HK1218137B - Methods for generating hepatocytes and cholangiocytes from pluripotent stem cells - Google Patents

Description

Method for generating hepatocytes and bile duct cells from pluripotent stem cells

Technical Field

The present invention relates to a method for generating functional hepatocytes from human pluripotent stem cells.

Background Art

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 research 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 body's elimination of xenobiotics.1-3 Given this role and the fact that individuals can vary in their ability to metabolize specific drugs, ⁴ obtaining functional hepatocytes from a representative population sample could have a significant impact on drug discovery and testing within the pharmaceutical industry. In addition to providing ^a new platform for drug testing, hPSC-derived hepatocytes could offer 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 the development of bioartificial liver devices using 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. Due to the fact that the regulatory pathways controlling hepatocyte maturation are poorly understood, reproducible and efficient generation of such cells has so far been challenging.

Given the potential therapeutic and commercial importance of functional human hepatocytes, significant efforts have been directed toward optimizing the generation of these cells from hPSCs. ^8-16 Nearly all approaches attempt to recapitulate the key stages of liver development in differentiation culture, including the induction of definitive endoderm, specification of endoderm to a hepatic fate, generation of liver progenitors termed hepatocytes, and differentiation of hepatoblasts into mature hepatocytes. ¹⁷ In most studies, differentiation is induced in a monolayer format, with the sequential addition of known pathway agonists and antagonists to modulate the early stages of development, including endoderm induction and hepatic specification. Through this strategy, it has been possible to optimize these early differentiation stages 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 While these early differentiation stages are reasonably well established, conditions have not been described that, for example, promote the maturation of hepatocytes into functional cells, as defined by the activity of stage I and stage II drug-metabolizing enzymes. Populations generated with the different protocols differed greatly in their maturation status and in most cases represented immature hepatocytes.

Summary of the Invention

One aspect of the present 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:

(a) specializing the induced endoderm cell population treated with a prolonged nodal agonist to obtain a cell population comprising 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;

(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.

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.

In some embodiments, a population of induced endoderm cells is induced from a pluripotent stem cell (PSC) such as an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC) by prolonged nodal agonist treatment. The pluripotent stem cell is optionally a human ESC (hESC) or a human iPSC (hiPSC).

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

In embodiments, the induced endoderm population resulting from 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.

In an embodiment, the specification step comprises contacting the induced endoderm population treated with an extended nodal agonist (e.g., activin) with a specification medium comprising 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.

In embodiments, the specification step comprises first contacting the extended nodal agonist-treated induced endoderm population 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 extended nodal agonist-treated induced endoderm population 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.

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

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

Inducing maturation and optionally further lineage specification and/or expansion may comprise 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 active conjugates and/or fragments thereof.

In one embodiment, inducing maturation and optionally further lineage specification and/or expansion further comprises activating a cAMP pathway within the cells of the aggregate to induce maturation of the hepatic progenitor cells and bile duct progenitor cells into hepatocytes and/or bile duct cells. In another embodiment, activating the cAMP pathway comprises exposing the aggregate to cAMP and/or a cAMP analog (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.

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

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

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 biliary progenitor cells and/or hepatocytes generated from a non-prolonged nodal agonist-treated induced endoderm cell population (e.g., from 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 induction of cAMP signaling. In other embodiments, at least 40%, 50%, 60%, 70%, 80% or 90% of the hepatocytes, optionally functional hepatocytes, are ASGPR-1+ cells.

In an embodiment, cholangiocyte fate is specified by treating aggregates of a cell population with a nodal agonist.

In an embodiment, the population of the cholangiocytes produced is a population of functional cholangiocytes. Compared to the cells of the cell population including liver progenitor cells and cholangiocyte progenitor cells and/or compared to the cell population produced from the aggregates never treated with nodal agonists, functional cholangiocytes include, for example, at least 1, at least 2 or 3 genes or proteins 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 cholangiocyte populations are CK19+ cholangiocytes. In other embodiments, at least 40%, 50%, 60%, 70%, 80% or 90% of the functional cholangiocytes are CFTR+ cholangiocytes.

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

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

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;

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

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;

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

e) inducing maturation, optionally further lineage specification and/or expansion of the hepatic 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.

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

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

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

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;

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

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

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 specialization medium comprising an FGF agonist and a BMP4 agonist and/or active conjugates and/or fragments thereof,

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

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.

In some embodiments, the inducing maturation, further lineage specification and/or expansion step further comprises activating a cAMP pathway within the aggregate to induce 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.

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.

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).

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

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;

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

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 specialization medium comprising at least one FGF agonist and one BMP4 agonist and/or active conjugates and/or fragments thereof, and

d) inducing the 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:

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

(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 culture, e.g., after about 24 to about 28 days of culture;

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

(iv) Optionally, activating the 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.

In embodiments, the maturation medium for the aggregated cells may include factors that promote hepatocyte maturation or factors that promote cholangiocyte development, or both.

In another embodiment, the aggregation of cells is 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, on day 26 (or optionally one or two days thereafter, for example, day 27, in embodiments using EBs), promotes the expression of an expanded albumin+/HNF4+ progenitor cell population. For example, it was demonstrated that when a Wnt agonist is added, up to a 10-fold expansion of the population can be achieved.

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.

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, to levels up to or greater than those found in adult hepatocytes. For example, adding an inhibitor of MEK/Erk added in the presence of cAMP to cultures 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 has been shown to also increase CYP1A2 levels. In an embodiment, the Wnt antagonist is XAV939. In another embodiment, the MEK/Erk antagonist is PD0325901.

In an embodiment, 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, e.g., where cholangiocyte maturation is preferred, induction of cAMP signaling is omitted.

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

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

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), optionally for about 4 to about 8 days, to provide an induced endoderm cell population;

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;

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

d) inducing the 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:

(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;

(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 culture, for example, after about 24 days to about 28 days of culture;

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

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 the cAMP analog and/or 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

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, e.g., within 6 days of the step of generating the aggregates, optionally after about 20 days to 40 days of culturing.

In embodiments, the method comprises aggregation, eg, after about 20 days of culturing and/or before 40 days of culturing.

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

(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.

Notch agonists can be, for example, any notch ligands that are bound to surfaces such as cells, plastics, ECM or beads. In one embodiment, the notch ligand is notch ligand delta. In one embodiment, inducing maturation, further lineage specialization and/or amplification include making a cell population including bile duct progenitor cells contact 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.

Optionally, contacting the cell population comprising bile duct progenitor cells with a notch agonist (e.g., a notch signal donor) comprises co-culturing the cell population comprising bile duct progenitor cells with a notch signal donor such as OP9, OP9delta and/or OP9 Jagged1 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 the bile duct progenitor cells to mature into bile duct cells, optionally functional bile duct cells, optionally, wherein the functional bile duct cells form branching, cystic, tubular or spherical structures.

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

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

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

In certain embodiments, the method further comprises 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% hepatocytes and/or cholangiocytes (e.g., optionally, functional hepatocytes and/or cholangiocytes).

The present 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.

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 indicate preferred embodiments of the present invention, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 1 shows the induction of the endoderm of hESC-derived embryoid bodies. Figure 1 (a) is a schematic diagram of the differentiation protocol. EBs were digested with trypsin on the 6th day and plated as a single layer 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 generating 3D aggregates, which were cultured in a hepatocyte culture medium supplemented with Dex for 8 days, and then in the culture medium supplemented with cAMP analogs, 8-br-cAMP for another 12 days (days 32-44). Figure 1 (b) shows flow cytometry analysis, which shows the ratio of CXCR4+, CKIT+ (CD1 17) and EPCAM+ cells in the populations induced by activin and activin/Wnt3a on day 6. Figure 1(c) shows intracellular flow cytometric analysis showing the proportions of SOX17+ and FOXA2+ cells in activin- and activin/Wnt3a-induced populations on day 6. The sizes of the SOX17+ and FOXA2+ populations were significantly greater in EBs induced with activin/Wnt3a (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); Student's t-test, n=4. Figure 1(d) shows RT-qPCR-based analysis of T, SOX17, GSC, and FOXA2 expression in EBs induced with activin and activin/Wnt3a. EBs were analyzed at the indicated time points. Columns represent the SD of the mean values of three independent experiments. FIG1(e) is a flow cytometric analysis showing the dynamics of the development of CXCR4+, CKIT+, SOX17+, and FOXA2+ populations in EBs induced with activin/Wnt3a. FIG1(f) is a flow cytometric analysis showing the proportion of CXCR4+, CKIT+, EPCAM+, Sox17+, and FOXA2+ cells in EBs induced with activin in neurobasal medium on day six.

Figure 2 (a) is an 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 values of three independent experiments. Values are determined relative to TBP and presented relative to expression in bFGF (20 ng/ml) cultures (values set to 1). ***P<0.001 compared to bFGF-treated cultures. In the scholar's t-test, n=3. (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 presented relative to expression in FGF10 (-) cultures (values set to 1). *P < 0.05, scholar's t-test, n = 3.

Figure 3 shows that the duration of activin signaling affects liver development. Figure 3 (a) is intracellular flow cytometry analysis, which shows the ratio of SOX17+ and FOXA2+ cells in the EBs induced by activin/Wnt3A and the monolayer populations obtained by EBs on the 6th day. The monolayer populations were directly cultured in the specified culture medium (-activin), or cultured for two days in activin (50ng/ml) and then cultured in the specified culture medium (+activin). After culturing for two or four days in the specified culture medium (-activin group for a total of 8 days and 10 days and + activation group for 10 days and 12 days), the populations were analyzed. The columns represent the standard deviation (SD) of the mean value of three independent experiments. Compared with untreated populations (73.3+/-7.5% relative to 45.9±3.7%), the ratio of SOX17+ on the 10th/12th day of activin treatment was significantly higher. Similarly, the proportion of FOXA2+ cells was significantly higher in activin-treated days 8/10 and 10/12 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) shows the total number of cells in activin-treated and non-treated monolayer cultures. Day 6 EB-derived cells were cultured directly in hepatic differentiation medium or cultured 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 RT-qPCR-based expression analysis of hepatic monolayer populations generated from activin-treated (black bars) and non-treated (grey bars) endoderm. Populations were analyzed for expression of designated endoderm (HEX, AFP, ALB, and HNF4a) and mesoderm (MEOX1, MESP1, CD31, and CD90) genes. Activin-treated populations (grey columns) were analyzed at days 12, 18, and 26 of total culture, while untreated populations (black columns) were analyzed at days 10, 16, and 24 of culture. The indicated expression levels are relative to TBP. The columns represent the standard deviation (SD) of the mean values of three independent experiments. FIG3 (e) is a flow cytometric analysis showing the proportions of CD31+, CD90+, and EPCAM+ cells in monolayer populations derived from endoderm that were treated with activin (day 26) and untreated (day 24). 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, Student's t-test, n=3). In contrast, a higher proportion of EPCAM+ cells was detected in populations derived from activin-treated endoderm compared to populations derived from non-treated cells (EPCAM: 90.7±2.7% vs. 56.8±7.3%, P<0.01, n=3). Figure 3(f) shows immunostaining analysis showing the proportion of albumin-positive cells in cultures derived from activin-treated (day 26) and non-treated (day 24) endoderm. Albumin was visualized using Alexa 488. Scale bar: 200 μm. (g) Intracellular flow cytometric analysis showing the proportion of albumin (ALB) and alpha-fetoprotein (AFP)-producing cells in monolayer cultures generated from endoderm of activin-treated (grey bars; day 26) and non-treated (black bars; day 24). The bars represent the standard deviation (SD) of the mean of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test; n = 3). AL: adult liver, FL: fetal liver.

Figure 4 shows that aggregates promote liver maturation. Figure 4 (a) is a phase contrast image of liver aggregates at day 28 of culture. Scale bar, 200 μm. Figure 4 (b) shows an RT-qPCR analysis of ALB, CPS1, TAT, G6P, and TDO expression in monolayer (black column) and 3D aggregate cultures (gray column) at day 32 of differentiation. Values are determined relative to TBP and presented relative to expression in adult liver (values set to 1). Figure 4 (c) is an RT-qPCR analysis of CYP7A1, CYP3A7, and CYP3A4 expression at day 32 of differentiation in monolayer (black column) and 3D aggregate cultures (gray column). 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 at 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 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 cultured for 2 days.

Figure 5 shows that cAMP signaling induces the maturation of hESC-derived hepatocyte-like cells. Figure 5 (a) is an 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. The expression level is relative to TBP. Figure 5 (b) is an intracellular flow cytometric analysis, which shows the ratio of alpha-fetoprotein (AFP) + and albumin (ALB) + cells (day 44) in liver aggregates cultured in the presence and absence of 8-Br-cAMP. Compared with the non-induced population (34.5 +/- 12.4% relative to 56.9 +/- 3.6%, P < 0.05, mean ± SD, n = 3), the frequency of AFP + cells was significantly lower in the population induced with cAMP. 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 an 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 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. The bars in all figures 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.

Fig. 6 shows that cAMP signaling increases the metabolic enzyme activity of hESC-derived hepatocytes. Fig. 6 (a) shows the expression of CYP3A7, CYP3A4, CYP1A2, CYP2B6 and UGT1A1 in the liver aggregates (the 44th day) cultured in the presence and absence of 8-Br-cAMP. The level of primary hepatocytes (PH) is shown as a control. Values are determined relative to TBP and are presented as fold changes relative to the expression (value is set to 1) in non-treated cells. Fig. 6 (b) shows RT-qPCR analysis, and RT-qPCR analysis shows the expression of PGC1a, TAT, HNF4a, CYP1A2 and CYP3A4 in the monolayer population (the 44th day) of untreated (-) and cAMP treatment (+). Values are determined relative to TBP and are presented as fold changes relative to the expression (value is set to 1) in non-treated cells. Figure 6(c) shows RT-qPCR analysis of CYP1A2 and ALB expression in cAMP-treated aggregates generated from untreated (-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 production of acetaminophen, the o-desethylated metabolite of phenacetin, was monitored by HPLC. Activity is shown 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 bupropion metabolite o-hydroxybupropion was measured by HPLC. Activity was measured per 50,000 cells (n=3). Figure 6(g) shows that the metabolism of sulfamethazine (SMZ) to N-acetylated SMZ indicates the presence of the second-stage enzymes NAT1 and/or NAT2. cAMP-treated aggregates and primary hepatocytes were incubated with SMZ (500 μM) for 48 hours, and N-acetylated SMZ was measured by HPLC. Activity was measured per 10,000 cells (n=3). Figure 6(h) is an HPLC analysis showing the generation of 4-methylumbelliferyl (4-MU) glucuronide (4-MUG) from 4-MU in cAMP-treated aggregates. cAMP-treated aggregates and primary hepatocytes were incubated with 4-MU for 48 hours. The formation of 4MUG was measured by HPLC. Activity was expressed per 10,000 cells (n=3). Columns in all figures represent the standard deviation (SD) of the mean values of samples from three independent experiments, *P<0.05, **P<0.01, ***P<0.001, Scholar's t-test, PH; primary hepatocytes.

Figure 7 shows hepatic differentiation from different pluripotent stem cell lines. Figure 7(a) is a flow cytometric analysis showing the proportion of CXCR4+, CKIT+, EPCAM+, SOX17+, and FOXA2+ cells in EBs generated from H9 hESCs, H1 hESCs, and 38-2 iPSCs on day 6 of activin/Wnt3a induction. 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. Different populations were analyzed at the following times: no activin: day 24, 2 days of activin: day 26, 4 days of activin: day 28 of differentiation. Figure 7(c) shows intracellular flow cytometric analysis showing the frequency of ALB and AFP-positive cells generated from different HPSC lines (no activin (-): day 24, 2 days of activin: day 26, 4 days of activin: day 28 of differentiation). Figure 7 (d) is a phase contrast image showing the morphology of H9-derived hepatocytes at day 26 of culture. Scale bar: 200 μm. Figure 7 (e) shows RT-qPCR analysis, which 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 columns 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, AL: adult liver, FL: fetal liver, PH: primary hepatocytes.

Figure 8(a)(b)(c) demonstrates that CHIR99021 can induce definitive endoderm cells. Flow cytometric analysis shows the proportions of CXCR4+, CKIT+, and EPCAM+ cells at day 6 in endoderm induced with (a) activin/wnt3a or (b) activin/CHIR99021, or at day 7 in monolayers induced with (c) activin/wnt3a or (d) activin/CHIR99021. (e), (f), and (g) Ectopic liver tissue in NSG mice. Photomicrographs of H&E-stained sections of the mesentery show clusters of hESC-derived hepatocytes (arrows) two months after transplantation. Magnification 5X. Intestine (arrow), transplanted cells (arrowhead). (f) High-magnification (10X) photomicrograph of the H&E-stained section from Figure 8(c). (g) Immunohistochemical staining shows the presence of hESC-derived cells in the mesenteric region two months after transplantation. Double staining for human albumin (Alexa 488 (green, shown as arrows) and CK19 (Cy3-labeled, red, shown as arrow heads) shows that the transplanted cells have the potential to differentiate into both hepatocyte and cholangiocyte lineages. hESC-derived hepatocyte-like cells are observed as albumin-positive cells (arrows), while biliary epithelial-like cells express CK19 and are found in tubular structures (arrow heads).

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

Figure 10 shows 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) tissue 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 and cultured 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. Cultures were maintained in culture medium containing HGF 20ng/ml and EGF 50ng/ml and in the presence or absence of GSI for 9 days. 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 extensively branched structures. The cells within the branches showed epithelial morphology and were organized around the lumen. These cells express CK19, which indicates that they are bile duct cells and that the branching structure may represent developmental bile ducts. Figure 10 (b) shows that activation of Notch signaling upregulates the expression of CK19 and the cystic fibrosis transmembrane conductance regulator (CFTR) in the ductal structure. The values shown are relative to cells cultured in the presence of GSI. The cells in Notch (-) co-culture (i.e., treated with GSI) maintain the characteristics of hepatocytes, as shown by the expression of albumin. Figure 10 (c) shows that the induction of the expression of CFTR in 3D co-culture is higher than those found in monolayer culture. The values shown are relative to cells cultured in the monolayer state.

FIG11 is a schematic diagram of a 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 at day 5 of induction were equivalent to cells in the EB at day 6. Additional activin treatment beyond 5 days in the monolayer was also necessary for the culture to generate hepatic progenitor cells and mature hepatocytes. (b) EB cultures were used to generate definitive endoderm. (c) Schematic diagram of the protocol for generating cholangiocytes. Definitive endoderm generated in monolayer culture was directed toward a hepatic fate, leading to the generation of hepatic progenitor cells (hepatocytes) that were cultured for 25 days. Hepatic progenitor cells were dissociated and aggregated with OP9/OP9 delta cells in low-density dishes for two days. Chimeric aggregates were embedded in collagen/Matrigel gels and cultured in culture medium supplemented with HGF (20 ng/ml) and EGF (50 ng/ml) in the presence or absence of pan Notch signaling inhibitors (e.g., notch antagonists) γ-secretase inhibitor (GSI) L-685, 458.

Figure 12 shows 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. By inducing 4 days with BMP4 and then inducing another 2 days with VEGF and bFGF to generate RFP (+) / CD34 (+) endothelial cells. The RFP (+) / CD34 (+) endothelial cells separated by FACS are seeded on the wells coated with type I collagen, and are cultivated with EGM-2 culture medium for 6 days in the presence of VEGF and bFGF. In order to generate chimeric aggregates, the RFP (+) / CD34 (+) cells cultured are trypsinized, dissociated and placed in Aggrewell plates with a cell density of 100 cells per well. After culturing for 2 days, the hepatocytes at day 25 are placed on RFP (+) / CD34 (+) endothelial aggregates with a cell density of 1000 cells per well. Scale bar 100 μm. (b) Phase contrast and fluorescence images show RFP-positive cells within endothelial/hepatic aggregates on day 33. No RFP was detected in hepatic aggregates generated without endothelial cells. Scale bar 100°. (c) Flow cytometry analysis shows the proportion of RFP-positive cells in endothelial/hepatic aggregates on day 33. (d) RT-qPCR analysis shows the expression of CYP3A4 in aggregates with and without endothelial cells on day 44. Values were determined relative to TBP and are presented as fold change relative to expression in adult liver samples (value set to 1).

Figure 13 shows the effect of 3D gel culture on the maturation of hPSC-derived hepatocytes. Aggregates composed of hepatoblasts or hepatocytes and endothelial cells (ends) were generated on the 25th day of culture, then cultured for an additional 7 days in a hepatocyte liquid culture medium supplemented with VEGF and bFGF, and subsequently cultured for 12 days 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 was presented as a multiple change relative to their levels in adult liver. The expression of AFP and CYP3A7 was presented as a multiple change relative to their levels in fetal liver. AL: adult liver, FL: fetal liver.

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

Figure 15. Notch signaling promotes bile duct cell development from hPSC-derived hepatocyte-like populations. In the presence or absence of γ-secretase inhibitors (GSI), antagonists of the Notch pathway, in the culture medium supplemented with HGF (20 ng/ml), EGF (50 ng/ml) and TGFbl (5 ng/ml) after co-culturing with OP9, ALB and CK19 based on RT-qPCR expression analysis. Cells were collected and analyzed on the 30th, 33rd and 36th days of culture. Values were determined relative to TBP and values were presented as fold changes relative to the expression levels (values were set to 1) in the 27th day hepatocyte aggregates. (b) Expression analysis of Notch target genes HES1, HES5 and HEY1 based on RT-qPCR in hepatocyte-derived cells after culture with or without OP9. Cells were determined on the 36th day 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) culture medium. Values were determined relative to TBP and values were presented as fold changes relative to the expression levels (values set to 1) in cells cultured on the 36th day on matrigel. The columns 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).

Figure 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 and mixed 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 tube, cyst, or sphere morphology 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 collected on day 44 and cells analyzed for gene expression indicative of hepatocyte (ALB, AFP, and CYP3A7) and cholangiocyte (CK19, Sox9, and CFTR) lineages. Values were determined relative to TBP and presented as fold change relative to expression levels in GSI-treated populations (values set to 1).

Figure 17. hPSC-derived bile duct cells form duct-like structures in vivo. (ab) Histological analysis of bile duct grafting in 8 weeks of matrigel plugs after the transplantation of hepatocyte-derived cells on the 25th day of co-cultivation with OP9 stromal cells for 9 days in a culture medium containing HGF, EGF and TGFb1. After co-cultivation, cells were dissociated and transplanted (10 ^e per recipient) into the mammary fat pad of immunodeficient NOD/SCID/IL2rg-/-(NSG) mice. Multi-duct structures were observed in the mammary fat pad 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 the mammary fat pad after transplantation. For these studies, bile duct cells were generated from HES2-RFP hESC, which express RFP from the ROSA locus. The bile duct cells generated after 9 days of co-culture with OP9 stromal cells were transplanted into the mammary fat pad of NSG mice. Due to the presence of RFP+ cells, the grafts developed 8 weeks after transplantation were analyzed by immunohistochemistry. RFP-positive cells were detected in all duct structures, confirming that the cells are of human origin and are derived from HES2-RFP cells. RFP+ structures are visible in the images at low (c) and high (d) magnifications.

Figure 18. hPSC-derived cysts generated in 3D gels contain functional CFTR protein. (a) Representative confocal microscopy images of cysts generated from H9 (hESC)- and Y2-1 (IPSC)-derived cholangiocytes, labeled with calcein and stimulated with forskolin/IBMX (F/I). Images were taken 24 hours after F/I stimulation. Scale bar, 500 μm. (b) Quantification of cyst swelling 24 hours after F/I stimulation in the presence or absence of a CFTR inhibitor. F/I-stimulated cyst swelling was quantified using SPEED Imaging software. Total cyst size was normalized to that before F/I stimulation. Values are from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test; n = 3).

Figure 19. Generate bile duct cells from iPSC of cystic fibrosis patients. (a) Phase contrast images of bile duct cell-like cells of F508 iPS cells (CF-iPS) with CFTR deleted on the 44th day of 3D gel culture in the presence or absence of forskolin. CF-iPS cell-derived hepatocytes and chimeric hepatocyte/OP9 aggregates were generated using the scheme shown in Figure 14A. After embedding collagen/matrigel culture, cyst formation was 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 bile duct cells formed 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 bile duct cells cultured for 7 and 14 days. CF-iPSC-derived bile duct cells were maintained under 3D gel conditions in the presence or absence of forskolin in the first week of a two-week culture period (left figure). Normal iPS cell-derived cholangiocytes were maintained in the first week of a two-week culture cycle in the presence or absence of a CFTR inhibitor (right panel). Adding forskolin increased the number of cyst structures, which developed from CF-iPSC-derived cholangiocytes at 7 and 14 days of culture (left panel). Adding a CFTR inhibitor to normal iPSC-derived cholangiocytes delayed the formation of cysts (right panel). (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 during 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 were smaller and typically contained an internal septum (lower panel).

Figure 20. Restoration of CFTR function in CF-iPSC-derived cholangiocytes by treatment with the small molecule correctors VX-809 and C4. Immunoblot analysis shows accumulation of the mature, complexly glycosylated form of CFTR (band C) in CF-iPSC-derived cholangiocytes treated with VX-809 and C4. The mutant form of the protein (band B) is predominant in uncorrected cells. Human bronchial epithelial cells (HBE) served as a positive control. (b) Representative confocal microscopy images of calcein-labeled and forskolin/IBMX (F/I)-stimulated cyst structures generated from CF-iPSCs derived from two individual patients (997 CFTRdel and C1 CFTRdel—both carrying the delta F508 mutation). Images were taken 24 hours after F/I stimulation. Scale bar 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. Cyst swelling in response to F/I stimulation was quantified using Velocimetry software. Total cyst size was normalized to that before F/I stimulation in each of three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test; n = 3).

Figure 21. Intracellular flow cytometric analysis showing the proportion of ALB+ and CK19+ cells in hepatocyte-derived populations 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 indicates isotype control.

Figure 22 shows liver specialization and differentiation of hepatoblasts from other hPSCs. RT-qPCR analysis shows expression of the indicated genes maintained in HES2 and Y2-1 iPS cell-derived hepatoblasts as indicated in Figure 14a. The expression of the indicated genes was analyzed on days 7, 13, 19, and 25 of culture. Values are 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.

Figure 23. 3D gels used to generate cystic structures from hPSC-derived cholangiocytes. (a) Schematic diagram of the differentiation protocol used to generate chimeric aggregates composed 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 on day 44 of culture in medium containing HGF, EGF, and TGFb1. In the presence of OP9, expression of Notch target genes was significantly upregulated. Values were determined relative to TBP and are presented as fold change relative to expression in cells cultured in the absence of OP9 (value set to 1). (c) Histological analysis (H&E stain) of cystic structures developed from H9-derived cholangiocytes cultured with OP9 cells in the presence (right panel) or absence (left panel) of GSI at day 44 of culture. (d) Immunoblot analysis shows the presence of the mature, complexly glycosylated form of the CFTR protein (band C) in structures generated from normal iPSC-derived cholangiocytes cultured in the presence or absence of OP9. Undifferentiated normal iPSCs served as a negative control. (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 on day 44 of culture. Values were determined relative to TBP and are presented as fold change relative to expression values detected in Caco-2 cells (a colorectal cancer cell line) (values set to 1). *P < 0.05, **P < 0.01, ***P < 0.001 (student's t-test; n = 3).

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

DETAILED DESCRIPTION

Described herein is a robust and reliable platform for efficiently generating hepatocytes and cholangiocytes from pluripotent stem cells (PSCs) through a series of steps described herein, as well as for generating metabolically functional hepatocytes and/or cholangiocytes. For example, it is demonstrated that one or more extended nodal (e.g., activin) signaling treatments, inducing aggregation and activating 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 fates, allows for the reproducible generation of hepatocyte and cholangiocyte lineage cells, including, for example, expanded hepatocytes and/or, with additional manipulation, functional and mature hepatocytes and cholangiocytes induced from embryoid bodies, definitive endoderm, or from monolayers.

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) designating a population of induced endoderm cells treated with a prolonged nodal agonist to obtain a cell population comprising hepatocytes and/or cholangiocyte progenitor cells by contacting the population of induced endoderm cells treated with a specialized medium comprising a combination of an FGF agonist and a BMP4 agonist and/or active conjugates and/or fragments thereof; 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 comprising hepatocytes and/or cholangiocytes, the inducing maturation step comprising generating aggregates of the cell population.

Aggregation is demonstrated herein to be important for promoting maturation.

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

The term "contacting" (e.g., contacting an endoderm cell population 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 as 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 to further differentiate the cells, as well as including culturing the cells under conditions known in the art, such as for culturing pluripotent (and/or differentiated) populations, such as those further described in the Examples.

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 of the three layers. Endoderm cells differentiate to first give rise to the embryonic gut and then to derived tissues, including the esophagus, stomach, intestines, rectum, colon, pharyngeal pouch-derived tonsils, thyroid, thymus, parathyroid glands, lungs, liver, gall bladder, and pancreas.

As used herein, "induced endoderm cell population" refers to a population of endoderm cells corresponding to the "definitive endoderm induction" stage as shown in Figure 1a. This population can be prepared, for example, from embryoid bodies (EBs) exposed to a nodal agonist such as activin, or optionally from 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 ^™ CHIR99021 Stemgent), 6-bromoindirubin-3'-oxime (BIO) (Cayman Chemical (cat: 13123)) or Stemolecule ^™ BIO (cat: 04003) from Stemgent). Alternatively, the induced endoderm cell population can be prepared from cells grown in a monolayer. The endoderm cell group of induction can be for example identified by flow cytometry and molecular analysis for one or more marks, and mark is such as surface marker CXCR4, CKIT and EPCAM and transcription factor SOX17 and FOXA2.The endoderm cell group of induction can also be for example identified by at least or greater than 70% of the population of co-expressing CXCR4 and CKIT or CXCR4 and EPCAM, 80%, 90% or 95%.The endoderm cell group of induction can also be for example identified by greater than 70% of the population expressing SOX17 and/or FOXA2, 80%, 90% or 95%.The endoderm cell group of induction can for example be in 2D (single layer) or 3D (aggregate of embryoid body or other forms) format.The endoderm population of induction can be derived from the induced pluripotent stem cell (iPSC) of for example hESC and the induced in example 1 display.

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

As described in Example 1 and shown in Figure 3a, culturing day 6 (day 5 when the method includes monolayer induction) cells in activin for an additional 2 days 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 "extended nodal agonist" treatment.

As used herein, "extended 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)). Extended 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 (as shown in Figure 3d) on day 26 of culture. Extended nodal agonist-treated induced endoderm populations are 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 an extended nodal agonist-treated induced endoderm population.

In an embodiment, the endodermal cell mass of the induction of the nodal agonist treatment of the endodermal cells obtained by inducing embryoid bodies (EBs). In another embodiment, the endodermal population of the induction of the nodal agonist treatment of the endodermal cells obtained by inducing a monolayer. In each case, the time of the endodermal population extension of induction is cultivated under the presence of a nodal agonist (e.g., activin).

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 distinct from "cell aggregates," which refer to clusters or clumps of cells.

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.

In some embodiments, the induced endoderm cell population (and/or prolonged nodal agonist-treated induced endoderm cell population) 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).

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, as determined using nude mouse teratoma formation. Pluripotency is also reflected in the expression of embryonic stem (ES) cell markers.

The term "progenitor cell" refers to a cell having a 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.

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

The term "embryonic stem cell" is used to refer to the pluripotent stem cells of the inner cell mass of an embryonic blastocyst (see, e.g., U.S. Patent Nos. 5,843,780 and 6,200,806). The distinguishing characteristics of embryonic stem cells define the embryonic stem cell phenotype. Thus, if a cell has one or more unique characteristics of an embryonic stem cell that allow the cell to be distinguished from other cells, the cell has the phenotype of an embryonic stem cell. Exemplary distinguishing characteristics of embryonic stem cells include, but are not limited to, gene expression profile, proliferation capacity, differentiation capacity, karyotype, response to specific culture conditions, etc.

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

In an embodiment, the specialization step comprises contacting the induced endoderm population treated with a prolonged nodal agonist with a specialized culture medium comprising an FGF agonist and BMP4. For example, the FGF agonist can be bFGF, FGF10, FGF2 or FGF4, active fragments thereof and/or combinations thereof. For example, the combination can be added sequentially to the cells.

In embodiments, the specification step comprises first contacting the extended nodal agonist-treated induced endoderm population with a specification medium comprising FGF10 and BMP4 for about 40 hours to about 60 hours, e.g., about 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, or about 60 hours, and then contacting the extended nodal agonist-treated induced endoderm population with a specification medium comprising bFGF and BMP4 for about 4 days to about 7 days, e.g., about 4 days, 5 days, 6 days, or about 7 days.

In one embodiment, the specialized culture medium includes Iscove's Modified Dulbecco's Medium (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).

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

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

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 comprising StemPRO-34 supplemented with bFGF, activin A, and BMP4.

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.

As used herein, the term "specialized medium" refers to a medium used to promote or facilitate the specialization of cells or cell groups. An example of a liver-specialized medium includes Iscove's modified Dulbecco's medium (IMDM), which is supplemented with 1% volume/volume B27 (Invitrogen: A11576SA) and ascorbic acid, MTG, a 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 medium can be used to specialize hepatocytes and bile duct cell lineages. At other stages and in other embodiments, the specialized medium includes one or more factors that promote the specialization of hepatocytes and/or bile duct cell development, for example, notch antagonists or notch agonists. The term "specialization" as 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 specific fate of cells can be reversed or converted to another fate. Specialization results in a cell's fate state that cannot be changed under typical conditions.

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

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 bile duct cell specialized cell populations).

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).

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-density plates. 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 the cell clusters. Aggrewells can optionally be used.

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, FIG9 f shows ALB expression on day 26 of induction using several different matrix-coated substrates.

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

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 active conjugates and/or fragments thereof. For example, prior to 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.

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 biliary progenitor cells into hepatocytes and/or biliary cells.

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

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, a combination of forskolin (FSK) (Sigma: 66575-29-9) + XAV939 + PD0325901 effectively increased CYP expression and induced the maturation of hepatic progenitor cells into hepatocytes. As used herein, "cAMP agonists" 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.

It has been found, 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). Therefore, in some embodiments, aggregates are incubated with cAMP analogs and/or agonists in the absence of OSM.

As used herein, the term "maturation" 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 the body). In one embodiment, the process by which immature hepatocytes or hepatic progenitor cells mature and functional hepatocytes are referred to as mature.

Figure 1a refers to "liver maturation A" and Figure 14a refers to differentiation of hepatoblasts. In both cases, the cell populations referred to are hepatoblasts that 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 the presence of EGF, TGB1, HGF, and EGF in combination with a Notch agonist (e.g., a Notch signal donor) such as OP9, OP9delta, and/or OP9 Jagged1 cells.

As used herein, the term "maturation medium" refers to a medium used to promote or facilitate the maturation of cells or cell populations, and 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), 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 hepatocyte maturation includes Hepatocyte Medium (HCM) without EGF (Lonza: CC-4182). The maturation medium may also optionally include a cAMP analog and/or a cAMP agonist, which, for example, induces expansion of the hepatocyte lineage and maturation of the hepatocyte lineage. The maturation medium may include factors that promote hepatocyte and/or cholangiocyte development, further lineage specification 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 bile duct cell lineage development and are added when bile duct cells are desired. Similarly, Notch antagonists promote hepatocyte lineage and can be added when hepatocytes are desired, for example, to inhibit bile duct cell development.

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.

For example, in embodiments, the maturation medium comprises a cAMP analog and/or a cAMP agonist and DEX, and optionally HGF is added to the aggregates, followed by culturing the pre-aggregation 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.

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

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

As used herein, the term "hepatoblast" refers to the progenitor cell with the ability to differentiate into cells of hepatocyte and cholangiocyte lineages (e.g., hepatocyte or cholangiocyte). Hepatoblast is a subset of the progenitor cells of, for example, hepatocyte and cholangiocyte, and hepatoblast can include immature hepatocyte and immature cholangiocyte (e.g., with specific cell fate and can only mature into hepatocyte or only mature into cholangiocyte cells). In certain embodiments, hepatoblast is limited by the expression of markers, 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, hepatoblast can produce cholangiocytes (e.g., CK19+ cells). As used herein, the term "liver progenitor cell" means the cell with the ability to differentiate into functional hepatocytes, and functional hepatocytes are, for example, albumin-positive and/or express CYP enzymes.

As used herein, the term "biliary progenitor cells" means cells that have the ability to differentiate into functional bile duct cells, eg, cells that are CK19 positive and/or express CFTR.

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 undergo maturation to acquire mature hepatocyte function. In some embodiments, immature hepatocytes are defined by the expression of markers such as Hex, alpha-fetoprotein, and albumin.

As used herein, "mature hepatocytes" refers to cells of the hepatocyte lineage that express CYP enzymes (e.g., CYP3A4 and CYP1A2) and albumin. Optionally, mature hepatocytes include functional or measurable levels of metabolizing enzymes, such as Phase I and Phase II drug metabolizing enzymes equivalent to those in 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 glucuronosyltransferase UGT1A1. For example, mature hepatocytes can be metabolically active hepatocytes. Cellular uptake of indocyanine green (ICG) is considered a characteristic of adult ^hepatocytes and is used clinically as a test substrate to assess liver function. For example, mature hepatocytes are ^ICG -positive hepatocytes. A hepatocyte population can be considered mature if 50%, 60%, 70%, 80%, 90%, or more of the hepatocytes in the population are ICG-positive. 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.

In embodiments, the hepatocytes are functional hepatocytes.

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

In embodiments, the hepatocytes, optionally functional hepatocytes, comprise increased expression of 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 population without prolonged nodal agonist treatment, 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.

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 increased levels 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, expression is increased by 1.15-fold to 6.1-fold compared to that found in primary hepatocytes (e.g., 6.1-fold (610%) for CYP1A2, 13.2-fold (1320%) for CYP3A4, 2-fold (200%) for CYP2B6, 1.52-fold (152%) for CYP2C9, 2-fold (200%) for UGT1A1, and 1.15-fold (115%) for CYP2D6). 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 comparable to or greater than those found in primary hepatocytes of similar stage, optionally at levels that are 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 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 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 at least 110%, 125%, 150%, 175%, 200%, 300%, 400%, or 500% of those found in primary hepatocytes.

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.

In further embodiments, the functional hepatocytes exhibit CYP1A2 activity in vitro. Optionally, the functional hepatocytes exhibit CYP1A2 activity that is equivalent to or greater than that found in primary hepatocytes, optionally at a level of at least 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, CYP1A2 activity is measured by incubating cells with phenacetin and monitoring the production of accumulated o-diethylated metabolites in the cells.

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 a level of at least 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, CYP2B6 activity is measured by incubating cells with bupropion and monitoring the formation of the metabolite o-hydroxybupropion in the cells.

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 sulfamethazine (SMZ) to N-acetyl SMZ.

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 production of 4-methylumbelliferone (4-MU) glucuronide (4-MUG) from 4-MU in the cells.

In embodiments, the hepatocytes, optionally functional hepatocytes, comprise increased expression of 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 without prolonged nodal agonist treatment, 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.

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

In an embodiment, bile duct cell fate is specified by treating aggregates of a cell population with a notch agonist.

The term "cholangiocyte" as used herein refers to cells that form bile ducts.

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 chloride bicarbonate anion exchanger 2 (Cl(-)/HCO(3)(-)AEs).

As used herein, the term "immature cholangiocyte" refers to a cell of the cholangiocyte lineage that must undergo maturation to acquire 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 for at least 1 day, at least 2 days, at least 3 days, or at least 4 days.

In embodiments, the cholangiocytes are functional cholangiocytes.

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 is a cholangiocyte lineage cell that expresses CK-19, MDR1, and/or CFTR. For example, a functional cholangiocyte expresses the MDR1 transporter and, when located in a cystic structure, can transport a tracer dye such as rhodamine 123 into the luminal space of the structure. As another example, as shown in FIG18 , the functional activity of CFTR can be evaluated, for example, using a forskolin-induced swelling assay on a cystic structure. 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 chloride bicarbonate anion exchanger 2 (Cl(-)/HCO(3)(-)AEs).

As used herein, the term "mature cholangiocytes" are cholangiocytes that express 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).

In an embodiment, the population of the cholangiocyte produced is a population of functional cholangiocytes. Compared to the cells of the cell population including hepatocytes and bile duct progenitor cells and/or compared to the population cells produced from the aggregates never treated with notch agonists, 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 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.

The term "expression" refers to the cellular processes involved in producing RNA and protein and, where appropriate, secreting the protein, including, but not limited to, 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.

The term "cell culture medium" (also referred to herein as "culture medium" or "medium") refers herein to a medium containing nutrients for culturing cells that maintain cell viability and support proliferation and, optionally, differentiation. Cell culture medium may comprise any suitable combination of salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum replacements, and other components such as peptide growth factors, vitamins, and the like. Cell culture media typically used for specific cell types are well known to those skilled in the art.

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.

Reference is made herein to various days of culture. One skilled in the art will recognize that the culture period can vary.

In certain embodiments, " the 5th day " as used herein generally refers to the endoderm cell group of the induction of for example PSC monolayer. The endoderm cell group of the induction derived from EB is cultivated for about 6 days, to arrive at a similar culture point that needs to be processed for about 24 hours to induce EB formation. Therefore, the 7th day monolayer induction culture is equivalent to the 8th day embryoid body induction culture, etc. At this stage, the endoderm population of induction can include cells expressing for example Foxa2 and SOX17. Similarly, " the 7th day " generally refers to the endoderm population of the induction 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 monolayer, or if derived from the 26th day of EB). Wherein, using monolayer cells, equivalent methods can be used using EB, and the culture period is generally delayed by 1 day. Figure 11 provides the example (Figure 11A) of the timetable using monolayer cells and the example (Figure 11B) using EB. Figure 11C and Figure 14A provide the example timetable for generating bile duct cells.

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., binding and activating FGF receptors). For example, FGF receptor activation can be assessed by measuring the activity of MEK/ERK, AKT and/or PI3K by immunoassay.

As used herein, the term "FGF" refers to any fibroblast growth factor, e.g., 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 active conjugates and fragments thereof, including naturally occurring active conjugates and fragments. In specific embodiments, the FGF is FGF10, FGF4, and/or FGF2.

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.

The concentration of FGF can be, for example, in the range of from about 1 ng to about 500 ng/ml, such as 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.

The concentration of FGF10 can be, for example, in the range of from about 1 ng to about 500 ng/ml, such as 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.

The concentration of bFGF can be, for example, in the range of from about 1 ng to about 500 ng/ml, such as 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.

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

As used herein, the term "BMP4" (e.g., Gene ID: 652) refers to bone morphogenetic protein 4, e.g., human BMP4, and active conjugates and fragments thereof, optionally including, for example, 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.

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

Thus, further aspects include a method of generating hepatocytes and/or bile duct cells from a population of pluripotent stem cells, the method comprising:

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;

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

c) specializing the induced endoderm cell population 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,

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;

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.

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

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

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;

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

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

e) specializing the induced endoderm cell population 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,

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

g) inducing maturation, further lineage specialization, and/or expansion of the hepatocytes and/or bile duct progenitor cells of the cell population to hepatocytes and/or bile duct cells, wherein the inducing maturation, further lineage specialization, and/or expansion comprises generating aggregates of the cell population. In some embodiments, the inducing maturation and optionally inducing further lineage specialization and/or expansion steps further comprise activating a cAMP pathway within the aggregate to induce the hepatocytes and/or bile duct progenitor cells of the cell population to mature into a population comprising hepatocytes and/or bile duct cells. In embodiments, the method comprises contacting the aggregates with a cAMP analog and/or a cAMP agonist.

Another aspect includes a method of generating functional hepatocytes and/or cholangiocytes from pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), the method comprising:

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;

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

c) specializing the induced endoderm cell population 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

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

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

(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 culture, for example, after about 24 days to about 28 days of culture;

(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.

In one embodiment, embryoid bodies (EBs) of pluripotent stem cells are formed by culturing the 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).

After EBs are formed, they can be cultured for another 3 to 10 days (optionally, 4 to 8 days or about 5 or 6 days) in an induction medium supplemented with a nodal agonist to induce an endoderm cell population. Optionally, the nodal agonist is activin A. EBs are also optionally incubated 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.

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, before the population of endoderm cells is contacted with at least one FGF agonist and one BMP4 agonist).

As mentioned, the cells are then treated to induce maturation, further lineage specialization and/or amplification, which may include, for example, aggregation and treatment with various maturation factors. These steps may, for example, generate a cell population that responds to cAMP activation. Prolonged nodal agonist treatment and aggregation are steps that generate cells that respond to cAMP activation.

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

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 the expansion of precursor populations.

For example, the aggregates include hepatoblasts, which can specialize into hepatocytes or cholangiocytes, as demonstrated.

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., the Wnt inhibitor XAV 939) and/or an inhibitor of MEK/Erk signaling (e.g., the MEK/Erk inhibitor PD0325901). Adding a Wnt antagonist and/or a MEK/Erk antagonist during activation of cAMP signaling enhances expression of CYP enzymes, e.g., to levels or above those in adult hepatocytes. For example, in the presence of cAMP, adding a MEK/Erk inhibitor, e.g., to cultures from about day 28 to about day 32, results in hepatocytes having increased levels of CYP3A4. Adding a MEK/Erk antagonist in combination with a Wnt antagonist has been shown to also increase 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).

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.

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

In another embodiment, inducing maturation, further lineage specification and/or expansion further comprises contacting the cell aggregates with a cAMP analog and/or a cAMP agonist and a Wnt agonist, such as a selective inhibitor of GSK3 (e.g., CHIR99021) or a TGF-β antagonist (e.g., inhibitor SB431542). Optionally, the cell aggregates are contacted with 0.1 μM to 10 μM (optionally, 0.2 μM to 4 μM) 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).

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 was demonstrated that, for example, up to a 10-fold expansion of this population can be obtained when a Wnt agonist is added.

In an embodiment, the 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 9 days. For example, such treatment results in expansion of hepatoblasts. In an embodiment, the method comprises 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.

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

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

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

Wnt antagonists such as XAV939 and/or MEK/Erk antagonists, e.g., PD0325901, added during the cAMP activation step, enhance the expression of CYP enzymes and promote the maturation of hepatocyte precursors;

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:

Notch agonist that promotes cholangiocyte lineage specification.

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

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 ( FIG. 7 e ).

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.

In an embodiment, the cell population includes hepatocytes and/or bile duct progenitor cells cultured in a maturation/specialization medium comprising HGF, OSM, and DEX, optionally co-cultured with endothelial cells (optionally, CD34+ positive endothelial cells) prior to cell aggregation. 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.

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.

In an embodiment, the cell group includes hepatocytes and/or bile duct progenitor cells cultured in a maturation/specialized culture medium comprising HGF, OSM and DEX, and the cell group is co-cultured with CD34+ positive endothelial cells to form chimeric aggregates, optionally using an aggregation container such as Aggrewells ^™ (e.g., a container that promotes the aggregation of a single cell type or mixed cell type) until chimeric aggregates are obtained, when using Aggrewells, e.g., about 1 day, about 2 days, or about 3 days. Aggregation can also be implemented using methods described herein or known in the art. As described in Example 8, endothelial cells can be added to the bottom of the container to coat the wells before the hepatocyte group. The hepatocyte group can be added as a single cell suspension, for example, hepatoblasts at day 25/26 can be added to the mixture cultured on top of the endothelial cells and in Aggrewells. After suitable aggregation, the chimeric liver/endothelial aggregates can then be removed from the Aggrewells and cultured. As shown in Figure 12b, the 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 information than liver aggregates generated without endothelial cells (Figure 12d). Because these levels were achieved without the addition of cAMP, endothelial cells can promote the maturation of hPSC-derived hepatocytes. In an embodiment, 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 the desired or preselected level of CYP3A4 information is obtained.

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.

In the Examples, aggregates cultured in collagen comprising a matrix were cultured in the presence of cAMP, PD0325901, and XAV939.

The combination of 3D aggregation, cAMP and PD/XAV is shown to promote the significant differentiation of human pluripotent stem cell-derived hepatocytes (Figure 9), (Figure 9d and Figure 9e). Some expression of AFP and fetal CYP3A7 was maintained. In Example 8, it was demonstrated that the combination of cAMP, PD and XAV in the treatment of liver endothelial chimeric aggregates and collagen gel to provide a source of extracellular matrix proteins promoted the further maturation of the population. As shown in Figure 13, when the aggregates were maintained as liquid culture, adding endothelial cells to the aggregates (ends) did not significantly affect the expression levels of ALB, CYP3A4, AFP or CYP3A7. In contrast, culturing the aggregates in collagen gel 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 livers.

In an embodiment, for example, within about 1 day to about 4 days after aggregation, cells are treated with a notch agonist. 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.

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:

(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.

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):

a) contacting pluripotent stem cells cultured as a monolayer or 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;

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

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

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:

(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 culture, e.g., after about 24 days to about 28 days of culture;

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

Wherein, when the Notch agonist is a Notch signal donor cell (optionally, OP-9, OP-Jagged1 and/or OP-9delta1 cell), the Notch signal donor cell is co-polymerized with the cell population.

In further embodiments, the aggregates are cultured (or co-cultured when Notch signaling donor cells are included) in a gelatinous matrix (optionally, a collagen-containing 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.

Furthermore, 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 hepatocyte properties.

In another embodiment, a method of generating hepatocyte lineage cells such as hepatoblasts, hepatocytes and/or cholangiocytes from pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), the method comprising:

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), optionally for about 4 to 8 days, to provide an induced endoderm cell population;

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

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

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

(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;

(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 culture, e.g., after about 24 days to about 28 days of culture;

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

IV)

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 formation, e.g., within 6 days of aggregation, optionally after about 27 days to about 36 days of culturing; or

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.

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 for up to any number of days between 5 and 90 days or between 5 and 60 days.

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 OP9, OP9delta and/or OP9 Jagged1 cells, and optionally co-culturing 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 the bile duct progenitor cells into bile duct cells, optionally, functional bile duct cells.

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, 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.

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 signaling including, but not limited to, 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 engineered to overexpress one or more notch ligands. OP-9-Jagged1 is engineered to overexpress recombinant/exogenous Jagged1 notch ligands, and OP9-delta1 is engineered to overexpress recombinant delta1 notch 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 act 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.

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.

As mentioned, hepatoblasts (e.g., at the 25th or 26th day stage as shown in Figures 1a and 14a) can be aggregated (also referred to as 3D aggregation) as described in step g), step g) comprising 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 comprising 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.

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.

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% Matrigel.

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.

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 to, for example, about 20%.

As demonstrated in Example 9, CFTR-expressing cholangiocyte branching and cystic structures can be generated using the methods described herein. CFTR is functional as shown using a swelling assay. Thus, in embodiments, the generated and/or isolated cholangiocytes are CFTR-expressing cholangiocytes.

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.

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.

As used herein, the term "HGF" refers to hepatocyte growth factor (gene ID: 3082), e.g., human HGF and its 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, 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 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.

As used herein, the term "TGFβ" refers to any of TGFb1, TGFb2, and TGFb3, for example, human TGFb1, TGFb2, and TGFb3, as well as active conjugates and fragments thereof, including naturally occurring active conjugates and fragments. As described below, when hepatoblasts were co-cultured with OP9 cells, TGFb1 promoted bile duct cell branching. TGFb2 and TGFb3 have also been tested and, under similar conditions, promoted branched structures.

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 used for bile duct cell specialization can be, for example, in the range of from about 5 ng/ml to about 10 ng/mL.

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 ^™ CHIR99021 Stemgent), 6-bromoindirubin-3'-oxime (BIO) (Cayman Chemical (cat: 13123)), or Stemolecule ^™ BIO (cat: 04003) from Stemgent. CHIR99021 is a selective inhibitor of GSK3. Contemplated 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-01 8L), 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.

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.

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.

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.

As used herein, the term "selective inhibitor" refers to an inhibitor that inhibits a selective entity or pathway at least 1.5X, 2X, 3X, 4X, or 10X more effectively than a related molecule. For example, a selective inhibitor of GSK-3 inhibits GSK-3 in the Wnt pathway more effectively than at least 1.5X, 2X, 3X, 4X, or 10X that is inhibited by, for example, LiCl, or inhibits other kinases, other GSKs, and/or GSK3 in other pathways 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 while having little effect on other kinases. Thus, a selective inhibitor can exhibit an IC50 that is 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 that is smaller in size than a full-length polypeptide, but is substantially homologous to the full-length polypeptide, is 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-fold, 2-fold, 3-fold, 4-fold or greater than 4-fold) the effective biological effect compared to a polypeptide that is a fragment of the full-length polypeptide.

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., wherein 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 greater than 100% of the effective biological effect, for example, 1.5-fold, 2-fold, 3-fold, 4-fold or greater than 4-fold greater effective biological effect (e.g., receptor activating activity), compared to the unlinked polypeptide or other molecule.

In one embodiment, the term "active fragments and conjugates" refers to fragments and conjugates of a molecule that retain the ability to activate its cognate receptor. 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.

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

As used herein, the term "Dex" 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.

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.

As mentioned, some embodiments of the present invention involve activating the cAMP pathway within the aggregates to induce hepatocyte and/or cholangiocyte maturation.

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.

As used herein, the term "activating the cAMP pathway" refers to inducing the pathway to convert ATP into cAMP, for example, increasing cAMP levels. 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 dissociate from the β and γ subunits. The Gα subunit, in turn, activates adenylate cyclase, which converts ATP into 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. A number of 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 are interchangeable.

In some embodiments of the present methods, the cAMP pathway is optionally activated by contacting the hepatic 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 hepatic 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.

Induction of maturation, further lineage specification and/or expansion may involve a series of steps.

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

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 as described above).

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.

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 cAMP analog and/or cAMP agonist is added, the amount of HGF in the culture medium is reduced (e.g., from 20 ng/ml of HGF to 10 ng/ml of HGF).

In another embodiment, the aggregates are cultured in HGF, Dex, and OSM for about 6, 7, 8, 9, 10, 11, or 12 days, at which time a cAMP analog and/or cAMP agonist is added. In one embodiment, OSM and optionally HGF are removed while the cAMP analog and/or cAMP agonist is 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) while the cAMP analog and/or cAMP agonist is added.

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.

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.

For example, maturity can be detected by determining the levels 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 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).

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).

In embodiments, the pluripotent stem cells are from a mammal, such as a human. In embodiments, the pluripotent stem cells are human iPSCs (hiPSCs).

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 (typically 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)).

In an embodiment, the method includes 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.

In one embodiment, obtaining the endoderm cell population includes forming embryoid bodies from pluripotent stem cell cultures. EBs are formed by any method known in the art, for example, the method described in Nostro, MC et al., ¹⁸ , wherein EBs are formed from small aggregates cultivated for 24 hours in a low-level BMP4 agonist.

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

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.

In another embodiment, EBs and/or monolayer cells 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 Stemolecule ^™ BIO in addition to activin A. For example, BIO, a specific inhibitor of GSK-3, has been shown to maintain pluripotency in human and mouse ESCs by activating Wnt signaling ⁵³ .

Optionally, the EBs and/or cell monolayers are exposed to from 10 ng/ml to 40 ng/ml of Wnt3a or from 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, the EBs and/or cell monolayers 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, the EBs are exposed to from about 0.1 μM to about 2 μM of CHIR-99021. In another embodiment, the cell monolayers are exposed to from about 1 μM to about 30 μM of CHIR-99021, for example, from about 1 μM to about 3 μM of CHIR-99021. Those skilled in the art will be able to determine equivalent useful amounts of other GSK-3 inhibitors.

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.

As described in the Examples and elsewhere, populations of induced endoderm cells are cultured with a nodal agonist, such as ActA, 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 from about 1 day to about 4 days, to produce extended nodal agonist-treated induced endoderm populations.

Optionally, the basal medium for inducing definitive endoderm is any medium known in the art for inducing definitive endoderm, optionally 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 further supplemented with a Wnt/β-catenin agonist, such as Wnt3a or a selective inhibitor of GSK-3 (such as CHIR-99021).

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

The definitive endoderm or induced endoderm cell population is optionally defined by expression of the 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.

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.

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

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.

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 making up the total cell population. Similarly, a "substantially pure" population of functional hepatocytes and/or bile duct cells 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 bile duct cells, or their progeny as defined by the terms herein. In some embodiments, the present invention includes a method for expanding a population of functional hepatocytes and/or bile duct cells, wherein the expanded population of functional hepatocytes and/or bile duct cells is a substantially pure population of functional hepatocytes and/or bile duct cells.

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 are used interchangeably.

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

Cells and components

As discussed above, functional hepatocytes and/or cholangiocytes can be separated using the methods described herein. Therefore, further aspects of the present application include populations of cells produced according to the methods described herein. In an embodiment, the population of cells is, for example, hepatoblasts, hepatocytes and/or cholangiocytes, for example, markers of mature and/or functional cells produced according to the 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.

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

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

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

In embodiments, the isolated, purified and/or enriched population is generated in vitro.

In embodiments, populations of cells are included in compositions with appropriate diluents.

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

In embodiments, functional hepatocytes and/or cholangiocytes are derived from iPS cells of a subject with a liver and/or biliary disease. 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 cystic fibrosis. In embodiments, the subject carries a mutation, for example, in a cystic fibrosis gene, such as deltaF508, 997CFTR del and/or C1 CFTR mutation. As shown in FIG9 , the described methods can generate a hepatoblast population from iPSCs generated/derived from a cystic fibrosis patient.

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

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).

use

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.

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

i) generating iPSCs from cells derived from or obtained from a subject affected by a liver and/or biliary disease; and

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

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.

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

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

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

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

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) hepatocyte maturation and/or cholangiocyte specification, 3) one or more properties of hepatoblasts, hepatocytes and/or cholangiocytes; and/or 4) restoration and/or amelioration of one or more defects in a cellular model of liver and/or biliary disease and comparing to a wild-type cell population and/or other control tested in the absence of the test compound.

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.

In embodiments, one or more properties of cholangiocytes include:

a) hepatoblast/cholangiocyte lineage differentiation capacity compared to wild-type iPSCs, optionally assessing I) the presence and/or number of branching structures and/or cysts; II) the expression level, form (mature and/or immature form) and/or expression pattern of cholangiocyte markers;

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

c) Transporter activity, optionally CFTR activity.

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

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). When tested with putative or known CFTR treatments, for example, to provide for the evaluation of new drugs/biological products and/or to assess patient-specific responses, restoration and/or improvement of this or another property can be assessed.

Another aspect includes functional CFTR assays, including:

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

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

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

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 with a specific CF gene mutation, G551D.

The cells described can also be used for cell transplantation. For example, mixed populations 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.

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.

For example, Yusa et al. (55) described correcting known genetic defects in iPSC-derived hepatocytes and re-implanting them. The corrected cells were re-implanted into mice and displayed functions that were not previously present in the diseased state. Yusa et al. found that the most suitable transposon for their purpose was piggyBac, a moth-derived DNA transposon that can be efficiently transposed into mammalian cells, including human embryonic stem (ES) cells. The mobile element is able to remove the transgene on either side of the piggyBac inverted repeat sequence without leaving any residual sequence. The generated iPSC-3-G5-A7 had a corrected A1AT, compared to the intact genome of the parental fibroblasts and expressed normal A1AT protein when differentiated into hepatocyte-like cells.

Transposons are optionally used. Other methods of introducing expression constructs include methods based on lentivirus and adenovirus. Effective systems for transferring genes into cells in vitro and in vivo are viral vectors, including herpes simplex virus, adenovirus, adeno-associated virus (AAV) and lentiviral vectors. Optional 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, a separate vector (each expressing one or more different transgenes) can also be delivered to cells.

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).

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

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).

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 hepatoblasts, hepatocytes and/or cholangiocytes according to the methods described herein.

In embodiments, the population of cells administered is a day 25/26 population of cells. In embodiments, the cells specialize to a hepatocyte or cholangiocyte fate.

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

As shown in, for example, Figures 8c) to 8e) and described in Example 3, hepatocytes derived from ESC transplantation engraft and are able to differentiate into cells of the hepatocyte and cholangiocyte lineages. Similarly, Example 9 demonstrates that CFTR-functional cholangiocytes can be generated in vitro and/or in vivo.

Thus, in an embodiment, a method of transplanting or treating a subject in need thereof 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 thereof, e.g., a subject with liver disease and/or biliary disease.

In embodiments, a therapeutically effective amount is administered.

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

In an embodiment, the obtained cells are derived from autologous cells, for example, iPSCs generated from the subject's blood and/or skin cells. In an embodiment, the PSCs can be iPSCs obtained, for example, from a biopsy, blood cells, skin cells, hair follicles, and/or fibroblasts.

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.

For example, drugs 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 clearance 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.

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, screening for inhibition of one or more of these isozymes can be performed, for example, using LC-MS/MS or fluorometry. CYP IC50 and/or _Kj can be determined.

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 that 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. CYP catalytic activity and mRNA levels can be measured relative to a control, with results expressed as fold induction.

Furthermore, in other embodiments, drug metabolites can be assessed, for example, a metabolite profile of a drug can be determined.

In an embodiment, different concentrations of a test agent are added to cells obtained using the methods described herein, and cell survival, 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 the specific toxicity of a drug to a patient.

In embodiments, functional hepatocytes and/or cholangiocytes are used in tissue engineering. For example, obtaining purified populations of functional hepatocytes and/or cholangiocytes allows for the generation of engineered constructs having a defined number of functional hepatocytes and/or cholangiocytes. In other examples, obtaining purified populations of functional hepatocytes and/or cholangiocytes allows for the generation of bioartificial liver devices.

Alternatives to whole-organ liver transplantation under investigation include transplantation using isolated cells, tissue engineering of implantable structures, 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 their clinical use. The limited availability of sources of functional human hepatocytes and/or cholangiocytes also hinders the development of such devices.

As mentioned in 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 can cause multiple auxiliary organ dysfunction through direct cytotoxicity (e.g., acute tubular necrosis due to jaundice), alterations in functional homeostasis (e.g., hepatorenal syndrome due to hemodynamic imbalance), 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.

Functional hepatocytes and/or albumin-producing hepatocytes can be obtained and used in the form of BAL. For example, if functional hepatocytes are obtained, it may not be necessary to perform albumin dialysis. ES/iPS-derived hepatocytes are able to produce 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 available from donated blood. The generation of albumin-secreting cells and/or together with higher liver function activity will be beneficial for establishing a BAL system.

In embodiments, the method is applied to patient-specific disease hiPSCs and used, for example, to model liver disease. For example, liver or other cells from a patient 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 implicated in or responsive to the disease in the patient's immune cells.

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.

As used herein, the term "subject" includes all members of the animal kingdom, including mammals, and appropriately refers to humans.

When applied to isolated cells, the terms "treating," "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 terms refer to providing medical or surgical care, health care, or management to an individual.

When applied to a subject, the term "treatment" as used herein refers to a method (including clinical results) intended to obtain a beneficial or desired result and includes medical procedures and applications, including, for example, drug intervention, surgery, radiotherapy, and natural therapy interventions and test treatments. Beneficial or desired clinical results can 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-worsening) 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" can also refer to prolonged survival compared to expected survival if not receiving treatment.

As used herein, the terms "administer," "introduce," and "transplant" 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 can be implanted directly into the liver, or alternatively, the cells can 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.

Reagent test kit

Another aspect includes a kit. The kit may 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.

In understanding the scope of the present invention, the term "comprise" and its derivatives as used herein are intended to be open terms that specify the presence of stated features, elements, components, groups, wholes and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, wholes and/or steps. The foregoing also applies to words with similar meanings, such as the terms "comprises", "having" and their derivatives. Finally, as used herein, terms of degree such as "substantially", "about" and "approximately" refer to a reasonable amount of deviation of the modified term such that the end result will not be significantly changed. These degree terms should be interpreted as including a deviation of at least ±5% of the modified term if the deviation would not negate the meaning of the word modified by the modified term.

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

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

In addition, the definitions and embodiments described in particular sections are intended to apply to the other embodiments described herein, and those skilled in the art will understand that they are appropriate. For example, in the following paragraphs, different aspects of the present invention are defined in more detail. Each aspect defined in this manner can be combined with any other one or more aspects unless there is a clear indication to the contrary. In particular, any feature indicated as preferred or advantageous can be combined with any other one or more features indicated as preferred or advantageous.

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 alternative variations are contemplated. Although specific terms are employed herein, such terms are intended to be descriptive rather than restrictive.

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

Examples

Example 1

Endoderm induction in EBs

Figure 1a illustrates the strategy for generating hepatocytes from hESCs using embryoid bodies (EBs). Similar to protocols using monolayer cultures, ¹⁶ it involves specialized steps that recapitulate key stages of liver development in the early embryo. As previously described, ¹⁸ EBs form 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, with populations defined by expression of surface markers CXCR4, CKIT, and EPCAM, as well as transcription factors SOX17 and FOXA2. As shown in Figure 1b, after five days of activin induction (six days of coculture), greater than 90% of the induced EB population co-expressed either CXCR4 and CKIT or CXCR4 and EPCAM. Intracellular flow cytometric analysis revealed that greater than 90% of the population expressed SOX17, and greater than 80% expressed FOXA2+.

Studies using a monolayer induction protocol have shown that Wnt signaling enhances the development of activin-induced endoderm, possibly due to enhanced anterior primitive streak formation ¹⁰ . Adding Wnt3A to activin-induced EB cultures resulted in a reproducible increase in the ratio of CXCR4+, SOX17+, and FOXA2+ cells within EBs ( FIG. 1 c ). Due to increased CXCR4 expression, the ratio 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 ( FIG. 1 d ). Adding Wnt did not accelerate the decline in Oct3/4 expression, but did result in an increase in T (short tail) expression on the third day and an increase in Goosecoid (GSC) expression on the fourth and fifth days ( FIG. 1 d ). As shown in the mouse ESC model ¹⁹ and monolayer hESC-derived cultures ¹⁰ , Wnt enhances the formation of primitive streak. Kinetic analysis shows a rapid and dynamic increase in the ratio of CKIT+CXCR4+, SOX17+ and FOXA2+ positive cells between the 3rd and 6th day of differentiation (Figure 1e). Endoderm induction in EB is affected by the basal medium used. StemPro34 supports more effective endoderm induction (Figure 1f) than the neural basal medium we used previously. The induction of highly enriched endoderm is an important first step in efficiently and reproducibly generating hepatocyte-like cells from hPSC. For example, it was found that the induction level of at least 90% of CXCR4+CKIT+ and 80% of SOX17+ cells resulted in the development of the best liver lineage.

The duration of nodal/activin signaling affects liver development

In order to specialize the CXCR4+CKIT+ population into the fate of the liver, the EB of the 6th day was dissociated and the cells were plated as a monolayer on a matrigel-coated plate, in the presence of FGF10 and BMP4, for 48 hours, then in the presence of bFGF and BMP4, for six days. Previously, it was demonstrated in mice ²⁰ and human ESC differentiation cultures ¹⁶ that FGF and BMP are important for human and mouse specialization. In Si-Tayeb et al., 80%-85% of the cells of FGF2 and BMP4 combination and generation express albumin. It was found that the combination of these two factors is necessary (Fig. 2 a) for optimizing liver induction under test conditions. The FGF10/BMP4 step was included because it was found that compared with the independent bFGF/BMP4 in the differentiation culture, the expression of albumin was increased (Fig. 2 b). Under these inductive conditions, a considerable amount of albumin-positive cells developed continuously in the culture between the 12th day and the 24th day of differentiation.

While the BMP4/FGF specification step promotes liver development, analysis of day 10 cultures showed that the proportion of SOX17+ and FOXA2+ cells within the culture had decreased significantly, from over 90% to approximately 50% (Figure 3a). Without being bound by theory, this decrease suggests that either the day 6 population was contaminated by non-endodermal cells that preferentially expanded in the presence of FGF and BMP4, or that the cells' endodermal fate had not yet been fixed, and therefore some adopted another fate under the conditions used. It has been 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 for human PSCs is unknown. The effect of increasing the duration of this signaling pathway was amplified in the human system by culturing the monolayer cells for two days before 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 3a). Without being bound by theory, since the total number of cells was lower in the treated population (Fig. 3b), it is possible that Activin signaling at this stage preferentially supports the survival of endoderm cells.

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

Aggregation promotes liver maturation

Although prolonged activin/nodal signal promotes liver development, the expression level of genes such as albumin and HNF4α is significantly lower in hESC-derived populations compared to adult livers, which indicates that the cells in the 26th day culture are still immature. Previous studies have shown that cell aggregation can maintain the differentiation phenotype of primary hepatocytes ^21-23 and a certain degree of maturation in promoting hESC-derived hepatocytes ²⁴ in culture. Next, the effect of aggregation on the maturation of the 26th day hepatoblast population of the endoderm derived from activin treatment is explored. Aggregates (Fig. 4a) are generated from monolayers by a combination of enzyme treatment and artificial dissociation and then cultured for six days in the presence of HGF, dexamethasone (Dex) and oncostatin M (OSM). Aggregation affected differentiation and resulted in increased expression of multiple genes involved in liver function, including ALB (albumin), CPS1 (carbamoylphosphatase synthase 1), TAT (tyrosine transaminase), G6P (glucose 6-phosphatase), and TDO (tryptophan 2,3-dioxygenase) (Figure 4b). In some cases, the levels were similar to those found in adult liver (ALB) or higher than those found in adult liver (TDO) (Figure 4b). Aggregation also increased the expression of several cytochrome P450 genes, including CYP7A1, CYP3A7, and CYP3A4. CYP3A4 levels were similar to those found in primary hepatocytes but much lower than those found in adult liver (Figure 4c). Expression of other P450 genes, including CYP1A2 and CYP2B6, and the phase II enzyme UGT1A1, was not induced to any significant levels.

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 culture, consistently producing populations containing greater than 50% positive cells (Figure 4d). Immunostaining showed that ASGPR-1 and E-cadherin were detected on cells in albumin day 32 aggregates. Overall, these findings suggest that the simple process of aggregation into 3-D structures promotes changes indicative of liver maturation.

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

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 acts with HNF4a to regulate the expression of many genes ^involved in hepatocyte function. To determine whether cAMP signaling could promote the 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 of culture. Treatment with 8-Br-cAMP significantly enhanced the expression of PGC1-a (15-fold), but not HNF4α, in hESC-derived hepatocytes (Fig. 5a). 8-Br-cAMP also induced the expression of G6P and TAT, on average, 25- and 33-fold, respectively, to levels similar to those in adult liver (Fig. 5a). In contrast, the expression levels of AFP and ALB were significantly increased by 8-Br-cAMP. MP down-regulated. Flow cytometry analysis confirmed the expression analysis of AFP and showed a reduction 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 mRNA levels decreased (Fig. 5b). Without being bound by theory, these differences may reflect differences in RNA relative to protein expression. Other tissues such as the pancreas also express PGC-1a. However, in contrast to the induction observed in hepatocytes, the expression of PGC1-a was not induced by cAMP signaling in hESC-derived insulin-positive pancreatic cells (Fig. 5c), suggesting that the response may be tissue-specific.

Cellular uptake of indocyanine green (ICG) is considered a characteristic ^of adult hepatocytes29 and is used clinically as a test substrate to ^assess liver function30. cAMP signaling dramatically increases the proportion of cells displaying this activity, as demonstrated by the observation that nearly all treated aggregates stained with ICG (Figure 5d).

Confocal microscopy was used to assess the co-expression of ALB and AFP or ALB and HNF4α in aggregates cultured in the presence or absence of 8-Br-cAMP on day 44. 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.

ELISA assays were used to measure albumin (ALB) secretion levels from hESC-derived monolayer and aggregate populations, as well as from HepG2 cells, Huh7 cells, and cryopreserved hepatocytes (PH, lot OSI). HNF4a protein levels were comparable in both aggregate populations, confirming PCR analysis. Albumin secretion from 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 detectable 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 uptake indocyanine green (ICG), a characteristic of adult hepatocytes, was enhanced by cAMP signaling (Stieger et al., 2012). ICG uptake and release were measured in cAMP-treated and untreated aggregates at day 44.

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

cAMP signaling also induced changes in the expression patterns of key stage I cytochrome P450 genes, specifically a decrease in the expression levels 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 6a). UGT1A1, an important stage II enzyme, was also significantly induced by 8-Br-cAMP (11-fold) (Figure 6a). The induction levels of CYP3A4 and CYP1A2 were significantly higher than those found in primary hepatocytes, while the levels of CYP2B6 were similar in both populations. UGT1A1 expression in the hESC-derived population did not reach the levels found in primary hepatocytes. Without being bound by theory, assuming that CYP1A2 expression is restricted to the liver and only detected after ^birth , these findings suggest that cAMP signaling promotes differentiation beyond the fetal stage of development.

The induction of P450 genes by cAMP signaling is only observed in cells in 3D aggregates, and the increase in the expression of CYP1A2 and CYP3A4 is almost undetectable when cAMP is added to monolayer culture (Fig. 6b). Inducing the expression of PGC1-a and TAT in monolayer format may be due to the fact that the promoter region of these genes comprises a cAMP response element binding protein (CREB) site. In order to further define the variable affecting cAMP reaction, the aggregation generated from the endoderm of extended activin processing is compared with those generated from the endoderm of no activin/nodal processing for two additional days. In the aggregates from non-processed populations (-Act), almost no induction of CYP1A2 and ALB is observed, which shows that cAMP signaling only has an effect (Fig. 6c) on highly enriched, appropriately patterned cells. For above-mentioned research, 8-Br-cAMP was included in the culture for 12 days (days 32-44). To determine whether changes in gene expression were dependent on continuous signaling, cells that were 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 6d). CYP1A2 expression was maintained after the shorter induction time, indicating that the higher levels of expression were not dependent on continuous signaling but rather reflected changes indicative of hepatocyte maturation.

To explore the functional activity of P450 enzymes, the ability of metabolizing isozymes to selectively label drugs was measured by high-performance liquid chromatography (HPLC). 8-Br-cAMP-treated cells expressed phenacetin, a selective substrate for o-deethylation of CYP1A2 (Figure 6e), at levels comparable to those observed in primary cultured hepatocytes. Untreated cells showed no detectable activity. CYP2B6 activity, measured by hydroxylation of bupropion, was also detected in 8-Br-cAMP-treated cells (Figure 6f), at levels comparable to those observed in primary cultured hepatocytes. Analysis of phase II metabolizing enzymes, including arylamine N-acetyltransferases NAT1 and/or NAT2 (Figure 6g) and UDP-glucuronosyltransferases (UGTs) (Figure 6h), revealed activity higher than that observed in primary cultured hepatocytes, suggesting that cAMP signaling induces upregulation of expression of a broad range of enzymes, consistent with population maturation. Together, these observations suggest that cAMP signaling promotes maturation of hESC-derived hepatocyte-like cells in 3-D aggregates.

In addition, the inducibility of the metabolic activity of two key enzymes, CYP1A2 and CYP3A4, was also assessed. Cells treated with 8-Br-cAMP were able to metabolize the CYP1A2 selective substrate phenacetin. Induction of cells with lansoprazole for 72 hours resulted in a 3.4-fold increase in this activity. Untreated (8-Br-cAMP) cells had low, non-inducible levels of activity. Two independent samples of primary hepatocytes showed levels below or equivalent to basal metabolic activity, but displayed higher levels of induction (18-fold and 9-fold). CYP3A4 activity was measured by the cells' ability to metabolize testosterone to 6β-hydroxytestosterone. 8-Br-cAMP-treated cells exhibited this activity. Addition of the CYP3A4 inducer rifampicin increased activity by 2.2-fold, indicating that this enzyme can also be induced in hESC-derived cells. While observing CYP1A2, CYP3A4 activity was barely detectable in uninduced cells. Primary hepatocytes showed low but significant levels of CYP3A4 induction.

Hepatic specification and maturation from other hPSC lines

To determine whether the method detailed above is widely applicable to different human pluripotent stem cell lines, the protocol was used to differentiate hESC cell lines H9 and H1 and induced pluripotent stem cell (iPSC) line 38-2 into liver fate. After the Wnt3a/activin induction step, EBs from the 6th day of all three cell lines contained a high proportion of CKIT+CXCR4+ and CKIT+EPCAM+ cells (Fig. 7a). Although the ratio of EPCAIVT cells was higher in all EBs, H9-derived cells expressed substantially higher levels of EPCAM than cells generated from other cell lines. The level of EPCAM expression was correlated with the degree of endoderm induction, as greater than 95% of the H9-derived population expressed SOX17+ and FOXA2+, while only 65-70% of the iPSC-derived cells expressed these transcription factors (Fig. 7a). These findings indicate that surface marker analysis alone is not sufficient to monitor endoderm development, and quantitative analysis of SOX17 and FOXA2 expression is necessary to measure the induction of this germ layer. As observed for HES2 cells, prolonged activin/nodal signaling enhances liver development in the CKIT+CXCR4+ population from each cell line (Fig. 7b). However, the duration of activin treatment required to generate significant levels of ALB expression varies 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. Due to 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 (Fig. 7c). H9-derived cells on day 26 of differentiation showed a cobblestone morphology very similar to that of cultured hepatocytes (Fig. 7d).

The addition of 8-Br-cAMP did induce the expression of significant levels 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 7e). 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 7e).

As observed with the HES2 cell line, H9-derived cells had lansoprazole-induced CYP1A2 activity. H9 and iPSC-derived cells also showed rifampicin-induced CYP3A4 activity. Induction of CYP1A2 activity was undetectable in iPSC-derived cells, which may reflect suboptimal differentiation of this population.

Microarray analysis of cAMP-stimulated hepatic populations

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 final analysis. Two-way unsupervised hierarchical cluster analysis showed that the three groups appeared 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 the clusters of highly expressed transcripts in each biological group. These clusters included 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 for most of the key P450 enzymes and those involved in many aspects of liver function, including gluconeogenesis, glucose homeostasis, and lipid metabolism. The highest-expressed clusters 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.

For a more detailed comparison of populations, the selected transcript subsets encoding proteins related to key aspects of liver function were analyzed. These include a subset 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 in 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, which 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 showed higher levels of ICG uptake. Taken together, these data from microarray analysis indicate that induction of hepatoblast-stage aggregates with cAMP leads to global expression changes indicative of hepatocyte maturation. Based on these analyses, hESC-derived hepatocytes generated by this method appear to represent a developmental stage at least equivalent to primary human hepatocytes.

discuss

In order for hPSC-derived hepatocytes to be useful for drug metabolism analysis and 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, many different studies have demonstrated 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 early stages of differentiation are reasonably well defined. In contrast, the factors and cellular interactions that control hepatocyte maturation are poorly understood, and consequently, only a few studies have reported on the development of metabolically competent cells. Duan et ^al.12 showed that it was possible to derive hepatocytes from H9 hESCs that displayed levels of CYP1A2, CYP3A4, CYP2C9, and CYP2D6 enzyme activity equivalent to those found in primary hepatocytes. The serum used by Duan et al. in their method included many factors, some of which varied between serum batches. The factors responsible were not identified. Although these findings indicate that relatively mature hESC-derived hepatocytes can be generated, the study does not provide details about the pathways that promote maturation, nor does it indicate that the strategy is widely applicable to other hPSC cell lines. As in Figure 6 of Duan et al., they measured the metabolic drugs in hepatocytes from human ES cells (H9). Phenacetin induces and provides an assessment of CYP1A2 activity, and midazolam, bufuralol, and diclofenac induce and provide an assessment of CYP3A4, CYP2B6, and CYP2D6, respectively. In the HES2 cell line, for example, described herein, the enzyme activity levels (CYP1A2 and CYP2B6) are almost identical compared to those seen in primary hepatocytes. In qPCR analysis, the levels of CYP1A2 and CYP2B6 expression in H9 cells are 5-8 times higher than those found in the 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, nearly identical or comparable levels of CYP2D6 expression were seen in cells generated using this method compared to primary hepatocytes.

Several other groups have shown that forced expression of specific transcription factors in hESC-derived populations, alone or in combination with co-culture with Swiss 3T3 cells, can promote maturation that results in the generation of cells expressing key metabolic enzymes ^32-34 . However, the major drawback of this approach is the need for viral transduction and the variation of these manipulations (including poor infection efficiency and differences in effective transcription factors for establishing appropriate expression levels) required for each experiment. When compared to primary hepatocytes, the expression levels in the hESC-derived populations generated by this approach are much lower than those found in primary hepatocytes.

At this, a deep understanding of the pathway regulating maturity is provided. Also show that the combination of 3D aggregation and cAMP signal conduction plays a key role in the maturation of hepatoblasts. In addition, activin/nodal signal conduction after endoderm induction is necessary for the optimal generation of liver progenitor cell group, and the progenitor cell group of enrichment is useful for the separation of mature population. The combination of these three different steps for example causes the generation of hepatocyte-like cells, which show expression profiles and the level of functional metabolic enzymes similar to those found in primary adult liver cells.

The observation that sustained activin/nodal signaling within the CXCR4+C-KIT+ population is beneficial for the generation of mature hepatocytes highlights the importance of appropriate 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 target population of EBs on day 6 contains greater than 95% CXCR4+CKIT+EPCAM+SOX17+ cells. Instead, without being bound by theory, it 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 BMPs or FGFs. In addition to reducing mesoderm contamination, the additional activin culture step may also play a role in properly patterning the endoderm to a ventral foregut fate, as this pathway is known to play a role in anterior-posterior patterning of the gut tube. ^{35, 36} It has been previously shown that sustained activin/nodal signaling also affects pancreatic development from hESCs. ¹⁸

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 maturation of primary fetal hepatocytes from both mice and humans. ^{24, 37, 38} More 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 cell maturation. However, the magnitude of these differences is difficult to interpret because comparisons were not made with fetal and adult liver controls. The present study has expanded on 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 are 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 a 3D structure, mimicking, to some extent, the cellular morphology of hepatocytes within the liver.

The second step in the maturation strategy is to optionally activate the cAMP pathway within the 3D aggregates 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 are induced in 2D monolayers as well as in 3D aggregates. PCR and microarray analysis clearly show that the effects of cAMP signaling go beyond the induction of target genes, as activation of the induced pathway alters gene expression patterns 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 the maturation of hepatoblasts. The fact that sustained cAMP signaling is not required to maintain elevated expression levels further supports this interpretation: the effect is one of maturation and not simply the induction and maintenance of specific gene expression. Some of the most notable changes in expression observed were detected for key drug-metabolizing enzymes, including CYP1A2, CYP3A4, and UGT1A1, at levels as high as or higher than those found in primary human hepatocytes. Transcript levels are indicative of function, as HES2-derived cells displayed levels of functional enzymes comparable to those in primary hepatocytes. Similar patterns of induction were observed in hepatocyte-like cells derived from two hESC lines and one hiPSC line, demonstrating that this maturation strategy is broadly applicable.

Endocrine hormones such as insulin and glucagon can affect cAMP levels in the adult liver, which have acute effects on glucose metabolism and chronic effects by regulating gene expression. Under fasting conditions, increased cAMP levels lead 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 the expression of PGC1-α is significantly upregulated in the mouse liver one day after birth ^43. This upregulation is believed to rapidly promote the maturation of neonatal hepatocytes. Without being limited by theory, the effects of cAMP signaling on hESC-derived hepatoblasts may be reproduced to some extent by the upregulation of PGC-1α expression, and the changes observed in the liver during fasting and/or in the hepatocyte lineage at birth lead to the generation of cells that display many characteristics of mature cells.

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 the routine production of hPSC-derived hepatocyte-like cells for drug metabolism analysis in the pharmaceutical industry. The 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.

Materials and methods:

HPSC culture and differentiation

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 Before generating embryoid bodies (EBs), hESCs were passaged onto Matrigel ^™ -coated 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 for another 3 days in induction medium A, 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 cultured in medium B, supplemented with bFGF (10 ng/ml), activin A (100 ng/ml), Wnt3a (25 ng/ml) and BMP4 (0.25 ng/ml). EBs were collected on day 6, dissociated into single cells and cultured for 2 days at a concentration of 4× ¹⁰⁵ cells on Matrigel ^™ -coated 12-well plates in the absence of WNT3A and with activin A at a concentration of 50 ng/ml. On day 8, medium B was replaced with liver-specific medium consisting of 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 medium was changed every two days. To promote maturation of HES2-derived hepatocytes, they were cultured in maturation medium A for 12 days. Maturation medium A consists of IMDM supplemented with 1% volume/volume 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 in six-well ultra-low-cluster plates at a concentration of 6×10 ⁵ cells per well in maturation medium A supplemented with a 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 consists 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 from day 8 to day 14 for culture, 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 an atmosphere of 5% _CO₂ , 5% _O₂ , and 90% _N₂ . Polymeric cultures were maintained in an ambient air atmosphere of 5% _CO₂ .

Flow cytometry

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

Immunostaining

Immunostaining was performed as previously described. 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 wash buffer with 0.2% Triton-X100 for 20 minutes. After an additional three 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. Isotype controls were concentration-matched to the primary antibody. To visualize the signal, cells were then incubated with donkey anti-goat Alexa 488 antibody (Invitrogen) or donkey anti-rabbit Cy3 antibody (Jackson ImmunoResearch) for 1 hour at room temperature. For SOX17 staining, cells were fixed, permeabilized, and blocked as described above. Cells were incubated overnight at 4°C with goat anti-SOX17 (R&D). Signals were visualized by incubating cells with donkey anti-goat Alexa488 (Invitrogen). For ASGPR-1 staining, aggregates were cultured on Matrigel ^™ -coated coverslips for 1 day. After attachment and spreading, cells were fixed with 4% PFA for 15 minutes at 37°C and then permeabilized with cold 100% methanol for 10 minutes. Cells were washed and blocked as described above. Fixed cells were incubated overnight at 4°C with goat anti-ASGPR-1 (Santa Cruz) and then incubated with rabbit anti-ALB (DAKO) for 1 hour at room temperature. Signals were visualized by incubating with donkey anti-goat Alexa488 and donkey anti-rabbit CY3 antibodies. Primary and secondary antibodies were diluted in DPBS + 2% BSA + 0.05% Triton-X100. Gold anti-fluorescence quenching with DAPI (Invitrogen) was used to counterstain cell nuclei. The stained cells were visualized using a fluorescence microscope (Leica CTR6000) and images captured using Leica Application Suite software.

Quantitative real-time PCR

Total RNA was prepared using a micro kit (Ambion) and treated with RNase-free DNase (Ambion). 500 ng to 1 μg of RNA was reverse transcribed into cDNA using random hexamers and oligo (dT) with III reverse transcriptase (Invitrogen). Quantitative PCR was performed on ep realplexes (Eppendorf) using the QuentiFast SYBR Green PCR kit ( ^Quiagen ) as previously described. Expression levels were normalized to the housekeeping gene TATA box binding protein (TBP). To measure the expression of UGT1A1, the delta-delta CT method was used to assess the expression of related genes relative to the levels of 8-Br-cAMP(-) treated cells. Total human adult and fetal liver RNA was purchased from Clontech. Two primary hepatocyte RNA samples were provided by Dr. Stephen C. Strom (University of Pittsburgh), and a third sample was purchased from Zenbio (Lot No. 2199). Two primary hepatocyte samples were cultured for 2 days and collected. One (HH1892) was isolated from a 1-year-old Caucasian male, while another (HH1901) was isolated from a 14-month-old male whose liver was transplanted due to cholestasis. The third sample (Zenbio: lot 2199) was isolated from a 48-year-old male Caucasian organ donor.

Indocyanine green uptake by hepatic aggregates

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 three times with PBS, and then examined using an inverted microscope (Leica).

Drug metabolism analysis by HPLC

Liver aggregates were incubated for 24 or 48 hours 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 metabolite levels were quantified using a separately optimized high-performance liquid chromatography assay. Hydroxybupropion levels were determined based on the HPLC method of Loboz et al. ⁴⁶ . 4-MU glucuronide was measured by high-performance liquid chromatography coupled with tandem mass spectrometry as previously described ⁴⁷ . Cryopreserved hepatocytes were thawed and plated on collagen-coated culture dishes at a density of 1×10 ⁴ cells per well for 24 or 48 hours. After 24 or 48 hours of culture, supernatants were collected and the activities of CYP1A2, CYP2B6, NAT1/2 and total UGTs were measured.

Chip processing and data analysis

Follow the operation criteria of standard Affymetrix company at the University Health Network Genomics Center, run RNA sample 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.The cDNA of 2.7 micrograms of amplification is then fragmented, labeled and hybridized to Affymetrix human gene ST 1.0 chip 18 hours (45 ℃, at 60RPM).Use gene chip fluidics station P450 fluid workbench to wash array, and use Affymetrix gene chip scanner 7G to scan array.After scanning, check each chip, and find to pass through Affymetrix quality control guide.Original CEL file is imported into Genspring software (Agilent, v11.5.1), and uses the ExonRMA16 algorithm summary probe level data based on HuGene-1_0-st0v1_na31_hg19_2010-09-03. In addition, each gene was normalized to the median value among all samples considered. All statistics were performed on Log2 transformed data. A total of 28,869 transcripts were represented on the array.

As a first step, transcripts were filtered to remove those whose expression was consistently below the 20th percentile across all three sample groups. Unsupervised hierarchical cluster analysis using Pearson's center distance under the average linkage rule was used to resolve overall similarities and differences between samples and groups. Directed statistical analysis between the three sample groups was performed using ANOVA with Benjamini and Hochberg false discovery rate (FDR, q < 0.05) ^48. To find sets of biologically meaningful differentially expressed transcripts, gene ontology (GO) analysis was performed using the corrected Benjamini and Yuketieli hypergeometric test at a significance level of q < 0.1 ^49. 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 liver-specific based on public information from the HOMER database ⁵⁵ .

Example 2

CHIR99021 is a selective inhibitor of GSK3, which 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. As shown in Figures 8a) and 8b), the proportion of CKIT+CXCR4+ and CXCR4+EPCAM+ cells induced with CHIR99021 instead of Wnt3a was greater than 95% of the population and comparable to that seen in activin/wnt3a induction.

Figures 8(a) and 8(b) demonstrate that CHIR99021 can induce definitive endoderm cells. Figure 8(a) is a flow cytometric analysis showing the proportion of CXCR4+, CKIT+, and EPCAM+ cells in activin/CHIR99021 embryoid bodies on day 6 of induction using activin/wnt3a. Figure 8(b) is a flow cytometric analysis showing the proportion of CXCR4+, CKIT+, and EPCAM+ cells in activin/CHIR99021 on day 6. Figures 8(c) and 8(d) show monolayers induced with (c) activin/wnt3a or (d) activin/CHIR99021 on day 7.

method

Induction of definitive endoderm using GSK3β inhibitors

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

For monolayer induction, hESCs 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). Prior to endoderm induction in monolayer culture, hESCs were passaged onto matrigel-coated surfaces (typically 12-well plates) for 1 day. 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 culture medium was replaced daily 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), and 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), and activin A (100 ng/ml). The culture medium was replaced every other day. As described above, by treatment with FGF and BMP pathway agonists, the definitive endoderm is specified to a hepatic fate on day 7.

Example 3

Ectopic liver tissue in NSG mice

Transplanted hESC-derived hepatocytes engrafted and generated cells expressing hepatocyte differentiation markers.

8( e ), FIG. 8( f ), and FIG. 8( g ) demonstrate the engraftment of ES-derived hepatocytes prepared using the method described in Example 1 , except that the GSK3 inhibitor CHIR99021 was used to prepare the transplanted cells described in Example 2 .

Figures 8(e), 8(f), (g), and 8(h) show ectopic liver tissue in NSG mice. Figure 8(e) is an exemplary H&E-stained micrograph of the mesenteric region, showing clusters of hESC-derived hepatocytes (arrows) 2 months after transplantation. Magnification is 5x. Intestine (arrow), implanted cells (arrowhead). Figure 8(f) shows a high-magnification (10x) micrograph of the H&E-stained section from Figure 8(e).

Figures 8(g) and 8(h) show the presence of hESC-derived cells in the mesenteric region 2 months after transplantation. Double staining for human albumin (Alexa 488: green) (shown as arrows) and CK19 (Cy3: red) (shown as arrowheads) shows that the transplanted cells have the potential to differentiate into hepatocyte and cholangiocyte lineages. hESC-derived hepatocyte-like cells were observed as albumin-positive cells (arrows), while cholangiocyte-like cells expressed CK19 and were found in duct-like structures (arrowheads).

method

Ectopic liver tissue in NSG mice

Six-week-old NSG mice were obtained from Jackson Laboratory (Bar Harbor, Maine, USA) and stored at UHN Animal Facility. Aggregates (day 27) included hESC-derived liver 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 and subjected to laparotomy with 1-3% isoflurane. The mesenteric region was exposed, and the 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.

Example 4

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

Inhibition of the Wnt and MEK/ERK pathways can increase the expression of genes related to phase I drug-metabolizing enzymes.

CYP3A4 gene expression in 3D liver aggregates cultured on 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 increased CYP3A4 gene expression (Figure 9d).

CYP1A2 gene expression in 3D liver aggregates cultured at day 44 was associated with inhibition of Wnt/β-catenin signaling (XAV939: 1 μM) and MEK/Erk signaling (PD032590: 1 μM) with small molecules. Together with 8-Br-cAMP, inhibition of Wnt and MEK/Erk signaling increased CYP1A2 gene expression (Figure 9d).

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

method

Induction of definitive endoderm using a GSK3β inhibitor.

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

For monolayer induction, hESCs 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). Prior to endoderm induction in monolayer culture, hESCs were passaged onto 12-well matrigel-coated plates for 1 day. On day 0, cells were incubated 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 culture medium was changed daily with 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), and 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), and activin A (100 ng/ml). The culture medium was changed every other day. On day 7, definitive endoderm cells began to differentiate using liver-specific medium as described above.

Example 5

Treatment with cAMP induces maturation of cell aggregates

Cell aggregates were generated as in Example 1.

Cell aggregates were cultured in HGF, Dex, and OSM until day 32, when cAMP was added. When cAMP analogs and/or cAMP agonists were added, OSM was removed. 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 aggregate survival.

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.

Example 6

Notch signaling pathway in hepatic progenitor cells influences cholangiocyte differentiation

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

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 ).

As shown in Figure 10b, increased expression of CK19 and cystic fibrosis transmembrane conductance regulator (CFTR) in OP9 co-cultures 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, albumin expression is seen, demonstrating that the cells retain hepatoblastic characteristics.

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 ).

The experiments described above demonstrate 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 influence cholangiocyte maturation.

Example 7

Exemplary maturation medium formulations

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

Basal culture medium:

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

Cytokines and growth factors:

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

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

Basal culture medium:

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)

Cytokines and growth factors:

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

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

Basal culture medium:

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)

Cytokines and growth factors:

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

Aggregation stage

From Day 26/Day 25 to Day 32/Day 31

Basal culture medium:

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.

Cytokines and growth factors:

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

Aggregation phase with cAMP

From Day 32/Day 31 to Day 44/Day 43

Basal culture medium:

EGF-free hepatocyte culture medium (HCM) (Lonza: CC-4182), 10 mM 8-Br-cAMP (Biolab: B007), and small molecules associated with inhibition of Wnt/β-catenin signaling (XAV93: 1 μM) and MEK/Erk signaling (PD032590: 1 μM).

Cholangiocyte Maturation Medium

Basal culture medium:

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

Cytokines and growth factors:

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

Example 8

Effects of endothelial cells on hESC-derived liver development.

In view of the fact that endothelial cells play an important role in liver development, this pedigree is assessed on the growth and/or mature impact of hESC-derived hepatocytes.For these researches, CD34+ endothelial cells are generated from hESC.For these researches, HES2hESC cell line is used, and HES2 hESC cell line is designed to express red fluorescent protein (RFP) cDNA from ROSA locus, so that we can follow the tracks of endothelial cells.By generating endothelial cells with the combination induction of BMP4, bFGF and VEGF for 6 days, now, CD34+ cells (also CD31+ and KDR+) are separated by FACS.The CD34+ cells of selecting are cultivated for 6 days in EGM2 endothelial cell growth medium, then use Aggrewells ^TM to be used for the generation of chimeric aggregates.Endothelial cells were added to Aggrewells 2 days before hepatocyte, to allow them to be coated in the bottom (Figure 12 a) of hole.At this moment, on the top of endothelial cells, add the single cell suspension of hepatoblasts in the 25th day, and mixture is cultivated 48 hours in Aggrewells. Aggregates are subsequently removed from Aggrewells and cultured for an additional 6 days, at which point they are collected and analyzed. As shown in Figure 12b, aggregates cultured with endothelial cells contain RFP+ cells, and more than those cultured alone. Flow cytometry analysis shows that RFP+ cells represent greater than 30% of the population (Figure 12c), which indicates that a large number have been integrated with the hepatocytes in the aggregates. qRT-PCR analysis shows that chimeric aggregates cultured for an additional 12 days express substantially higher levels of CYP3A4 messages (Figure 12d) than liver aggregates without endothelial cells. Importantly, these levels are achieved without the addition of cAMP, indicating that endothelial cells can promote the maturation of hPSC-derived hepatocytes. These results indicate that interacting with embryonic endothelial cells affects the survival and maturation of hESC-derived hepatocytes.

Maturation of hESC-derived hepatocytes in collagen gels.

The combination of 3D aggregation, cAMP and PD/XAV does promote the significant differentiation of human pluripotent stem cell-derived hepatocytes (Figure 9), (Figure 9d and Figure 9e). The cells do retain some expression of AFP and fetal CYP3A7, indicating that they may not be fully mature. In order to promote the 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 gel to provide a source of extracellular matrix proteins. As shown in Figure 13, when the aggregates were maintained in liquid culture, adding endothelial cells to the aggregates (ends) did not significantly affect the expression levels of ALB, CYP3A4, AFP or CYP3A7. In contrast, the cultivation of aggregates in collagen gels had a significant effect on AFP and CYP3A7 expression, because both were reduced to almost undetectable levels, similar to those found in adult livers. This finding suggests that signal transduction pathways, cell interactions and the extracellular environment all play a role in the maturation of hPSC-derived hepatocytes. This suggests that it is possible to generate cells that express very little AFP (if any). This expression pattern suggests that these cells have progressed to a stage equivalent to hepatocytes in the adult liver.

Example 9

The development of protocols for efficiently 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 comprising hepatocytes is particularly important because hepatocytes and bile duct cells, which constitute the biliary system of the organ, are the main targets for the adverse effects of drugs and the main targets for a range 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 protocol that promotes the generation of cells that, despite low efficiency and lack of metabolic function, display 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 to simulate genetic liver diseases that affect hepatocyte function.

Diseases involving the bile duct are a common cause 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 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 address these unmet needs.

Success in deriving cholangiocytes from hPSCs will depend on the ability to accurately mimic the embryonic development of this lineage in differentiated cultures. 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.

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 regulating the swelling of cysts after stimulating the cAMP pathway with forskolin. Cysts generated from iPSCs of cystic fibrosis patients show defects in forskolin-induced swelling assays that can be rescued by adding CFTR correctors. Together, these findings indicate that it is possible to generate cholangiocytes and bile duct-like structures from hPSCs and to use these derived cell types to simulate various aspects of cystic fibrosis biliary disease in vitro.

result

Characterization of the hepatoblast stage of development in hPSC differentiation cultures

In order to generate bile duct cells from hPSC, it is necessary to first characterize the hepatoblast stage of development in differentiation culture. For these studies, we use a modified version of the scheme (Figure 14a), which is developed for generating functional hepatocytes from hPSC ¹ . The main difference from our previous method is: the step of endoderm induction is carried out in a single layer rather than in a 3D embryoid body (EB). This change has led to the acceleration of endoderm development in culture, due to the generation of a population (Figure 14b) comprising CXCR4+CKIT+ and EPCAM+ cells greater than 90% by the 3rd day of differentiation. Suitable populations were not detected until the 5th day of EB differentiation 1. In order to specialize endoderm to liver fate, cultures were treated with a combination of bFGF and BMP4 on the 7th day of differentiation.

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

Notch signaling promotes cholangiocyte development from hPSC-derived hepatocyte-like populations.

To explore the effect of Notch signaling on bile duct cell development, we co-cultured hepatoblast populations (day 25) with OP9 stromal cells known to express different Notch ligands including ^Jagged14,5 . When cultured as single cell suspensions on the matrix, hPSC-derived cells did not survive well. 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 the majority of cells in the aggregates before co-culture were ALB+AFP+CD19+NOTCH2+, indicating that they retained hepatoblast characteristics. This aggregation step appears to select hepatoblasts, because after 48 hours of culture, aggregates generated from the day 25 population, which only included 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 that formed aggregates no longer expressed ALB, suggesting that they had undergone the initial stages of bile duct cell specialization. As shown in studies in mice, HGF, EGF, and TGFβ1 signaling play a ^role in bile duct development. We next added these factors, either alone or in combination, to the cultures to determine whether activation of these pathways would promote the further development of CK19+ ^clusters . Adding EGF or TGFβ1, or a combination of the two, resulted in an increase in the size of CK19+ aggregates. In contrast, HGF alone had little effect. Interestingly, the combination of EGF and HGF, or EGF, HGF, and TGFβ1, induced dramatic morphological changes and promoted the formation of branched structures including CK19+ cells. RT-qPCR Figure 15a and flow cytometry analysis (Figure 21) confirmed the immunostaining findings and demonstrated the complete absence of ALB+ cells after co-culture with OP9.

Adding a gamma secretase inhibitor (GSI), an antagonist of the Notch pathway, hindered the downregulation of ALB expression, reduced the proportion of CK19+ cells in culture, and inhibited the development of branched structures, indicating that these effects were mediated by Notch signaling (Figure 15a). After 9 days of culture on OP9, Notch targets HES1, HES5, and HEY1 were upregulated. (Figure 15b) This increase in expression was prevented by the addition of a gamma secretase inhibitor, indicating that co-culturing with OP9 effectively activated the Notch pathway (Figure 15b). Analysis of the differentiation potential of two other hPSC cell lines (ESC HES2 and iPSC Y2-1) showed similar temporal patterns of expression of TBX3 and hepatoblast markers, suggesting that transitions through these stages are characteristic of in vitro liver development. (Figure 22) After co-culturing with OP9 stromal cells, aggregates derived from the two cell lines generated branched structures including CK19+ALB- cells. As observed with H9-derived populations, the downregulation of ALB expression and the 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 the combination of HGF, EGF, and TGFpi signaling promotes morphological changes that lead to the formation of branched structures, likely reflecting early stages of duct morphogenesis.

Three-dimensional culture promotes cholangiocyte maturation

In order to determine whether the branches observed in the OP9 co-culture indicate the initial stage of bile duct development, we next set up a culture system to promote the growth of 3D cell structures. By this method, chimeric aggregates including hESC-derived cells and OP9 stromal cells (GFP+) on day 25 were cultured in a culture medium mixture, which included 1.2 mg/ml of collagen, 40% matrigel and HGF, EGF and TGFpi (Figure 23a). Within 2 weeks of cultivation under these conditions, the aggregates experienced huge morphological changes and formed tubular structures, hollow capsules or a mixture of the two (Figure 16a, Figure 16b). Capsules were the most abundant structures in these cultures (Figure 16b). Compared with populations derived from aggregates without OP9 cells, the expression of Notch target genes Hes1, Hes5 and Hey1 was upregulated in populations developed from chimeric aggregates, indicating that Notch signaling was active in culture (Figure 23b). Histological analysis showed that the tubular and cystic structures had a ductal morphology with an inner lumen and included epithelial cells that expressed CK19+ and E-cadherin+ but did not express ALB-. ZO-1 (occluded zone 1), a marker of tight junctions, was also expressed and found to be restricted to the top surface of the structure, indicating that the cells had acquired gene expression polarity, a characteristic of mature epithelial ducts. The cells in the tubes 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 mainly detected on the top surface of the tubular structure. Immunoblot analysis confirmed the presence of the protein in the population generated from the chimeric aggregates (Figure 23d). The levels of CFTR information and protein were quite low in cells derived from aggregates cultured without OP9, indicating that its expression depends on Notch signaling (Figure 23d, Figure 23e). The lack of liver markers (including ALB, AFP and CYP3A7) and the increase in the expression of cholangiocyte markers CK19, SOX9 and CFTR in these structures were confirmed by RT-qPCR analysis (Figure 16c). After culturing under these conditions, similar CK19+CFTR+ tubular and cystic structures were developed from iPSC-derived aggregates. In short, these findings illustrate that when cultivated in a mixture of matrigel and collagen, hepatoblast populations can generate tubular structures that express the markers found in mature bile ducts.

The addition of GSI prevented the formation of tubular structures and cysts and promoted the development of dense aggregates (Figure 16a, Figure 16b spheroids) that expressed ALB and low levels of CK19, indicating that, in the absence of Notch signaling, cells with hepatoblastic properties persisted in culture. The 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 bile duct cell development (CK19, Sox9, and CFTR) (Figure 16c).

hPSC-derived cholangiocytes form tubular structures in vivo.

In order to evaluate the developmental potential of hPSC-derived cholangiocytes in vivo, we transplanted the hPSC-derived cholangiocytes (10 ⁶ cells) in the matrigel plug into the mammary fat pad of immunodeficient NOD/SCID/IL2rg-/-(NSG) mice. For these studies, we used cells dissociated from branching structures, which were generated by co-culturing the 25th day hepatoblast population derived from HES2-RFPhESC with OP9 matrix. These hESCs were designed to express RFP ⁹ from the ROSA locus. After 6 to 8 weeks of transplantation, multiple tubular structures (Figure 17 a and Figure 17 b) were detected in the matrigel plug. The cells in the duct were RFP+, indicating that they were the origin of the human race, derived from HES2-RFP cells (Figure 17 c, Figure 17 d). In addition, the cells expressing CK19 and CFTR showed that they displayed the characteristic of cholangiocytes. As observed with the structure generated in vitro, CFTR expression was separated from the top surface of the duct. Teratoma was not observed in any transplanted animal.

hPSC-derived cholangiocytes are functional

As a first step in evaluating the functionality of hPSC-derived cholangiocytes, we assessed 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 into 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 the movement of the dye, reflecting active transport, is likely via the MDR transporter.

To demonstrate CFTR functional activity, we next performed a forskolin-induced swelling assay on cystic structures. Using this assay, CFTR function was increased by activating the cAMP pathway through the addition of forskolin/IBMX, which leads to fluid transport and swelling of the cysts. Swelling could be visualized after staining with calcein green, a cell-permeable fluorescent dye (Figure 18a). When measured 24 hours later, the addition of forskolin and IBMX to the culture 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). Adding a CFTR inhibitor (CFTR _inh -172) blocked the forskolin/IBMX-induced swelling, indicating that the increase in cyst size was CFTR-dependent. Findings from these assays indicate that cells in hPSC-derived cysts/tubular structures display characteristics of functional cholangiocytes found in the hepatic bile duct.

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

In order to demonstrate the purposes of this system in vitro simulation disease, we next analyzed the cyst formation from iPSC generated from two different cystic fibrosis patients carrying common F508 deletion (e.g., deltaF508). The hiPSC cell line generates hepatoblast populations (Figure 24a and Figure 24b) with kinetics similar to those observed for wild-type hPSC. Although hepatoblast development does not change, the cyst formation from the patient's iPSC is clearly impaired because only branched structures (Figure 19a) are observed in the gel after two weeks of cultivation. Cyst formation (Figure 19a, Figure 19b) from patient cells can be induced by adding forskolin in the first week of a biweekly culture period (variation). However, many cysts developed from patient iPSC are not completely hollow, but comprise branched tubular structures (Figure 19c). After a longer culture period, higher frequencies of hollow cysts (typically those developed from normal iPSC) are detected, indicating that the maturation of mutant cells is postponed. Adding 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).

Next, 2 days before the CFTR functional assay, we used chemical correctors VX-809 and Corr-4a to assess the functional restoration of F508 del CFTR in cholangiocyte-like cells derived from cystic fibrosis patient iPSCs. These two molecules are used to correct the folding defects of the mutant CFTR protein. Adding the corrector 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 different patches in the cysts derived from patients treated with the corrector. This pattern may reflect the incomplete rescue of the transport defect of the mutant protein. Immunoblot analysis also shows the effect of adding the corrector. Most CFTR in normal cells is the larger mature form identified by the upper band (C) in the HBE in lane (lane) in Figure 20a. Patient-derived cells contain much less CFTR protein, and most of it is a smaller immature form. Adding 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 swelling assay induced by forskolin/IBMX (Figure 20b, Figure 20c). Patient-specific cysts showed little swelling in the absence of the corrector. However, with the addition of the corrector, 24 hours after induction with forskolin/IBMX and VX770, cysts generated from patient C1 increased by approximately 2.18+/-0.52 times, while those from patient 997 increased by 1.64±0.08 times. In summary, these findings illustrate 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 defects.

discuss

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

Studies in mice have shown that the Notch pathway is important for inducing bile duct cell fate decisions in vivo, involving interactions between Jagged 1 on portal mesenchymal cells and Notch2 on hepatocytes.

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, a counter-effect is observed. Previous reports have shown that generating bile duct-like tubular structures from human ES cells (although inefficient) results in structures with polarity and rhodamine 123 uptake.

Notch signaling provided by OP9 promoted the engraftment of human ES cell-derived cholangiocyte-like 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.

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

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

Furthermore, patient-specific cholangiocyte-like ductal structures can be efficiently obtained and used to validate existing or new therapeutics for other serious 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.

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

All publications, patents, and patent applications are herein incorporated 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, including, for example, accession numbers and/or biomarker sequences (e.g., proteins and/or nucleic acids) provided in the tables or elsewhere, are incorporated by reference in their entirety.

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Claims (15)

1. A method for generating hepatocytes and/or bile duct cells from a human induced pluripotent stem cell population, the method comprising: a) The human induced pluripotent stem cells, either as a monolayer culture or formed into embryoids, are contacted with an induction medium for 4-8 days to provide an induced endoderm cell population, the induction medium comprising ActA and Wnt/β-catenin agonist. b) Expose the induced endoderm cell population to a nodal agonist for 1-4 days to provide an extended nodal agonist-induced endoderm cell population. c) The induced endoderm cell population treated with the extended nodal agonist was contacted with a specialized culture medium for 4-10 days to obtain an expanded hepatocyte population; the specialized culture medium included an FGF agonist selected from FGF2, FGF4 and FGF10 and a BMP4 agonist selected from BMP4, BMP2 and BMP7. d) The expanded hepatocyte population was cultured in a maturation medium containing HGF, Dex and OSM for 10 to 14 days; e) The hepatocyte population obtained by enzyme treatment and artificial dissociation step d); f) Generate aggregates of the dissociated hepatocyte population and culture the aggregates in a maturation medium comprising HGF, Dex and OSM; as well as g) The aggregates obtained in step f) are cultured using a cAMP analogue for 6 to 10 days. 2. The method of claim 1, wherein the induced endoderm population from the prolonged nodal agonist treatment comprises at least 80%, 85%, 90%, or 95% CXCR4 and CKIT positive cells and/or at least 70%, 75%, or 80% SOX17 ⁺ cells. 3. The method according to claim 1 or 2, wherein the FGF agonist is FGF2. 4. The method according to claim 1 or 2, wherein the aggregate of the hepatocyte population comprises at least 70%, 80%, 85%, or 90% albumin-positive cells. 5. The method according to claim 1 or 2, wherein the cAMP analog is selected from 8-bromoadenosine-3',5'-cyclic monophosphate, butyryl-cAMP, adenosine 3',5'-cyclic monothiophosphate, SP-cAMP, 8-bromoadenosine-3',5'-cyclic monothiophosphate, and Sp-8-Br-cAMP. 6. The method according to claim 1 or 2, wherein the aggregates of the hepatocyte population are further cultured for 6 to 10 days using a Wnt agonist. 7. The method according to claim 1 or 2, wherein the Wnt/β-catenin agonist is Wnt3a. 8. The method according to claim 1 or 2, wherein, during step g), the aggregates of the hepatocyte population are further contacted with one of the following: a) Wnt/β-catenin agonists and TGFβ antagonists to promote the expansion of albumin+/HNF4+ progenitor cell populations; or b) Wnt antagonists and Mek/Erk antagonists to promote CYP enzyme expression. 9. The method according to claim 8, wherein the TGFβ antagonist is SB431542, the Wnt antagonist is XAV939, and the Mek/Erk antagonist is PD0325901. 10. The method according to claim 1 or 2, wherein the maturation culture medium further comprises a Notch antagonist that promotes hepatocyte lineage specialization. 11. The method of claim 10, wherein the method generates functional hepatocytes; Among these, the functional hepatocytes, compared to cell populations including hepatic progenitor cells and/or bile duct progenitor cells, Increased expression and/or activity of at least one protein selected from the group consisting of ALB, CPS1, G6P, TDO, CYP7A1, CYP3A7, CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2D6, NAT2, and UGT1A1; and/or Increased expression of at least one gene selected from the group consisting of ALB, CPS1, G6P, TDO, CYP7A1, CYP3A7, CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2D6, NAT2, and UGT1A1 was observed. 12. The method of claim 11, wherein at least 40%, 50%, 60%, 70%, 80%, or 90% of the functional hepatocytes are ASGPR-1 ⁺ cells. 13. The method of claim 10, wherein the Notch antagonist is a γ-secretase inhibitor L695,458. 14. The method according to claim 1 or 2, wherein the aggregates of the hepatocyte population are embedded in a gel/matrix and subjected to 3D culture. 15. The method of claim 1 or 2, further comprising: enriching or separating a population of cells, said population comprising 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 95% hepatocytes, bile duct cells, functional hepatocytes, or functional bile duct cells.