were observed in the gel after two weeks of culture ( Fig.19 a). Cyst formation from patient cells can be induced by adding forskolin during the first week of a biweekly culture period (variation) Fig.19 a. Fig.19 b) However, many cysts developed from patient iPSCs were not completely hollow but contained branching tubular structures ( Fig.19c). A higher frequency of hollow cysts (typical of those developed from normal iPSCs) was detected after longer culture periods, indicating that maturation of mutant cells was delayed. Addition of a CFTR inhibitor to cultures of normal iPSC-derived cholangiocytes also delayed cyst formation, indicating that the generation of these structures is, in part, dependent on functional CFTR (Fig. 19b).

[0543] Next, we evaluated functional restoration of F508 del CFTR in cholangiocyte-like cells from cystic fibrosis patient iPSCs using the chemical correctors VX-809 and Corr-4a 2 days prior to the CFTR functional assay. These two molecules are used to correct the folding defect of the mutant CFTR protein. The addition of the correctors did not improve cyst formation, but did result in the accumulation of detectable levels of CFTR at the apical site of the lumen. However, unlike the uniform distribution of CFTR in the lumen of wild-type cysts, the protein appeared in distinct patches in patient-derived cysts treated with the correctors. This pattern may reflect incomplete rescue of the transport defect of the mutant protein. Immunoblot analysis also showed the effect of adding the correctors. Most CFTR in normal cells is transported through Fig. 20 The lane in a identifies the larger mature form of the upper band in HBE (C). Patient-derived cells contain much less CFTR protein, and most of it is the smaller immature form. Addition of the corrector dramatically increases the proportion of mature protein in patient cells. To determine whether the corrector affects CFTR function, we next subjected treated and untreated cysts to a forskolin / IBMX-induced swelling assay ( Fig. 20 b. Fig. 20 c). Patient specific cysts showed little swelling in the absence of correctors. However, with the addition of correctors, cysts generated from patient C1 increased approximately 2.18+ / -0.52-fold, while those from patient 997 increased 1.64±0.08-fold 24 hours after induction with forskolin / IBMX and VX770. In summary, these findings show that it is possible to mimic aspects of CFTR dysfunction in patient specific iPSC-derived cholangiocytes, and that the correctors used to treat these patients can rescue the defects.

[0544] discuss

[0545] A system is described for directed differentiation of hPSCs into functional cholangiocyte-like cells that self-organize into tubular structures in vitro and in vivo.

[0546] Studies in mice have shown that the Notch pathway is important for inducing cholangiocyte fate decisions in vivo, involving Jagged 1 interacting with portal interstitial cells and Notch2 on hepatocytes.

[0547] Notch signaling provided by OP9 cells successfully manipulates fate decisions not only in monolayers but also in three-dimensional gel cultures. In addition to G-secretase inhibitors, when Notch signaling is affected, an inverse effect is observed. Previous reports have shown that the generation of cholangiocyte-like tubular structures from human ES cells (although inefficient) leads to the display of polarity and rhodamine 123 uptake functions.

[0548] Notch signaling provided by OP9 promoted engraftment of cholangiocyte-like cells from human ES cells, and the cells formed RFP-positive tubular structures in the mouse mammary fat pad. These structures were not observed in the absence of OP9. In conclusion, the OP9 co-culture system effectively provides notch signaling to induce cholangiocyte-like cells from human PSC-derived hepatoblasts in vitro and in vivo.

[0549] Hepatocyte maturation from hPSC is shown to be enhanced by the three-dimensional culture environment herein. Similarly, the maturation of cholangiocyte lineage cells is also promoted by a three-dimensional gel culture system. When hepatocyte aggregates are stimulated by Notch signaling via OP9 cells, gene expression and maturation associated with the cholangiocyte lineage are significantly increased. In addition, functional activity as cholangiocytes was detected in vitro.

[0550] Human IPS-derived cholangiocyte-like ductal structures display functional CFTR activity. These cells can be used for drug screening in a patient-specific manner.

[0551] Furthermore, patient-specific cholangiocyte-like ductal architecture can be efficiently obtained and used to validate existing or new therapeutics for other severe biliary diseases, such as the monogenic conditions progressive familial intrahepatic cholestasis (PFIC types 1, 2, and 3) and Alagille syndrome, as well as the more common and complex biliary diseases, biliary atresia and primary sclerosing cholangitis.

[0552] While the present application has been described with reference to what are presently considered to be preferred embodiments, it should be understood that the application is not limited to the disclosed embodiments, but rather is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0553] All publications, patents and patent applications are incorporated herein by reference in their entirety, just as if each individual publication, patent or patent application was specifically and individually incorporated by reference in its entirety. Specifically, the sequences associated with each accession number provided herein include, for example, accession numbers and / or biomarker sequences (e.g., proteins and / or nucleic acids) provided in a table or anywhere else, which are incorporated by reference in their entirety.

[0554] References:

[0555] 1.Gebhardt,R.et al.New hepatocyte in vitro systems for drugmetabolism:metabolic capacity and recommendations for application in basicresearch and drug development,standard operation procedures.Drug Metab Rev35,145-213(2003).

[0556] 2.Guillouzo,A.Liver cell models in in vitro toxicology.Environ HealthPerspect 106Suppl 2,511-532(1998).

[0557] 3.Hewitt,N.J.et al.Primary hepatocytes:current understanding of theregulation of metabolic enzymes and transporter proteins,and pharmaceuticalpractice for the use of hepatocytes in metabolism,enzyme induction,transporter,clearance,and hepatotoxicity studies.Drug Metab Rev39,159-234(2007).

[0558] 4.Byers,J.et al.An estimate of the number of hepatocyte donorsrequired to provide reasonable estimates of human hepatic clearance from invitro experiments.Drug Metab Lett 1,91-95(2007).

[0559] 5.Kawasaki,S.et al.Living related liver transplantation in adults.AnnSurg 227,269-274(1998).

[0560] 6.Hashikura,Y.et al.Successful living-related partial livertransplantation to an adult patient.Lancet 343,1233-1234(1994).

[0561] 7.Miro,J.M.,Laguno,M.,Moreno,A.&Rimola,A.Management of end stageliver disease(ESLD):what is the current role of orthotopic livertransplantation(OLT)?J Hepatol 44,S140-145(2006).

[0562] 8.Cai,J.et al.Directed differentiation of human embryonic stem cellsinto functional hepatic cells.Hepatology 45,1229-1239(2007).

[0563] 9.Duan,Y.et al.Differentiation and enrichment of hepatocyte-likecells from human embryonic stem cells in vitro and in vivo.Stem Cells 25,3058-3068(2007).

[0564] 10.Hay,D.C.et al.Highly efficient differentiation of hESCs tofunctional hepatic endoderm requires ActivinA and Wnt3a signaling.ProcNatlAcadSci U S A 105,12301-12306(2008).

[0565] 11.Basma,H.et al.Differentiation and transplantation of humanembryonic stem cell-derived hepatocytes.Gastroenterology136,990-999(2009).

[0566] 12.Duan,Y.et al.Differentiation and characterization of metabolicallyfunctioning hepatocytes from human embryonic stem cells.Stem Cells 28,674-686(2010).

[0567] 13.Sullivan,G.J.et al.Generation of functional human hepatic endodermfrom human induced pluripotent stem cells.Hepatology 51,329-335(2010).

[0568] 14.Touboul,T.et al.Generation of functional hepatocytes from humanembryonic stem cells under chemically defined conditions that recapitulateliver development.Hepatology 51,1754-1765(2010).

[0569] 15.Chen,Y.F.et al.Rapid generation of mature hepatocyte-like cellsfrom human induced pluripotent stem cells by an efficient three-stepprotocol.Hepatology 55,1193-1203(2012).

[0570] 16.Si-Tayeb,K.et al.Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells.Hepatology 51,297-305(2010).

[0571] 17.Si-Tayeb,K.,Lemaigre,F.P.&Duncan,S.A.Organogenesis and developmentof the liver.Dev Cell 18,175-189(2010).

[0572] 18.Nostro,M.C.et al.Stage-specific signaling through TGFβfamilymembers and WNT regulates patterning and pancreatic specification of humanpluripotent stem cells.Development 138,861-871(2011).

[0573] 19.Gadue,P.,Huber,T.L.,Paddison,P.J.&Keller,G.M.Wnt and TGF-βsignaling are required for the induction of an in vitro model of primitivestreak formation using embryonic stem cells.Proc Natl Acad Sci U S A 103,16806-16811(2006).

[0574] 20.Gouon-Evans,V.et al.BMP-4 is required for hepatic specification ofmouse embryonic stem cell-derived definitive endoderm.Nat Biotechnol24,1402-1411(2006).

[0575] 21.Borel Rinkes,I.H.,Toner,M.,Sheeha,S.J.,Tompkins,R.G.&Yarmush,M.L.Long-term functional recovery of hepatocytes after cryopreservation in athree-dimensional culture configuration.Cell Transplant 1,281-292(1992).

[0576] 22.Roberts,R.A.&Soames,A.R.Hepatocyte spheroids:prolonged hepatocyteviability for in vitro modeling of nongenotoxic carcinogenesis.Fundam ApplToxicol 21,149-158(1993).

[0577] 23.Sargiacomo,M.et al.Long-term cultures of human fetal liver cells:athree-dimensional experimental model for monitoring liver tissuedevelopment.JHepatol 28,480-490(1998).

[0578] 24.Miki,T.,Ring,A.&Gerlach,J.Hepatic differentiation of humanembryonic stem cells is promoted by three-dimensional dynamic perfusionculture conditions.Tissue Eng Part C Methods 17,557-568(2011).

[0579] 25.Dankel,S.N.,Hoang,T.,Flageng,M.H.,Sagen,J.V.&Mellgren,G.cAMP-mediated regulation of HNF-4alpha depends on the level of coactivator PGC-lalpha.Biochim Biophys Acta 1803,1013-1019(2010).

[0580] 26.Benet,M.,Lahoz,A.,Guzman,C.,Castell,J.V.&Jover,R.CCAAT / enhancer-binding protein alpha(C / EBPalpha)and hepatocyte nuclear factor 4alpha(HNF4alpha)synergistically cooperate with constitutive androstane receptor totransactivate the human cytochrome P450 2B6(CYP2B6)gene:application to thedevelopment of a metabolically competent human hepatic cell model.J Biol Chem285,28457-28471(2010).

[0581] 27.Bell,A.W.&Michalopoulos,G.K.Phenobarbital regulates nuclearexpression of HNF-4alpha in mouse and rat hepatocytes independent of CAR andPXR.Hepatology 44,186-194(2006).

[0582] 28.Arpiainen,S.et al.Coactivator PGC-lalpha regulates the fastinginducible xenobiotic-metabolizing enzyme CYP2A5 in mouse primaryhepatocytes.Toxicol Appl Pharmacol 232,135-141(2008).

[0583] 29.Stieger,B.,Heger,M.,de Graaf,W.,Paumgartner,G.&van Gulik,T.Theemerging role of transport systems in liver function tests.Eur J Pharmacol675,1-5(2012).

[0584] 30.Huet,P.M.&Villeneuve,J.P.Determinants of drug disposition inpatients with cirrhosis.Hepatology 3,913-918(1983).

[0585] 31.Hines,R.N.&McCarver,D.G.The ontogeny of human drug-metabolizingenzymes:phase I oxidative enzymes.J Pharmacol Exp Ther300,355-360(2002).

[0586] 32.Nagamoto,Y.et al.The promotion of hepatic maturation of humanpluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3cell sheets.Biomaterialx 33,4526-4534(2012).

[0587] 33.Takayama,K.et al.Efficient generation of functional hepatocytesfrom human embryonic stem cells and induced pluripotent stem cells byHNF4alpha transduction.Mol Ther20,127-137(2012).

[0588] 34.Takayama,K.et al.Efficient and directive generation of twodistinct endoderm lineages from human ESCs and iPSCs by differentiationstage-specific SOX17 transduction.PLoS One 6,e21780(2011).

[0589] 35.Green,M.D.et al.Generation of anterior foregut endoderm from humanembryonic and induced pluripotent stem cells.Nat Biotechnol 29,267-272(2011).

[0590] 36.Spence,J.R.et al.Directed differentiation of human pluripotentstem cells into intestinal tissue in vitro.Nature 470,105-109(2011).

[0591] 37.Koyama,T.,Ehashi,T.,Ohshima,N.&Miyoshi,H.Efficient proliferationand maturation of fetal liver cells in three-dimensional culture bystimulation of oncostatin M,epidermal growth factor,and dimethylsulfoxide.Tissue Eng Part A 15,1099-1107(2009).

[0592] 38.Ring,A.et al.Hepatic maturation of human fetal hepatocytes infour-compartment three-dimensional perfusion culture.Tissue Eng Part CMethods 16,835-845(2010).

[0593] 39.Montoliu,L.,Blendy,J.A.,Cole,T.J.&Schutz,G.Analysis of the cAMPresponse on liver-specific gene expression in transgenic mice.Fundam ClinPharmacol 8,138-146(1994).

[0594] 40.Leopold,J.A.et al.Aldosterone impairs vascular reactivity bydecreasing glucose-6-phosphate dehydrogenase activity.NatMed 13,189-197(2007).

[0595] 41.Liu,Y.et al.A fasting inducible switch modulates gluconeogenesisvia activator / coactivator exchange.Nature 456,269-273(2008).

[0596] 42.Herzig,S.et al.CREB regulates hepatic gluconeogenesis through thecoactivator PGC-1.Nature 413,179-183(2001).

[0597] 43.Lin,J.et al.PGC-1βin the regulation of hepatic glucose and energymetabolism.J Biol Chem 278,30843-30848(2003).

[0598] 44.Kennedy,M.,D′Souza,S.L.,Lynch-Kattman,M.,Schwantz,S.&Keller,G.Development of the hemangioblast defines the onset of hematopoiesis inhuman ES cell differentiation cultures.Blood 109,2679-2687(2007).

[0599] 45.Nostro,M.C.,Cheng,X.,Keller,G.M.&Gadue,P.Wnt,activin,and BMPsignaling regulate distinct stages in the developmental pathway fromembryonic stem cells to blood.Cell Stem Cell 2,60-71(2008).

[0600] 46.Loboz,K.K.,Gross,A.S.,Ray,J.&McLachlan,A.J.HPLC assay forbupropion and its major metabolites in human plasma.J Chromatogr B AnalytTechnol Biomed Life Sci 823,115-121(2005).

[0601] 47.Gagne,J.F.et al.Common human UGT1A polymorphisms and the alteredmetabolism of irinotecan active metabolite 7-ethyl-10-hydroxycamptothecin(SN-38).Mol Pharmacol 62,608-617(2002).

[0602] 48.Klipper-Aurbach,Y.et al.Mathematical formulae for the predictionof the residualβ cell function during the first two years of disease inchildren and adolescents with insulin-dependent diabetes mellitus.MedHypotheses 45,486-490(1995).

[0603] 49.Benjamini,Y.,Drai,D.,Elmer,G.,Kafkafi,N.&Golani,I.Controlling thefalse discovery rate in behavior genetics research.Behav Brain Res 125,279-284(2001).

[0604] 50.Zhang,F.&Chen,J.Y.HOMER:a human organ-specific molecularelectronic repository.BMC Bioinformatics 12 Suppl 10,S4(2011).

[0605] 51.Allen,J.W.Hassanein,T&Bhatia S.N.Advances in Bioartificial LiverDevices.Hepatology 34:447-455(2001).

[0606] 52.Stange,J.Extracorporeal liver support.Organogeneis 7:1,64-73(2011).

[0607] 53.Sato,N et al.Maintenance of pluripotency in human and mouseembryonic stem cells through activation of Wnt signaling by a pharmacologicalGSK-3 specific inhibitor.Nature Medicine 10:55-63(2004).

[0608] 54.Takebe,T et al.Vascularized and functional human liver from aniPSC-derived organ bud transplant.Nature 499:481-4(2013).

[0609] 55.Yusa et al Targeted gene correction of alphal-antityrpsindeficiency in induced pluripotent stem cells Nature 478:391-6(2011).

[0610] 56.Joung,JK and Jeffry D Sander TALENs:a wildely applicabletechnology for targeted genome editing Nature Reviews 14:49-55(2013).

[0611] 57.Ran FA et al Genome engineering using the CRISPR-Cas9 systemNature Protocols 8:2281-2308(2013).

[0612] 58.Gaj et al.ZFN,TALEN and CRISPR / CAs based methods for genomeengineering.Trends in Biotechnology 31:397-405(2013).

Claims

1. A method for generating bile duct cells from a population of human induced pluripotent stem cells (hiPSCs), the method comprising: a) contacting the human induced pluripotent stem cells cultured as a monolayer or formed into embryoid bodies (EBs) with an induction medium comprising ActA and a Wnt / β-catenin agonist for 4 to 8 days to provide an induced endoderm cell population; b) contacting the induced endoderm cell population with a nodal agonist for 1-4 days to provide a prolonged nodal agonist-treated induced endoderm cell population; c) contacting the extended nodal agonist-treated induced endoderm cell population with a specialized medium for 4-10 days to obtain an expanded hepatoblast population; the specialized medium comprising an FGF agonist selected from FGF2, FGF4 and FGF10 and a BMP4 agonist selected from BMP4, BMP2 and BMP7; d) culturing the expanded hepatoblast population with a maturation medium comprising hepatocyte growth factor (HGF), dexamethasone (DEX) and oncostatin M (OSM) for 10 to 14 days; e) dissociating the hepatoblast population obtained in step d) by enzyme treatment and manual dissociation; and f) generating aggregates of said dissociated hepatoblast population, and culturing said aggregates with a maturation medium comprising hepatocyte growth factor (HGF), dexamethasone (DEX) and oncostatin M (OSM); wherein the method is performed in the absence of a cAMP signaling activator, Therein, the fate of the bile duct cells is specified by including a Notch agonist in the maturation medium.

2. The method according to claim 1, wherein: The prolonged nodal agonist-treated induced endoderm population comprises at least 80% CXCR4 and cKIT positive cells and / or at least 70% SOX17+ cells.

3. The method according to claim 1, wherein: The induced endoderm population after prolonged nodal agonist treatment includes at least 80% CXCR4 and cKIT positive cells and / or at least 75% SOX17 + cell.

4. The method according to claim 1, wherein: The induced endoderm population after prolonged nodal agonist treatment comprises at least 80% CXCR4 and cKIT positive cells and / or at least 80% SOX17 + cell.

5. The method according to claim 1, wherein: The induced endoderm population after prolonged nodal agonist treatment comprises at least 85% CXCR4 and cKIT positive cells and / or at least 70% SOX17 + cell.

6. The method according to claim 1, wherein: The induced endoderm population after prolonged nodal agonist treatment comprises at least 85% CXCR4 and cKIT positive cells and / or at least 75% SOX17 + cell.

7. The method according to claim 1, wherein: The induced endoderm population after prolonged nodal agonist treatment comprises at least 85% CXCR4 and cKIT positive cells and / or at least 80% SOX17 + cell.

8. The method according to claim 1, wherein: The induced endoderm population after prolonged nodal agonist treatment includes at least 90% CXCR4 and cKIT positive cells and / or at least 70% SOX17 + cell.

9. The method according to claim 1, wherein: The induced endoderm population after prolonged nodal agonist treatment includes at least 90% CXCR4 and cKIT positive cells and / or at least 75% SOX17 + cell.

10. The method according to claim 1, wherein: The induced endoderm population after prolonged nodal agonist treatment includes at least 90% CXCR4 and cKIT positive cells and / or at least 80% SOX17 + cell.

11. The method according to claim 1, wherein: The induced endoderm population after prolonged nodal agonist treatment comprises at least 95% CXCR4 and cKIT positive cells and / or at least 70% SOX17 + cell.

12. The method according to claim 1, wherein: The induced endoderm population of the prolonged nodal agonist treatment comprises at least 95% of CXCR4 and cKIT positive cells and / or at least 75% of SOX17 + cell.

13. The method according to claim 1, wherein: The induced endoderm population after prolonged nodal agonist treatment comprises at least 95% CXCR4 and cKIT positive cells and / or at least 80% SOX17 + cell.

14. The method according to any one of claims 1 to 13, wherein: Aggregates of the hepatoblast population comprise at least 70% albumin positive cells.

15. The method according to any one of claims 1 to 13, wherein: Aggregates of the hepatoblast population comprise at least 80% albumin positive cells.

16. The method according to any one of claims 1 to 13, wherein: Aggregates of the hepatoblast population comprise at least 85% albumin positive cells.

17. The method according to any one of claims 1 to 13, wherein: Aggregates of the hepatoblast population comprise at least 90% albumin positive cells.

18. The method according to any one of claims 1 to 13, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 5 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

19. The method according to any one of claims 1 to 13, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 8 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

20. The method according to any one of claims 1 to 13, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 9 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

21. The method according to any one of claims 1 to 13, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 10 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

22. The method according to any one of claims 1 to 13, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 11 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

23. The method according to any one of claims 1 to 13, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 12 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

24. The method according to any one of claims 1 to 13, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 13 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

25. The method according to any one of claims 1 to 13, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 14 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

26. The method of claim 14, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 5 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

27. The method of claim 14, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 8 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

28. The method of claim 14, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 9 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

29. The method of claim 14, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 10 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

30. The method of claim 14, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 11 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

31. The method of claim 14, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 12 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

32. The method of claim 14, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 13 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

33. The method of claim 14, wherein: The hepatoblasts are co-cultured with a Notch agonist for at least 14 days to induce further lineage specification, inducing maturation of hepatocytes into cholangiocytes or functional cholangiocytes, or both.

34. The method of claim 18, wherein: Compared to hepatoblast populations not treated with Notch agonists, the functional cholangiocytes included increased expression of: (a) at least one gene selected from Sox9, CK19 and CFTR (cystic fibrosis transmembrane conductance regulator); (b) at least one protein selected from the group consisting of Sox9, CK19 and CFTR; or (c) Both (a) and (b).

35. The method of claim 26, wherein: Compared to hepatoblast populations not treated with Notch agonists, the functional cholangiocytes included increased expression of: (a) at least one gene selected from Sox9, CK19 and CFTR (cystic fibrosis transmembrane conductance regulator); (b) at least one protein selected from the group consisting of Sox9, CK19 and CFTR; or (c) Both (a) and (b).

Images