US20160002595A1 - Methods for generating hepatocytes and cholangiocytes from pluripotent stem cells - Google Patents

Methods for generating hepatocytes and cholangiocytes from pluripotent stem cells Download PDF

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US20160002595A1
US20160002595A1 US14/768,409 US201414768409A US2016002595A1 US 20160002595 A1 US20160002595 A1 US 20160002595A1 US 201414768409 A US201414768409 A US 201414768409A US 2016002595 A1 US2016002595 A1 US 2016002595A1
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Gordon Keller
Shinichiro Ogawa
Anand Ghanekar
Christine BEAR
Binita M. KAMATH
Mina Ogawa
James SURAPISITCHAT
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Hospital for Sick Children HSC
University Health Network
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University Health Network
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Definitions

  • the disclosure relates to methods for producing functional hepatocytes from human pluripotent stem cells.
  • hPSCs human pluripotent stem cells
  • hiPSCs induced pluripotent stem cells
  • hPSC-derived hepatocytes can offer potential new therapies for patients with liver disease.
  • liver transplantation provides an effective treatment for end-stage liver disease, a shortage of viable donor organs limits the patient population that can be treated with this approach 5-7 .
  • Hepatocyte transplantation and bio-artificial liver devices developed with hPSC-derived hepatocytes represent alternative life-saving therapies for patients with specific types of liver disease. These applications are, however, dependent on the ability to generate mature metabolically functional cells from the hPSCs. Reproducible and efficient generation of such cells has been challenging to date, due to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood.
  • An aspect of the disclosure includes a method of producing hepatocyte lineage cells from an extended nodal agonist treated induced endodermal cell population, the method comprising:
  • the hepatocyte and/or cholangiocyte lineage cells are hepatoblasts.
  • the method produces an expanded population of hepatoblasts.
  • the hepatocyte lineage cells are mature hepatocytes or the cholangiocyte lineage cells are mature cholangiocytes.
  • the extended nodal agonist treated induced endodermal cell population is induced from pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).
  • PSCs pluripotent stem cells
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • the pluripotent stem cells are optionally human ESCs (hESCs) or human iPSCs (hiPSCs).
  • the extended nodal agonist treated induced endoderm population is, in an embodiment, obtained by inducing endoderm cells in embryoid bodies (EBs). In another embodiment, the extended nodal agonist treated induced endodermal population is obtaining by inducing endoderm cells that are in a monolayer. In each case, the induced endodermal population is cultured in the presence of a nodal agonist, for example activin, for an extended period to produce an extended nodal agonist treated induced endodermal population.
  • a nodal agonist for example activin
  • the extended nodal agonist treated induced endodermal population comprises at least, 80%, 85%, 90%, 95 CXCR4+ and cKIT+ positive cells and/or at least 70%, 75%, 80% SOX17+ cells.
  • the specifying step comprises contacting an extended nodal agonist treated (e.g. activin treated) induced endodermal population with specification media comprising a FGF and BMP4.
  • the FGF can for example be bFGF, FGF10, FGF2 or FGF4 or combinations thereof. The combinations can for example be added sequentially.
  • the specifying step comprises first contacting an extended nodal agonist treated induced endodermal population with specification media comprising FGF10 and BMP4 for approximately 40 to 60 hours, optionally approximately 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 hours and then contacting the extended nodal agonist treated induced endodermal population with specification media comprising bFGF and BMP4 for about 4 to 7 days, optionally about 4, 5, 6 or 7 days.
  • 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, 25, 26, 27, or 28 days in culture.
  • aggregates are generated from a monolayer of the cell population comprising hepatocyte and/or cholangiocyte progenitors by enzymatic treatment and/or manual dissociation.
  • the cell population comprising hepatocyte and/or cholangiocyte progenitors and/or the aggregates are cultured in the presence of hepatocyte growth factor (HGF), dexamethasone (DEX) and/or Oncostatin M (OSM) and/or active conjugates and/or fragments thereof.
  • HGF hepatocyte growth factor
  • DEX dexamethasone
  • OSM Oncostatin M
  • inducing maturation, and optionally further lineage specification and/or expansion further comprises activating the cAMP pathway within the cells of the aggregates to induce the maturation of the hepatocyte and cholangiocyte progenitors into hepatocytes and/or cholangiocytes.
  • activating the cAMP pathway comprises contacting the aggregates with cAMP and/or a cAMP analog (e.g.
  • a maturation media comprising a cAMP agonist and DEX and optionally HGF is added to the aggregates subsequent to culturing the pre-aggregate population in a maturation media comprising HGF, DEX and OSM, for example for about 10, 11, 12, 13 or 14 days.
  • the population of hepatocytes produced is a population comprising functional hepatocytes.
  • 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 protein 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 hepatocyte and/or cholangiocyte progenitors, and/or hepatocytes produced from a non-extended nodal agonist treated induced endodermal cell population (e.g.
  • At least 40%, 50%, 60%, 70%, 80% or 90% of the hepatocytes, optionally functional hepatocytes, are ASGPR-1+ cells.
  • cholangiocyte fate is specified by treating aggregates of the cell population with a notch agonist.
  • the population of cholangiocytes produced is a population of functional cholangiocytes.
  • the functional cholangiocytes comprise for example increased expression of at least 1, at least 2 or 3 genes or proteins selected from Sox9, CK19 and CFTR (Cystic fibrosis transmembrane conductance regulator) compared to the cells of the cell population comprising hepatocyte and cholangiocyte progenitors and/or compared to a population cells produced from aggregates not treated with a notch agonist.
  • at least 40%, 50%, 60%, 70%, 80% or 90% of the population of cholangiocytes are CK19+ cholangiocytes.
  • at least 40%, 50%, 60%, 70%, 80% or 90% of the functional cholangiocytes are CFTR+ cholangiocytes.
  • the method can be applied to an endodermal cell population grown in a monolayer.
  • a further aspect includes a method of producing hepatocytes and/or cholangiocytes from a pluripotent stem cell population, the method comprising:
  • the endodermal population can also be comprised in embryoid bodies.
  • a further aspect of the disclosure provides a method of producing hepatocytes and/or cholangiocytes from a pluripotent stem cell population, the method comprising:
  • the inducing maturation, further lineage specification and/or expansion step further comprises activating the cAMP pathway within the aggregates to induce the maturation of hepatocyte and/or cholangiocyte progenitors of the cell population into a population comprising hepatocytes and/or cholangiocytes.
  • the method comprises contacting the aggregates with a cAMP analog and/or cAMP agonist.
  • the monolayer or EBs are contacted with a nodal agonist in induction media for at least about 1 day, 2 days, 3 days or about 4 days.
  • the EBs are cultured with a nodal agonist for at least 36, 38, 42, 44, 46, 48, 50, 52, 56, 58 or 60 hours or for at least about 1 day, 2 days, 3 days or about 4 days.
  • a nodal agonist for at least 36, 38, 42, 44, 46, 48, 50, 52, 56, 58 or 60 hours or for at least about 1 day, 2 days, 3 days or about 4 days.
  • PSCs pluripotent stem cells
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • the aggregated cell maturation media can comprise factors which promote hepatocyte maturation or factors which promote cholangiocyte development or both.
  • aggregated cells are upon aggregation treated with a wnt agonist such as CIHR 99021, optionally in the presence of a TGFbeta antagonist such as SB431542.
  • a wnt agonist such as CIHR 99021
  • TGFbeta antagonist such as SB431542.
  • activation of the Wnt pathway and SMAD pathway at for example day 26 or optionally one or two days later e.g. day 27, in embodiments using EBs
  • promotes expansion of an albumin+/HNF4+ progenitor population promotes expansion of an albumin+/HNF4+ progenitor population. It is demonstrated for example that up to a 10 fold expansion of said population can be obtained when a wnt agonist is added.
  • the aggregated cells are treated with a wnt agonist and optionally a TGFbeta antagonist (such as SB431542) for about 6 to about 12 days, preferably about 8 to about 10 days, optionally for about 9 days.
  • a wnt agonist and optionally a TGFbeta antagonist such as SB431542
  • a Wnt antagonist such as XAV939 (also referred to as XAV for short) and/or a Mek/Erk antagonist, for example PD0325901 (also referred to as PD for short) is added during the cAMP activation step.
  • Addition of a Wnt antagonist and/or a MEK/Erk antagonist during activation of cAMP signaling enhances expression of CYP enzymes, for example up to levels or greater than levels seen in adult liver cells.
  • an inhibitor of MEK/Erk added in the presence of cAMP for example, added to about day 28 to about day 32 cultures, results in hepatocytes with increased levels of CYP3A4 a.
  • Addition of a MEK/Erk antagonist in combination with a Wnt antagonist is shown to also increase levels of CYP1A2.
  • the Wnt antagonist is XAV939.
  • the MEK/Erk antagonist is PD0325901.
  • the cells are treated with a notch agonist. Addition of a notch agonist at such stages promotes cholangiocyte maturation. In some embodiments, for example where cholangiocyte maturation is preferred, inducing cAMP signaling is omitted.
  • Notch antagonist such as gamma-secretase inhibitor (GSI) L695,458
  • GPI gamma-secretase inhibitor
  • the method comprises approximately 1 to about 4 days after aggregation, treating the cells with a notch antagonist, for example in embodiments where hepatocyte differentiation is desired.
  • the method of producing hepatocytes and/or cholangiocytes from pluripotent stem cells comprises:
  • the method comprises aggregating for example after about 20 days of culture and/or before 40 days of culture
  • the disclosure also provides a method of inducing maturation, further lineage specification and/or expansion of cholangiocyte progenitors into cholangiocytes, the inducing maturation, further lineage specification and/or expansion comprising:
  • the notch agonist can for example be any notch ligand bound to a surface such as a cell, plastic, ECM or bead.
  • the notch ligand is notch ligand delta.
  • inducing maturation, further lineage specification and/or expansion comprises contacting a cell population comprising cholangiocyte progenitors with a notch signaling donor (e.g.
  • notch agonist such as OP9, OP9delta, and/or OP9 Jagged1 cells and optionally in the presence of EGF, TGFbeta1, HGF and EGF, and/or HGF, TGFbeta1 and EGF for at least or about 5 to about 90 days, to induce the maturation of cholangiocyte progenitors into functional cholangiocytes.
  • contacting a cell population comprising cholangiocyte progenitors with a notch agonist comprises co-culturing the cell population comprising cholangiocyte progenitors with a notch signaling donor such as OP9, OP9delta, and/or OP9 Jagged1 cells and optionally in maturatoin media comprising EGF, TGFbeta1, HGF and EGF, and/or HGF, TGFbeta1 and EGF, for at least or about 5 to at least or about 90 days, optionally for at least or about 5 to at least or about 60 days, at least or about 30 days, at least or about 25 days, 2 at least or about 1 days and/or at least or about 14 days to induce the maturation of cholangiocyte progenitors into cholangiocytes, optionally functional cholangiocytes, optionally wherein the functional cholangiocytes form branched, cyst, tubular or sphere type structures.
  • a notch signaling donor such as OP9, OP9
  • the application provides a method comprising:
  • the method further comprises enriching or isolating a hepatocyte and/or cholangiocyte population of cells.
  • the hepatocyte and/or cholangiocyte population of cells comprises 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 disclosure also provides the use of the population 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 disease.
  • FIG. 1 shows endoderm induction in hESC-derived embryoid bodies.
  • FIG. 1( a ) is a schematic representation of the differentiation protocol. EBs are trypsinized at day six and plated as a monolayer in the presence of activin for two days to generate appropriate-staged definitive endoderm. The hepatic lineage is specified from this endoderm population by culture in the presence of BMP4 and FGF.
  • FIG. 1( b ) shows flow cytometric analyses showing the proportion of CXCR4+, CKIT+ (CD117) and EPCAM+ cells in day six activin- and activin/Wnt3a-induced populations.
  • FIG. 1( b ) shows flow cytometric analyses showing the proportion of CXCR4+, CKIT+ (CD117) and EPCAM+ cells in day six activin- and activin/Wnt3a-induced populations.
  • FIG. 1( c ) shows intracellular flow cytometric analyses showing the proportion of SOX17+ and FOXA2+ cells in day six activin- and activin/Wnt3a-induced populations.
  • FIG. 1( d ) shows RT-qPCR based analyses of T, SOX17, GSC and FOXA2 expression in activin and activin/Wnt3a-induced EBs. EBs were analyzed at the indicated time points. Bars represent SD of the mean of three independent experiments.
  • FIG. 1( e ) is a flow cytometric analysis showing the kinetics of development of the CXCR4+, CKIT+, SOX17+ and FOXA2+ populations in the activin/Wnt3a induced EBs.
  • FIG. 1( f ) is a flow cytometric analysis showing the proportion of CXCR4+, CKIT+, EPCAM+, Sox17+ and FOXA2+ cells in day six EBs induced with activin in with neural based media.
  • FIG. 2 ( a ) RT-qPCR analysis of albumin expression in monolayer cultures specified with the indicated cytokines.
  • Cells were treated with the different factors (bFGF 10 ng/ml; BMP4 50 ng/ml; HGF 20 ng/ml; or bFGF 20 ng/ml plus BMP4 50 ng/ml) from 6 days to day 12 and then cultured with DEX, HGF and OSM and analyzed at day 24. Bars represent the standard deviation (SD) of the mean of three independent experiments. Values are determined relative to TBP and presented relative to expression in bFGF (20 ng/ml) culture, which is set a one. ***P ⁇ 0.001 as compared with the culture treated bFGF.
  • SD standard deviation
  • FIG. 3 shows that the duration of activin signaling affects hepatic development.
  • FIG. 3( a ) is an intracellular flow cytometric analysis showing the proportion of SOX17+ and FOXA2+ cells in day six activin/Wnt3A-induced EBs as well as in monolayer populations derived from them.
  • the monolayer populations were cultured either directly in the specification media (-activin) or for two days in activin (50 ng/ml) and then in the specification media (+activin).
  • Populations were analyzed following two or four days culture in the specification media (total days eight and 10 for the -activin group and days 10 and 12 for the +activin group). Bars represent standard deviation (SD) of the mean of three independent experiments.
  • FIG. 3( b ) depicts total cell number in activin treated and non-treated monolayer cultures. Day six EB-derived cells were cultured directly in hepatic differentiation media or in the presence of activin for two days and then in hepatic differentiation media. FIG.
  • FIG. 3( c ) is a flow cytometric analysis showing the proportion of CXCR4 and CKIT positive cells in populations at days 8, 10 and 12 culture generated from non-treated cell and activin-treated endoderm.
  • FIG. 3( d ) shows RT-qPCR based expression analyses of hepatic monolayer populations generated from activin-treated (Black bars) and non-treated (Grey bars) endoderm. The populations were analyzed for expression of the indicated endoderm (HEX, AFP, ALB, and HNF4a) and mesoderm (MEOX1, MESP1, CD31 and CD90) genes.
  • FIG. 3( e ) is a flow cytometric analysis showing the proportion of CD31 + CD90 + and EPCAM + cells in monolayer populations derived from activin treated (day 26) and non-treated (day 24) endoderm.
  • 3( f ) shows immunostaining analyses showing the proportion of albumin positive cells in cultures generated from activin treated (day 26) and non-treated (day 24) endoderm. Albumin is visualized with Alexa 488. Scale bars: 200 ⁇ m.
  • FIG. 4 shows that aggregation promotes hepatic maturation.
  • FIG. 4( a ) is a phase-contrast image of hepatic aggregates at day 28 of culture. Scale bar, 200 ⁇ m.
  • FIG. 4( b ) shows RT-qPCR based analyses of ALB, CPS1, TAT, G6P and TDO expression in monolayer (black bar) and 3D aggregate cultures (grey bar) at day 32 of differentiation. Values are determined relative to TBP and presented relative to expression in adult liver, which is set a one.
  • FIG. 4 shows that aggregation promotes hepatic maturation.
  • FIG. 4( a ) is a phase-contrast image of hepatic aggregates at day 28 of culture. Scale bar, 200 ⁇ m.
  • FIG. 4( b ) shows RT-qPCR based analyses of ALB, CPS1, TAT, G6P and TDO expression in monolayer (black bar) and 3D aggregate cultures (grey
  • FIG. 4( c ) is a RT-qPCR based analysis for CYP7A1, CYP3A7 and CYP3A4 expression at day 32 of differentiation in monolayer (black bar) and 3D aggregate culture (grey bar). Expression levels are relative to TBP.
  • FIG. 5 shows that cAMP signaling induces maturation of hESC-derived hepatocyte-like cells.
  • FIG. 5( a ) is a RT-qPCR analysis of PGC1-a, HNF4a, AFP, ALB, G6P, and TAT expression in hepatic aggregates cultured in the presence and absence of 8-Br-cAMP. Expression levels are relative to TBP.
  • FIG. 5( b ) is an intracellular flow cytometric analysis showing the proportion of alpha-fetoprotein (AFP) + and albumin (ALB) + cells (day 44) in hepatic aggregates cultured in the presence and absence of 8-Br-cAMP.
  • AFP alpha-fetoprotein
  • ALB albumin
  • FIG. 5( c ) is a RT-qPCR analysis of PGC1- ⁇ expression in cAMP treated pancreatic aggregates and hepatic aggregates generated from HES2, H9 and 38-2 cells.
  • FIG. 5( d ) shows ICG uptake at day 44 in non-treated and cAMP-treated aggregates. Bar in all graphs represent the standard deviation (SD) of the mean of the values from three independent experiments. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, Student's t-test, AL: adult liver, FL: fetal liver.
  • FIG. 6 shows that cAMP signaling increases metabolic enzyme activity in hESC-derived hepatocytes.
  • FIG. 6( a ) shows expression of CYP3A7, CYP3A4, CYP1A2, CYP2B6 and UGT1A1 in hepatic aggregates (day 44) cultured in the presence and absence of 8-Br-cAMP.
  • the levels in primary hepatocytes (PH) are shown as a control. Values are determined relative to TBP and presented as fold change relative to expression in non-treated cells, which is set at one.
  • FIG. 6 shows that cAMP signaling increases metabolic enzyme activity in hESC-derived hepatocytes.
  • FIG. 6( a ) shows expression of CYP3A7, CYP3A4, CYP1A2, CYP2B6 and UGT1A1 in hepatic aggregates (day 44) cultured in the presence and absence of 8-Br-cAMP.
  • FIG. 6( b ) shows RT-qPCR analyses showing expression of PGC1a, TAT, HNF4a, CYP1A2 and CYP3A4 in untreated ( ⁇ ) and cAMP-treated (+) monolayer populations (day 44). Values are determined relative to TBP and presented as fold change relative to expression in non-treated cells, which is set at one.
  • FIG. 6( c ) shows RT-qPCR analyses of CYP1A2 and ALB expression in cAMP treated aggregates (day 44) generated from non-treated (-Act) or extended activin treated (+Act) endoderm.
  • FIG. 6( d ) shows RT-qPCR analyses of CYP1A2 expression in aggregates cultured for six (cAMP+/ ⁇ ) or 12 days in 8-Br-cAMP (cAMP+).
  • FIG. 6( f ) shows that hESC-derived hepatic cells display CYP2B6 activity in vitro.
  • FIG. 6( g ) shows that metabolism of sulfamethazine (SMZ) to N-acetylated SMZ indicates the presence of the Phase II enzyme(s) NAT1 and/or NAT2.
  • SMZ sulfamethazine
  • FIG. 6( h ) is an HPLC analysis showing generation of 4-MU glucuronide (4-MUG) from 4-methylumbelliferone (4-MU) by the cAMP-treated aggregates indicative of Total UGT activity.
  • SD standard deviation
  • FIG. 7 shows hepatic differentiation from different pluripotent stem cell lines.
  • FIG. 7( a ) is a flow cytometric analysis showing the proportion of CXCR4+, CKIT+, EPCAM+, SOX17+ and FOXa2+ cells in activin/Wnt3a induced day six EBs generated from H9 hESCs, H1 hESCs and 38-2 iPSCs.
  • FIG. 7( b ) shows RT-qPCR analyses of albumin expression in monolayer cultures generated from H9, H1 and 38-2-derived endoderm treated with activin for varying periods of time. The different populations were analyzed at the following times: no activin: day 24, 2 day activin: day 26, 4 day activin: day 28 of differentiation.
  • FIG. 7 shows hepatic differentiation from different pluripotent stem cell lines.
  • FIG. 7( a ) is a flow cytometric analysis showing the proportion of CXCR4+, CKIT+, EPCAM+, SOX17+
  • FIG. 7( c ) shows intracellular flow cytometric analyses showing the frequency of ALB and AFP positive cells generated from the different hPSC lines (No activin ( ⁇ ): day 24, 2 day activin: day 26, 4 day activin: day 28 of differentiation).
  • FIG. 7( d ) is a phase contrast image showing morphology of H9-derived hepatic cells at day 26 of culture. Scale bar: 200 ⁇ m.
  • FIG. 7( e ) shows RT-qPCR analyses showing CYP3A7, CYP3A4, CYP1A2, CYP2B6 and UGT1A1 in H9- and iPSC (38-2)-derived hepatic aggregates (day 44) cultured in the presence and absence of 8-Br-cAMP.
  • FIG. 8 ( a ) (b) (c) demonstrate that CHIR99021 can induce definitive endoderm cells.
  • Flow cytometric analysis showing the proportion of CXCR4+, CKIT+ and EPCAM+ cells at day six of embryoid body induction with either (a) activin/wnt3a or (b) activin/CHIR 99021 or at day seven of monolayer induction with (c) activin/wnt3a or (d) activin/CHIR 99021.
  • FIG. 9 depicts factors that influence hepatic progenitor proliferation and maturation.
  • FIG. 9 ( a ) shows expansion of the hepatic progenitor population by Wnt signaling and Smad signaling. The fold increase in the number of ALB+AFP+ cells following 9 days of culture of H9-derived day 27 hepatic progenitors in different concentrations of CHIR99021 (0.3 ⁇ M, 1 ⁇ M and 3 ⁇ M) and TGFbeta inhibitor SB431542 (6 ⁇ M) is shown.
  • FIGS. 9 ( b ) and ( c ) depicts immunofluorescent staining showing the presence of ALB positive cells (b) and double positive of ALB and HNF4 ⁇ (c) following 9 days (day 36) of culture of H9-derived day 27 hepatic progenitor cells.
  • FIGS. 9 ( d ) and ( e ) shows that inhibition of Wnt/ ⁇ -catenin and MEK/Erk signaling increases expression of CYP3A4 (erkinhib+camp enough) and CYP1A2 in day 44 aggregates (all three).
  • the Wnt inhibitor XAV 939 (1 ⁇ M) and the MEK/Erk inhibitor PD0325901 (1 ⁇ M) were added alone or together to the aggregate cultures at day 30 of differentiation together with 8-Br-cAMP. Shown is the expression of CYP3A4 ( d ) and CYP1A2 ( e ) relative to the levels found in adult liver. Addition of the MEK/ErK inhibitor together with cAMP induced levels of CYP3A4 expression comparable to those found in the adult liver whereas addition of both the Wnt and MEK/ErK inhibitors with cAMP induced the highest levels of CYP1A2 expression.
  • FIG. 9 ( f ) shows expression of ALB in day 26 hepatocyte-like cells culture on different extra cellular matrix (ECM). Values shown are relative to cells cultured on gelatin, which is set to 1.
  • FIG. 10 shows that the notch signaling pathway in hepatic progenitor cells influences the differentiation of the cholangiocyte lineage.
  • H9-derived day 25 hepatic progenitor cells were mixed (aggregated) with OP9-delta 1 stromal cells at a ratio of 5:1, in low cluster culture dishes for 48 hours.
  • the chimeric aggregates were embedded in a mixture of type 1 collagen (80%) and Matrigel (20%) to establish a 3D co-culture.
  • the culture was maintained in media containing the HGF 20 ng/ml and EGF 50 ng/ml and in the presence or absence of GSI for 9 days.
  • the aggregate morphology was maintained in cultures treated with GSI. These aggregates contained hepatocyte-like cells that express albumin. In the absence of GSI, the aggregates developed extensive branched structures. The cell within the branches displayed an epithelial morphology and were organized around an inner lumen. These cells expressed CK19, suggesting that they were cholangiocytes and that the branched structures may represent developing bile ducts.
  • FIG. 10( b ) shows that activation of Notch signaling upregulates expression of CK19 and the cystic fibrosis transmembrane conductance regulator (CFTR) in the ductal structures. Values shown are relative to cells cultured in the presence of GSI. The cells in the Notch ( ⁇ ) co-culture (i.e., treated with GSI) retained the characteristics of hepatocytes as demonstrated by the expression of Albumin.
  • FIG. 10 ( c ) shows that the expression of CFTR in 3D co-culture were higher induced than those found in monolayer culture. Values shows are relative to cells cultured in the monolayer condition.
  • FIG. 11 is a schematic representation of hepatocyte/cholangiocyte differentiation protocol.
  • b EB cultures used to generate definitive endoderm.
  • hepatic progenitor cells were dissociated and aggregated with OP9/OP9 delta cells in low cluster dishes for two days.
  • the chimeric aggregates were embedded in a Collagen/Matrigel gel and cultured in the medium supplemented with HGF (20 ng/ml) and EGF (50 ng/ml) in the presence or absence of the inhibitor of pan Notch signaling (e.g. notch antagonist), gamma-secretase inhibitor (GSI) L-685, 458.
  • pan Notch signaling e.g. notch antagonist
  • GSI gamma-secretase inhibitor
  • FIG. 12 demonstrates that hESCs-derived endothelial cells enhance hepatic maturation.
  • RFP(+)/CD34(+) endothelial cells were generated by induction of EBs with BMP4 for 4 days and then with VEGF and bFGF for an additional 2 days.
  • FACS isolated RFP(+)/CD34(+) endothelial cells were plated on collagen type I coated wells and cultured with EGM-2 medium in the presence of VEGF and bFGF for 6 days.
  • the cultured RFP(+)/CD34(+) cells were trypsinized, dissociated and placed into Aggrewell plates at a cell density of 100 cells per well. Following 2 days of culture, the day 25 hepatoblasts cells were placed onto the RFP(+)/CD34(+) endothelial aggregates at a cell density of 1000 cells per well. Scale bar 100 um (b) phase contrast and fluorescent images showing RFP positive cells within endothelial/hepatic aggregates at day 33. RFP is not detected in hepatic aggregates generated without the endothelial cells.
  • FIG. 13 demonstrates the effect of 3D gel culture on maturation of hPSC-derived hepatocytes.
  • Aggregates consisting of hepatoblasts or hepatoblasts and endothelial cells (end) were generated at day 25 of culture and then cultured for an additional 7 days in liquid in hepatocyte culture medium supplemented VEGF and bFGF and followed by 12 days of culture in the same medium supplemented with cAMP, PD0325901 (PD) and XAV939.
  • day 32 chimeric aggregates were embedded in a Collagen type 1 gel and cultured in the presence of cAMP, PD0325901 and XAV939 for 12 days.
  • FIG. 14 Characterization of the hepatoblast stage of development in hPSC differentiation cultures.
  • AL Adult liver
  • FL fetal liver.
  • FIG. 15 Notch signaling promotes cholangiocyte development from the hPSC-derived hepatoblast-like population.
  • HGF 20 ng/ml
  • EGF 50 ng/ml
  • TGFb1 5 ng/ml
  • GSI gamma-secretase inhibitor
  • FIG. 16 Three-dimensional culture promotes cholangiocyte maturation: Morphology of chimeric aggregates consisting of day 25 hESC-derived cells and OP9 stromal cells (GFP+). H9-derived day 25 hepatoblast were mixed (aggregated) with OP9 stromal cells at a ratio of 4:1, in low cluster culture dishes for 48 hours. The chimeric aggregates were embedded in a mixture of type 1 collagen (1.2 mg/ml) and Matrigel (40%) to establish a 3D gel culture. The cultures were maintained over 2 weeks in the media containing of HGF, EGF and TGFb1 in the presence or absence of GSI.
  • GFP+ OP9 stromal cells
  • FIG. 17 hPSC-derived cholangiocytes form duct-like structures in vivo.
  • a-b Histological analyses of a cholangiocyte graft in a Matrigel plug 8 weeks following transplantation of day 25 hepatoblast-derived cells cocultured with OP9 stromal cells for 9 days in media containing of HGF, EGF and TGFb1. Following co-culture, the cells were dissociated and transplanted (10 6 per recipient) into the mammary fat pad of immunodeficient NOD/SCID/IL2rg ⁇ / ⁇ (NSG) mice. Multiple duct structure were visualized in mammary fat pad at low (a) and high magnification images (b) (H&E staining).
  • FIG. 18 hPSC-derived cyst structures generated in 3D gels contain functional CFTR protein
  • F/I Representative confocal microscopy images of calcein-green-labeled and forskolin/IBMX (F/I) stimulated cyst structures generated from H9 (hESC)- and Y2-1 (iPSC)-derived derived cholangiocytes. Image was taken 24 hours after Ell stimulation. Scale bar 500 ⁇ m
  • FIG. 19 The generation of cholangiocytes from cystic fibrosis patient iPSCs.
  • CF-iPSCs Phase contrast images of cholangiocyte like cells derived from CFTR deleted F508 iPS cells (CF-iPSCs) at day 44 of 3D gel culture in the presence or absence of forskolin.
  • CF-iPSCs-derived hepatoblasts and chimeric hepatoblast/OP9 aggregates were generated using the protocol shown in FIG. 14 a . After embedding in collagen/Matrigel culture, cyst formation was induced from the aggregates by the addition of forskolin for the first week of the two-week culture period (left).
  • CF-iPSCs derived cholangiocytes formed branched ductal structure rather than hollow cysts (right).
  • CF-iPSCs derived cholangiocytes were maintained in the 3D gel conditions in the presence or absence of forskolin for the first week of the two weeks culture period (left graph).
  • Normal iPS cells-derived cholangiocytes were maintained in the presence or absence of CFTR inhibitor for the first week of the two-week culture periods (right graph).
  • FIG. 20 Restoration of CFTR function in the CF-iPSC-derived cholangiocytes by treatment with the small molecule correctors VX-809 and C4.
  • Western blot analysis shows the accumulation of mature complex 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 was predominant in the uncorrected cells.
  • Human bronchial epithelial cells (HBE) were used as a positive control.
  • FIG. 21 Intracellular flow cytometric analysis showing the proportion of ALB+ and CK19+ cells in the hepatoblast-derived population following 9 days of coculture with OP9.
  • Cells were cultured in media containing of HGF, EGF and TGFb1 in the presence or absence of GSI.
  • Ctrl shows isotype control.
  • FIG. 22 Hepatic specification and differentiation of hepatoblast from other hPSCs.
  • RT-qPCR analyses showing expression of indicated genes in HES2 and Y2-1 iPS cells-derived hepatoblast cells maintained as indicated in FIG. 14 a .
  • the expression of the indicated genes was analyzed on days, day 7, 13, 19 and 25 of culture. Values are determined relative to TBP and presented as fold change relative to expression in fetal liver, which is set at one.
  • AL Adult liver
  • FL fetal liver.
  • FIG. 23 3D gels used for the generation of cystic structures from hPSC-derived cholangiocytes.
  • FIG. 24 Generation of definitive endoderm and hepatoblasts from cystic fibrosis patient iPSCs.
  • Flow cytometric analyses showing the development of the CXCR4+, CKIT+, and EPCAM+ populations from CF-iPS cells (C1 del CFTR) at day 7 of monolayer culture.
  • (b) RT-qPCR analyses showing expression of indicated genes in the CF-iPSC-derived hepatoblast population maintained in the culture conditions outlined FIG. 14 a . The expression of indicated gene was analyzed on day 7s, 13, 19 and day 25 of culture. Values are determined relative to TBP and presented as fold change relative to expression in fetal liver, which is set at one.
  • AL Adult liver
  • FL fetal liver.
  • Described herein is a robust and reliable platform for the efficient generation of hepatocytes and cholangiocytes from pluripotent stem cells (PSCs) through a series of steps described herein and for the generation of metabolicaly functional hepatocytes and/or cholangiocytes. It is demonstrated for example that one or more of extended nodal (e.g.
  • activin signaling treatment, inducing aggregation and activtating cAMP signaling for example in combination with FGF agonist induction and BMP4 agonist induction optionally in combination with one or more steps that increases expansion of a particular cell population and/or specific fate permits the reproducible generation of hepatocyte and cholangiocyte lineage cells including for example expanded hepatoblasts and/or with further manipulation, functional and mature hepatocytes and cholangiocytes from definitive endoderm induced in embryoid bodies or from monolayers.
  • An aspect of the present disclosure includes a method of producing hepatocyte or cholangiocyte lineaage cells such as hepatoblasts, hepatocytes and/or cholangiocytes from an extended nodal agonist treated induced endodermal cell population, the method comprising: (a) specifying the extended nodal agonist treated induced endodermal cell population to obtain a cell population comprising hepatocyte and/or cholangiocyte progenitors by contacting the extended nodal agonist treated induced endodermal cell population with specification media comprising a combination of a FGF agonist and a BMP4 agonist and/or active conjugates and/or fragments thereof to obtain a cell population comprising hepatocyte and/or cholangiocyte progenitor, and (b) inducing maturation, further lineage specification and/or expansion of the hepatocyte and/or cholangiocyte progenitors of the cell population to obtain an expanded population of hepatocytes and/or a population comprising he
  • the hepatocyte and/or cholangiocyte progenitors comprise hepatoblasts and/or immature hepatocytes and/or immature cholangiocytes.
  • contacting e.g. contacting an endodermal cell population with a component or components
  • incubating the component(s) and the cell together in vitro e.g., adding the compound to cells in culture
  • the step of contacting can be conducted in any suitable manner.
  • the cells may be treated in adherent culture, or in suspension culture, the components can be added temporally substantially simultaneously (e.g. together in a cocktail) or sequentially (e.g. within 1 hour, 1 day or more from an addition of a first component).
  • the cells can also be contacted with another agent such as a growth factor or other differentiation agent or environments to stabilize the cells, or to differentiate the cells further and include culturing the cells under conditions known in the art for example for culturing the pluripotent (and/or differentiated) population for example as further described in the Examples.
  • another agent such as a growth factor or other differentiation agent or environments to stabilize the cells, or to differentiate the cells further and include culturing the cells under conditions known in the art for example for culturing the pluripotent (and/or differentiated) population for example as further described in the Examples.
  • endoderm and “definitive endoderm” as used herein refer to one of the three primary germ cell layers in the very early embryo (the other two germ cell layers are the mesoderm and ectoderm). The endoderm is the innermost of the three layers.
  • An endoderm cell differentiates to give rise first to the embryonic gut and then to derivative tissues including esophagus, stomach, intestine, rectum, colon, pharyngeal pouch derivatives tonsils, thyroid, thymus, parathyroid glands, lung, liver, gall bladder and pancreas.
  • the “induced endodermal cell population” as used herein refers to a population of endoderm cells corresponding to “definitive endoderm induction” stage for example as shown in FIG. 1 a .
  • This population can be for example prepared from embyroid bodies (EB) that have been exposed to a nodal agonist, such as activin, or opitionally from EB that have been exposed to a nodal agonist and a wnt/beta-catenin agonist such as Wnt3a or a GSK-3 selective inhibitor such as CHIR-99021 (StemoleculeTM CHIR99021 Stemgent), 6-bromo-Indirubin-3′-Oxime (BIO) (Cayman Chemical (cat:13123)), or StemoleculeTM BIO from Stemgent (cat:04003).
  • a nodal agonist such as activin
  • a wnt/beta-catenin agonist such as Wnt3a
  • GSK-3 selective inhibitor such as CHIR-
  • the induced endodermal cell population can be prepared from cells grown in a monolayer.
  • the induced endodermal cell population can for example be identified by flow cytometric and molecular analysis for one or more markers such as surface markers CXCR4, CKIT and EPCAM and the transcription factors SOX17 and FOXA2.
  • the induced endodermal cell population can also for example be identified by at least or greater than 70, 80, 90 or 95% of the population co-expressing CXCR4 and CKIT or CXCR4 and EPCAM.
  • the induced endodermal cell population can also for example be identified by greater than 70, 80, 90 or 95% of the population of the population expressing SOX17 and/or FOXA2.
  • the induced endodermal cell population can for example be in a 2D (monolayer) or 3D (Embryoid Body or other form of aggregates) format.
  • the induced endodermal population can be derived for example from hESCs as well as an induced pluripotent cell (iPSC) as demonstrated in Example 1.
  • the induced endoderm cell population is for example treated with a nodal agonist extended period of time to provide an extended nodal agonist treated induced endoderm cell population.
  • culturing day 6 cells (day 5 when the method comprises monolayer induction) for two additional days in activin prior to specifying with FGF/BMP4 results in a higher proportion of SOX17+FOXA2+ cells as measured at day 12 compared to cells not cultured for two additional days in activin (e.g. an example of a nodal agonist).
  • This step is also referred to herein as an “extended activin” treatment and is an example of an “extended nodal agonist” treatment.
  • extended nodal agonist treated induced endoderm cell population refers to an induced endodermal cell population that has been treated with a nodal agonist such as activin for an extended period, for example from about 1 to about 4 or about 1, 2, 3 or 4 additional days (e.g. “the extended period” which is in addition to the endoderm induction phase which can comprise treatment with a nodal agonist).
  • the extended nodal agonist treatment as demonstrated herein resulted in higher levels of expression of genes indicative of hepatic progenitor (hepatoblast) development, including HEX, AFP, ALB and HNF4 ⁇ at day 26 of culture (as shown in FIG. 3 d ).
  • the extended nodal agonist treated induced endoderm population is obtained by inducing endoderm cells in embryoid bodies (EBs) or by inducing endoderm cells that are in a monolayer, and wherein the induced endodermal population is cultured in the presence of a nodal agonist, for example activin, for an extended period to produce an extended nodal agonist treated induced endodermal population.
  • EBs embryoid bodies
  • a nodal agonist for example activin
  • the extended nodal agonist treated induced endodermal cell population is, in an embodiment, obtained by inducing endoderm cells in embryoid bodies (EBs). In another embodiment, the extended nodal agonist treated induced endodermal population is obtaining by inducing endoderm cells that are in a monolayer. In each case, the induced endodermal population is cultured in the presence of a nodal agonist, for example activin, for an extended period.
  • EBs embryoid bodies
  • the induced endodermal population is subsequently dissociated, for example in embodiments where the induced endodermal cell population is derived from EBs.
  • dissociated cells or “dissociated cell populations” refers to cells that are not in 3D aggregates, for example, physically separated from one another. Dissociated cells are distinguished from “cell aggregates” which refers to clusters or clumps of cells.
  • the induced endodermal population comprises at least 80%, 85%, 90% CXCR4+ and cKIT+ positive cells and/or at least 70%, 75%, 80% SOX17+ cells.
  • the induced endodermal cell population (and/or the extended nodal agonist treated induced endodermal cell population) is produced from pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).
  • PSCs pluripotent stem cells
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • the pluripotent stem cells are optionally human ESCs (hESCs) or human iPSCs (hiPSCs).
  • Pluripotent refers to a cell with the capacity, under different conditions, to differentiate to more than one differentiated cell type, and for example the capacity to differentiate to cell types characteristic of the three germ cell layers. Pluripotent cells are characterized by their ability to differentiate to more than one cell type using, for example, a nude mouse teratoma formation assay. Pluripotency is also evidenced by the expression of embryonic stem (ES) cell marker.
  • ES embryonic stem
  • progenitor cell refers to cells that have a cellular phenotype that is at an earlier step along a developmental pathway or progression than a fully differentiated cell relative to a cell which it can give rise to by differentiation. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.
  • stem cell refers to an undifferentiated cell which is capable of proliferation, self-renewal and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiable, daughter cells.
  • the daughter cells can for example be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential.
  • embryonic stem cell is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see, for example, U.S. Pat. Nos. 5,843,780, 6,200,806). Such cells can also be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, U.S. Pat. Nos. 5,945,577, 5,994,619, 6,235,970).
  • the distinguishing characteristics of an embryonic stem cell define an embryonic stem cell phenotype. Accordingly, a cell has the phenotype of an embryonic stem cell if it possesses one or more of the unique characteristics of an embryonic stem cell such that that cell can be distinguished from other cells. Exemplary distinguishing embryonic stem cell characteristics include, without limitation, gene expression profile, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular culture conditions, and the like.
  • the method of producing hepatocytes and/or cholangiocytes from an extended nodal agonist treated induced endodermal cell population comprises: (a) specifying the extended nodal agonist treated endodermal cell population to a cell population comprising hepatocyte and/or cholangiocyte progenitors by contacting the induced endodermal cell population with specification media comprising a FGF agonist and a BMP4 agonist and/or active conjugates and/or fragments thereof.
  • the specifying step comprises contacting an extended nodal agonist treated induced endodermal population with specification media comprising a FGF agonist and BMP4.
  • the FGF agonist can for example be bFGF, FGF10, FGF2 or FGF4, active fragments and/or combinations thereof. The combinations can be added to the cells for example sequentially.
  • the specifying step comprises first contacting an extended nodal agonist treated induced endodermal population with specification media comprising FGF10 and BMP4 for about 40 to about 60 hours for example about 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or about 60 hours and then contacting the extended nodal agonist treated induced endodermal population with specification media comprising bFGF and BMP4 for about 4 to about 7 days, for example about 4, 5, 6 or about 7 days.
  • the specification media comprises Iscove's Modified Dulbecco's Medium (IMDM) supplement with 1% vol/vol B27 supplement (Invitrogen: A11576SA), ascorbic acid, MTG, FGF10 (50 ng/ml) (for example from day 8 to day 10, bFGF (20 ng/ml) (for example from day 10 to day 14), and BMP4 (50 ng/ml).
  • IMDM Iscove's Modified Dulbecco's Medium
  • B27 supplement Invitrogen: A11576SA
  • ascorbic acid MTG
  • FGF10 50 ng/ml
  • bFGF (20 ng/ml) for example from day 10 to day 14
  • BMP4 50 ng/ml
  • the endodermal cell population 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.
  • the endodermal cell population is incubated in cell culture medium comprising BMP4 and FGF10 or bFGF.
  • the endodermal cell population is incubated in cell culture medium comprising Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 1% vol/vol B27, ascorbic acid, monothioglycerol, BMP4 and FGF10 or bFGF.
  • IMDM Iscove's Modified Dulbecco's Medium
  • the endodermal cell population is dissociated and the monolayer cells are then contacted with FGF and a BMP4 agonist.
  • the monolayer cells are contacted with activin for 1 to 4 days, optionally 1, 2, 3 or 4 days prior to being contacted with FGF and a BMP4 agonist such as BMP4.
  • the monolayer cells are incubated in cell culture medium comprising activin A, optionally medium comprising StemPRO-34 supplemented with bFGF, activin A and BMP4.
  • the specifying step comprises contacting cells with a specification media that comprises one or more factors that promote maturation, further lineage specification and/or expansion.
  • hepatic specification media refers to culture medium that is used to promote or facilitate specification of a cell or a cell population.
  • a hepatic specification media includes Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 1% vol/vol B27 (Invitrogen: A11576SA), and ascorbic acid, MTG, a BMP4 agonist and at least one FGF agonist selected from FGF10, bFGF, FGF4 and FGF2.
  • the specification media comprises one or more factors that promote specification of hepatocyte and/or cholangiocyte development, for example a notch antagonist or a notch agonist.
  • a notch antagonist or a notch agonist for example a notch antagonist or a notch agonist.
  • the term “specifying” as used herein means a process of committing a cell toward a specific cell fate, prior to which the cell type is not yet determined and any bias the cell has toward a certain fate can be reversed or transformed to another fate. Specification induces a state where the cell's fate cannot be changed under typical conditions.
  • Specification of the induced endoderm along a hepatic fate can for example be confirmed by measuring hepatic and/or cholangiocyte expressed genes, including for example Tbx3 ALB, AFP, CK19, Sox9, NHF6beta and Notch2 as demonstrated for example in Example 9.
  • hepatic and/or cholangiocyte expressed genes including for example Tbx3 ALB, AFP, CK19, Sox9, NHF6beta and Notch2 as demonstrated for example in Example 9.
  • Notch 2 expression is upregulated in HGF/DEX/OSM treated hepatoblasts.
  • Detection of Notch2 protein and/or expression can be used to confirm that the cell population can be specified to cholangiocytes, for example Notch2 can be detected as described in Example 9.
  • differentiated is a relative term and a “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell it is being compared with.
  • stem cells can differentiate to lineage-restricted precursor cells (such as an induced endodermal progenitor cell), which in turn can differentiate into other types of precursor cells further down the pathway and then mature to an end-stage functional cell, which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
  • lineage-restricted precursor cells such as an induced endodermal progenitor cell
  • end-stage functional cell which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
  • differentiation includes steps for producing an induced endodermal population and specified cell populations, for example a hepatocyte or cholangiocyte specified cell population.
  • the aggregates are generated from a cell population comprising at least 70%, 80%, 85%, or at least 90% albumin positive cells
  • the aggregates are generated after 24, 25, 26, 27, or 28 days in culture (for example where the day PSCs are obtained is considered day 0).
  • aggregates are generated by enzymatic treatment and/or manual dissociation.
  • aggregates are generated by dissociating cells with collagenase and/or TrypleLE.
  • the cells are subsequently cultured in ultra-low cluster dishes.
  • monolayer cultures can be broken apart mechanically by pipetting, or can be dissociated enzymatically and aggregated with an incubation in low attachment plates or by shaking the population of cells. Aggrewells can optionally be used.
  • the cells are gown on matrix coated plates, optionally on Matrigel coated plates.
  • matrix coated plates that support the attachment of hepatoblasts, hepatocytes and/or cholangiocytes can also be used, for example laminin, fibronectin and collagen coated plates.
  • FIG. 9 f for example demonstrates ALB expression at day 26 of induction using several different matrix coating substrates.
  • the cell population comprising hepatocyte and/or cholangiocyte progenitors and/or the aggregates are cultured in the presence of hepatocyte growth factor (HGF), dexamethasone (DEX) and/or Oncostatin M (OSM) and/or active conjugates and/or fragments thereof.
  • HGF hepatocyte growth factor
  • DEX dexamethasone
  • OSM Oncostatin M
  • the cell population comprising hepatocyte and/or cholangiocyte progenitors can be cultured in a maturation media comprising HGF, DEX and/or OSM for about 10, 11, 12, 13 or 14 days prior to aggregation and/or subsequent to aggregation the aggregates can be cultured in a maturation media comprising HGF, DEX and/or OSM for about 6, 7, 8, 9, or 10 days.
  • addition of about 10 ng/mL HGF promotes survival of aggregates.
  • inducing maturation, and optionally inducing further lineage specification and/or expansion further comprises activating the cAMP pathway within the cells of the aggregates to induce the differentiation and/or maturation of the hepatocyte and cholangiocyte progenitors into hepatocytes and/or cholangiocytes.
  • the extended nodal agonist treatment and the aggregation of cells for example at day 25 for monolayer induced cells and day 26 for EB induced cells, produce a population which is for example capable of responding to cAMP signaling.
  • activation of cAMP increases CYP expression and hepatocyte maturation.
  • activating the cAMP pathway comprises contacting the aggregates with a cAMP agonist analog such as 8-bromoadensoine-3′5′′-cyclic monophosphate (8-Br-cAMP), dibutyryl-cAMP, Adenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS) and/or 8-Bromoadenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer (Sp-8-Br-cAMPS)) and/or any other cAMP agonist, such as cholera toxin, forskolin, caffeine, theophylline and pertussis toxin.
  • a cAMP agonist analog such as 8-bromoadensoine-3′5′′-cyclic monophosphate (8-Br-cAMP), dibutyryl-cAMP, Adenosine-3′,5′-cyclic monophosphorothioate, Sp-i
  • cAMP agonists include, cAMP, cAMP analogs that activate cAMP as well as molecules such as cholera toxin, forskolin, caffeine, theophylline and pertussis toxin which activate cAMP.
  • IBMX which is a phosphodiesterase inhibitor, (phosphodiesterases are cAMP inhibitors) can also be used in some embodiments, for example in combination with forskolin.
  • aggregates are cultured with cAMP analogs and/or agonists in the absence of OSM.
  • maturation means a process that is required for a cell (e.g. hepatoblast) to become more specialized and/or attain a fully functional state, for example its functional state in vivo.
  • a cell e.g. hepatoblast
  • maturation the process by which immature hepatocytes or hepatic progenitors become mature, functional hepatocytes is referred to as maturation.
  • FIG. 1 a refers to “hepatic maturation A” and FIG. 14 a refers to hepatoblast differentiation.
  • the cell population referred to in both cases is a hepatoblast cell population, that can produce hepatocytes if continued to be cultured for example in the presence of DEX optionally in combination with HGF and OSM and/or cAMP or produce cholangiocytes if cultured in combination with a Notch agonist (e.g. a notch signal donor), such as OP9, OP9 delta and/or OP9 Jagged1 cells in the presence of EGF, TGB1 HGF and EGF.
  • a Notch agonist e.g. a notch signal donor
  • maturation media refers to culture medium that is used to promote or facilitate maturation of a cell or a cell population and which can comprise maturation factors, as well as cell expansion inducers and lineage inducers.
  • a maturation media for inducing hepatocyte maturation includes Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 1% vol/vol B27 (Invitrogen: A11576SA) as well as ascorbic acid, Glutamine, MTG and optionally Hepatocyte growth factor (HGF), Dexamethasone (Dex) and/or Oncostatin M.
  • IMDM Iscove's Modified Dulbecco's Medium
  • HGF Hepatocyte growth factor
  • Dex Dexamethasone
  • Oncostatin M Oncostatin
  • a maturation media for inducing hepatocyte includes Hepatocyte culture medium (HCM) (Lonza: CC-4182) without EGF.
  • HCM Hepatocyte culture medium
  • the maturation media optionally also comprises a cAMP analog and/or cAMP agonist which for example induces expansion of hepatocyte lineage cells and their maturation.
  • Maturation media can comprise factors that promote hepatocyte and/or cholangiocyte development, further lineage specification and/or expansion and/or further lineage selection.
  • Wnt antagonist alone or in combination with TGFbeta antagonists and/or MEK/Erk antagonists promote hepatocyte maturation.
  • Notch agonists for example, are demonstrated herein to induce cholangiocyte lineage development and are added when cholangiocytes are desired.
  • Notch antagonists promote hepatocyte lineages and can be added when hepatocytes are desired, for example to inhibit cholangiocyte development.
  • Different maturation medias can be used sequentially (e.g. a monolayer maturation media (used for example pre-aggregation), an aggregates maturation media (used for example post aggregation); a hepatocyte maturation media that for example comprises factors that promote hepatocyte development and a cholangiocyte maturation media that for example promotes cholangiocyte development.
  • a monolayer maturation media used for example pre-aggregation
  • an aggregates maturation media used for example post aggregation
  • a hepatocyte maturation media that for example comprises factors that promote hepatocyte development
  • a cholangiocyte maturation media that for example promotes cholangiocyte development.
  • a maturation media comprising a cAMP analog and/or cAMP agonist and DEX and optionally HGF is added to the aggregates subsequent to culturing the pre-aggregate population in the maturation media comprising HGF, DEX and OSM, for example for about 10, 11, 12, 13 or 14 days.
  • hepatocyte refers to a parenchymal liver cell. Hepatocytes make up the majority of the liver's cytoplasmic mass and are involved in protein synthesis and storage, carbohydrate metabolism, cholesterol, bile salt and phospholipid synthesis and the detoxification, modification and excretion of exogenous and endogenous substances.
  • primary hepatocyte is a hepatocyte that has taken directly from living tissue (e.g. biopsy material) and established for growth in vitro.
  • hepatoblast refers to a progenitor cell which has the capacity to differentiate into cells of the hepatic and cholangiocyte lineages e.g. a hepatocyte or a cholangiocyte.
  • Hepatoblasts are for example a subset of hepatocyte and cholangiocyte progenitors which can comprise immature hepatocytes and immature cholangiocytes (e.g. cells which have a specified cell fate and can mature to a hepatocyte only or a cholangiocyte only).
  • hepatoblast cells are defined by expression of markers such as Hex, HNF4, alpha-fetoprotein (AFP) and albumin (ALB).
  • hepatoblasts can give rise to cholangiocyte cells (e.g. CK19+ cells) when notch signaling is activated in for example day 28 hepatoblast containing cultures.
  • hepatocyte progenitor as used herein means cells that have the capacity to differentiate into functional hepatocytes which are for example albumin positive and/or expresses CYP enzymes.
  • cholangiocyte progenitor means cells that have the capacity to differentiate into functional cholangiocytes which are for example CK19 positive and/or express CFTR.
  • immature hepatocyte refers to a hepatocyte lineage cell that expresses albumin but that does not express appreciable levels of functional CYP3A4 and/or CYP1A2 enzyme.
  • immature hepatocytes must undergo maturation to acquire the functionality of mature hepatocytes.
  • immature hepatocyte cells are defined by expression of markers such as Hex, alpha-fetoprotein and albumin.
  • a “mature hepatocyte” as used herein means a hepatocyte lineage cell that express CYP enzymes for example CYP3A4 and CYP1A2 and albumin.
  • mature hepatocytes include functional, or measurable, levels of metabolic enzymes such as Phase I and Phase II drug-metabolizing enzymes for example comparable to adult cells.
  • Phase I drug-metabolizing enzymes include but are not limited to cytochromes P450 CYP1A2, CYP3A4 and CYP286.
  • Phase II drug-metabolizing enzymes include but are not limited to arylamine N-acetyltransferases NAT1 and NAT2 and UDP-glucuronosyltransferase UGT1A1.
  • the mature hepatocyte can be a metabolically active hepatocyte.
  • Cellular uptake of Indocyanine green (ICG) is considered to be a characteristic of adult hepatocytes 29 and is used clinically as a test substrate to evaluate hepatic function 30 .
  • a mature hepatocyte is for example an ICG positive staining hepatocyte.
  • the population of hepatocytes can be considered a mature 50%, 60%, 70%, 80%, 90% or more of the hepatocytes are ICG.
  • a mature hepatocyte expresses increased albumin compared to an “immature hepatocyte” for example at least 5%, 10%, 25%, 50%, 75%, 100% or 200% more albumin, than an immature hepatocyte.
  • the hepatocyte is a functional hepatocyte.
  • the term “functional hepatocyte” as used herein refers to a hepatocyte cell that displays one or more of characteristics of an adult hepatocyte (e.g. a mature hepatocyte) and/or an immature hepatocyte that is committed to a hepatic fate and is more differentiationed than a starting cell (e.g. compared to an endodermal population cell, a hepatocyte precursor or an immature hepatocyte), which for example expresses albumin and/or increased albumin compared to a starting cell.
  • functional hepatocytes are mature hepatocytes and include functional, or measurable, levels of metabolic enzymes such as Phase I and Phase II drug-metabolizing enzymes for example comparable to adult cells.
  • the functional hepatocyte can be a metabolically active hepatocyte.
  • Cellular uptake of Indocyanine green (ICG) is considered to be a characteristic of adult hepatocytes 29 and is used clinically as a test substrate to evaluate hepatic function 30 .
  • a functional hepatocyte is for example an ICG positive staining hepatocyte.
  • the population of hepatocytes can be considered a functional 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.
  • a functional hepatocyte is an albumin secreting hepatocyte and a 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.
  • 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 protein 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 hepatocyte and/or cholangiocyte progenitors, and/or hepatocytes produced from a non-extended nodal agonist treated induced endodermal cell population, produced without aggregation and/or cAMP signaling induction.
  • at least 40, 50, 60, 70, 80 or 90% of the hepatocytes, optionally functional hepatocytes are ASGPR-1+ cells.
  • functional hepatocytes display nucleic acid or protein levels of CYP1A2, CYP2B6, CYP3A4, CYP2C9 and/or CYP2D6 that are comparable or higher than those found in primary mature hepatocytes, optionally levels that are increased at least 1.1, 2, 3, 4, or 5 fold or any 0.1 increment between 1.1 fold and 5 fold, optionally increased at least 50% to 100%, 75% to 125%, 85% to 115%, 90% to 110% or 95% to 105%. For example, increased expression of 1.15 fold to 6.1 fold (e.g.
  • CYP1A2 6.1 folds (610%), CYP3A4 13.2 folds (1320%), CYP 2B6 2 folds (200%), CYP2C9 1.52 folds 152%, UGT1A1 2 folds (200%), CYP2D6 1.15 folds (115%)) of those found in primary hepatocytes.
  • hepatocytes display nucleic acid or protein levels of ALB, HNF4, AFP, CPS1, G6P, TDO1, NAT1, NAT2 and/or UGT1A1 that are comparable or higher to those found in primary hepatocytes of similar stage, optionally 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.
  • functional hepatocytes display nucleic acid or protein levels of CYP1A2, CYP2B6, CYP3A4, CYP2C9 and/or CYP2D6 that are higher than those in hepatoblasts and/or immature hepatocytes, optionally levels that are at least 110%, 125%, 150%, 175%, 200%, 300%, 400% or 500% of those found in primary hepatocytes.
  • functional hepatocytes display nucleic acid or protein levels of ALB, CPS1, G6P, TDO1, NAT1, NAT2 and/or UGT1A1 that are higher than those in hepatoblasts and/or immature hepatocytes, optionally levels that are at least 110%, 125%, 150%, 175%, 200%, 300%, 400% or 500% of those found in primary hepatocytes.
  • functional hepatocytes express the receptor asialo-glycoprotein receptor 1 (ASGPR1).
  • ASGPR1 receptor asialo-glycoprotein receptor 1
  • at least 40, 50, 60, 70, 80 or 90% of the hepatocytes, optionally functional hepatocytes, are ASGPR-1+ cells.
  • functional hepatocytes display CYP1A2 activity in vitro.
  • functional hepatocytes display CYP1A2 activity is comparable or higher than those found in primary hepatocytes, optionally 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.
  • CYP1A2 activity is measured by incubating cells with phenacetin and monitoring the generation of O-deethylated metabolite accumulation in the cells.
  • functional hepatocytes display CYP2B6 activity in vitro.
  • functional hepatocytes display CYP2B6 activity that is comparable or higher than those found in primary hepatocytes, optionally 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.
  • CYP2B6 activity is measured by incubating cells with bupropin and monitoring the formation of the metabolite O-hydroxy-bupropion in the cells.
  • hepatocytes display NAT1 and/or NAT2 activity in vitro.
  • hepatocytes display NAT1 and/or NAT2 activity that is comparable or higher than those found in primary hepatocytes, optionally levels that are 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 those found in primary hepatocytes.
  • NAT1 and/or NAT2 activity is indicated by the metabolism of sulfamethazine (SMZ) to N-acetylated SMZ.
  • SMZ sulfamethazine
  • hepatocytes display UGT activity in vitro.
  • hepatocytes display UGT activity that is comparable or higher than those found in primary hepatocytes, optionally 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.
  • UGT activity is indicated by the generation of 4-MU glucuronide (4-MUG) from 4-methylumbelliferone (4-MU) in the cells.
  • 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 protein 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 hepatocyte and/or cholangiocyte progenitors, and/or hepatocytes produced from a non-extended nodal agonist treated induced endodermal cell population, produced without aggregation and/or cAMP signaling induction.
  • at least 40, 50, 60, 70, 80 or 90% of the hepatocytes, optionally functional hepatocytes are ASGPR-1+ cells.
  • functional hepatocytes display a global gene expression profile that is indicative of hepatocyte maturation.
  • functional hepatocytes display a global gene expression profile that is more similar to a primary hepatocyte than a global gene expression profile of a hepatoblast and/or a immature hepatocyte.
  • Global gene expression profiles are obtained by any method known in the art, for example microarray analysis.
  • cholangiocyte fate is specified by treating aggregates of the cell population with a notch agonist.
  • cholangiocyte refers to the cells that make bile ducts.
  • cholangiocyte precursor refers to cells which have the capacity to differentiate into a cholangiocyte cell (e.g. hepatoblasts), as well as immature cholangiocytes that can mature to functional cholangiocytes.
  • cells of the cholangiocyte lineages are defined by expression of markers such as CK19, secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl( ⁇ )/HCO(3)( ⁇ ) AEs).
  • immature cholangiocyte refers to a cholangiocyte lineage cell which must undergo maturation to acquire the functionality of mature cholangiocytes.
  • immature cholangiocyte cells express CK19 and/or Sox9, optionally including early Notch agonist treated cells, optionally treated for at least 1 day, at least 2 days, at least 3 days, or at least 4 days.
  • the cholangiocyte is a functional cholangiocyte.
  • cholangiocyte refers to cholangiocyte cells that display one or more of the characteristics of adult cholangiocytes (e.g. mature cholangiocyte) and/or are CK-19, MDR1 and/or CFTR expressing cholangiocyte lineage cells.
  • functional cholangiocytes express the MDR1 transporter and can when in cystic structures, transport a tracer dye such as rhodamine123, into the structure luminal space.
  • CFTR functional activity can be assessed for example using a forskolin induced swelling assay on cystic structures, as shown for example in FIG. 18 .
  • the population can be considered a functional population if for example at least 25%, 30%, 35%, 40%, 45% 50%, 60%, 70%, 80%, 90% or more of the cells express secretin reseptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), CK-19 and/or chloride bicarbonate anion exchanger 2 (Cl( ⁇ )/HCO(3)( ⁇ ) AEs).
  • SR secretin reseptor
  • CFTR cystic fibrosis transmembrane conductance regulator
  • CK-19 chloride bicarbonate anion exchanger 2
  • mature cholangiocytes are cholangiocytes that express specified transporter or cell membrane receptor activity, such as secretin reseptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and optionally chloride bicarbonate anion exchanger 2 (Cl( ⁇ )/HCO(3)( ⁇ ) AEs).
  • SR secretin reseptor
  • CFTR cystic fibrosis transmembrane conductance regulator
  • chloride bicarbonate anion exchanger 2 Cl( ⁇ )/HCO(3)( ⁇ ) AEs
  • the population of cholangiocytes produced is a population of functional cholangiocytes.
  • the functional cholangiocyte comprises for example increased expression of at least 1, at least 2 or 3 genes or proteins selected from Sox9, CK19 and CFTR (Cystic fibrosis transmembrane conductance regulator) compared to the cells of the cell population comprising hepatocyte and cholangiocyte progenitors and/or compared to a population cells produced from aggregates not treated with a notch agonist.
  • at least 40, 50, 60, 70, 80 or 90% of the population of cholangiocytes are CK19+ cholangiocytes.
  • at least 40, 50, 60, 70, 80 or 90% of the functional cholangiocytes are CFTR+ cholangiocytes.
  • RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene.
  • cell culture medium (also referred to herein as a “culture medium” or “medium”) as referred to herein is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation and optionally differentiation.
  • the cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, vitamins etc.
  • Cell culture media ordinarily used for particular cell types are known to those skilled in the art.
  • the suitable culture medium can include a suitable base culture medium including for example DMEM (Life Technologies), IMDM, RPMI, CMRL and/or any other media that supports the growth of endodermal cells to provide for example a base culture medium composition to which components and optionally other agents can be added.
  • DMEM Life Technologies
  • IMDM IMDM
  • RPMI RPMI
  • CMRL any other media that supports the growth of endodermal cells to provide for example a base culture medium composition to which components and optionally other agents can be added.
  • day 5 refers generally to induced endoderm cell populations derived from for example PSC monolayers. Induced endoderm cell populations derived from EBs are cultured for about 6 days to arrive at a similar culture point as they require treatment for about 24 hours to induce EB formation. Hence, day 7 monolayer induction cultures are equivalent to day 8 embryoid body induction cultures etc. At this stage the induced endodermal population can comprise of cells that express for example Foxa2 and Sox17. Similarly, “day 7” generally refers to induced endodermal populations that have been extended nodal agonist treated for two days (e.g. which would be day 8 for EB methods).
  • Day 25 generally refers to the stage at which cells are aggregated (if derived from monolayers or Day 26 if derived from EBs). Where monolayer cells are used, equivalent methods can be used with EBs with the culture periods typically delayed 1 day.
  • FIG. 11 provides an example of a schedule using monolayer cells ( FIG. 11A ) and an example using EBs ( FIG. 11B ). An example schedule for the generation of cholangiocytes is provided in FIGS. 11C and 14A .
  • FGF agonist means a molecule such as a cytokine, including for example FGF, or a small molecule, that activates a FGF signalling pathway, e.g binds and activates a FGF receptor.
  • FGF receptor activation can be assessed by measuring MEK/ERK, AKT and/or PI3K activity by immuno detection.
  • FGF refers to any fibroblast growth factor, for example human FGF1 (Gene ID: 2246), FGF2 (also known as bFGF; Gene ID: 2247), FGF3 (Gene ID: 2248), FGF4 (Gene ID: 2249), FGF5 (Gene ID: 2250), FGF6 (Gene ID: 2251), FGF7 (Gene ID: 2252), FGF8 (Gene ID: 2253), FGF9 (Gene ID: 2254) and FGF10 (Gene ID: 2255) optionally including active conjugates and fragments thereof, including naturally occurring active conjugates and fragments.
  • FGF is FGF10, FGF4 and/or FGF2.
  • active conjugates and fragments of FGF include conjugates and fragments of a fibroblast growth factor that bind and activate a FGF receptor and optionally activate FGF signalling.
  • the concentration of FGF can for example range 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.
  • the FGF concentration 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 for example range 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.
  • the FGF10 concentration 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 for example range 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.
  • the bFGF concentration 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 BMP4 agonist is selected from the group BMP4, BMP2, and BMP7.
  • BMP4, BMP7 and BMP2 for example share the same receptors in embryo development.
  • BMP4 (for example Gene ID: 652) as used herein refers to Bone Morphogenetic Protein 4, for example human BMP4, as well as active conjugates and fragments thereof, optionally including naturally occurring active conjugates and fragments, that can for example activate BMP4 receptor signaling.
  • concentration of BMP for example, BMP4 can for example range 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.
  • the BMP concentration for example the BMP4 concentration 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 method can be applied to an endodermal cell population grown in a monolayer.
  • a further aspect includes a method of producing hepatocytes and/or cholangiocytes from a pluripotent stem cell population, the method comprising:
  • the endodermal population can also be comprised in embryoid bodies. Accordingly a further aspect comprises a method of producing hepatocytes and/or cholangiocytes from a pluripotent stem cell population, the method comprising:
  • Another aspect includes a method of producing 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:
  • PSCs pluripotent stem cells
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • embryoid bodies (EBs) of the pluripotent stem cells are formed by culturing the pluirpotent 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.
  • BMP4 optionally 1-5 ng/ml BMP4 or about 5 ng/ml BMP4
  • the EBs may be recultured in induction medium supplemented with a nodal agonist for 3 to 10 days, optionally 4 to 8 days or about 5 or 6 days to induce an endodermal cell population.
  • the nodal agonist is optionally Activin A.
  • the EBs of are also optionally contacted with a wnt/beta-catenin agonist such as Wnt3a or a GSK-3 selective inhibitor such as CHIR-99021 to induce an endodermal cell population.
  • the EBs are cultured in the presence of a nodal agonist such as Activin A for an additional 1 to 4 days (e.g. the extended nodal agonist treatment) (i.e., prior to contacting the endodermal cell population with a combination of at least one FGF agonist and one BMP4 agonist).
  • a nodal agonist such as Activin A for an additional 1 to 4 days (e.g. the extended nodal agonist treatment) (i.e., prior to contacting the endodermal cell population with a combination of at least one FGF agonist and one BMP4 agonist).
  • cells are then treated to induce maturation, further lineage specification and/or expansion including for example aggregation and treatment with various maturation factors. These steps for example can generate a population of cells that is responsive to cAMP activation. Extended nodal agonist treatment and aggregation are steps that generate cells responsive to cAMP activation.
  • Cells responsive to cAMP activation are for example after 26 days of culture for monolayer based methods.
  • the aggregated cell maturation/specification media can comprise factors which promote hepatocyte maturation or factors which promote cholangiocyte development or both and/or which increase expansion of a precursor population.
  • the aggregates comprise hepatoblasts which as demonstrated can be specified to hepatocytes or cholangiocytes.
  • inducing maturation, further lineage specification and/or expansion further comprises contacting the cell aggregates with i) a cAMP signaling activator (e.g. cAMP analog and/or agonist) and/or ii) an antagonist of Wnt/beta-catenin signaling (for example, Wnt inhibitor XAV 939) and/or an inhibitor of MEK/Erk signaling (for example, MEK/Erk inhibitor PD0325901).
  • Wnt/beta-catenin signaling for example, Wnt inhibitor XAV 939
  • MEK/Erk signaling for example, MEK/Erk inhibitor PD0325901
  • Addition of a Wnt antagonist and/or a MEK/Erk antagonist during activation of cAMP signaling enhances expression of CYP enzymes, for example up to levels or greater than levels seen in adult liver cells.
  • wnt antagonists include for example XAV939, IWP2, DKK1, XXX (IWP2 (STEMGENT 04-0034), Dkk-1 (R&D, 5439-DK-010)), IWR-1 endo (Calbiochem 681699-10).
  • Wnt signaling include Dickkopf (Dkk) proteins, Wnt Inhibitory Factor-1 (WIF-1), and secreted Frizzled-Related Proteins (sFRPs) and can be used in an embodiment.
  • Dkk Dickkopf
  • WIF-1 Wnt Inhibitory Factor-1
  • sFRPs secreted Frizzled-Related Proteins
  • the MEK/Erk antagonist is selected from PD0325901, U0126 (Promega V1121), PD 098059 (Sigma-Aldrich P215-1MG).
  • the cell aggregates are contacted with 0.1 to 10 ⁇ M, optionally 0.5 to 2 ⁇ M or about 1 ⁇ M XAV 939 and/or PD0325901.
  • Concentrations of other inhibitors/activators are for example concentrations that give similar activation/inhibition to inhibitors activators described herein.
  • inducing maturation, further lineage specification and/or expansion further comprises contacting the cell aggregates with both a cAMP analog and/or cAMP agonist and wnt agonist such as a GSK3 selective inhibitor (for example, CHIR99021) or a TGF- ⁇ antagonist (for example, inhibitor SB431542).
  • a GSK3 selective inhibitor for example, CHIR99021
  • a TGF- ⁇ antagonist for example, inhibitor SB431542
  • the cell aggregates are contacted with 0.1 to 10 ⁇ M, optionally 0.2 to 4 ⁇ M CHIR99021 and/or about 2 to 10 ⁇ M or about 6 ⁇ M SB431542.
  • TGF- ⁇ inhibitors include SB431542 (Sigma-Aldrich S4317-5MG), SB 525334 (Sigma-Aldrich S8822-5MG), and A83-01 (Tocris, 2929).
  • the aggregated cells are treated with a wnt agonist and optionally a TGF ⁇ antagonist (such as SB431542) for about 6 to about 12 days, preferably about 8 to about 10 days, optionally 9 days.
  • a TGF ⁇ antagonist such as SB431542
  • Such treatment results in expansion of the hepatoblasts.
  • the method comprises producing an expanded population of hepatoblasts. These cells can be used to produce more differentiated population of cells including mature hepatocytes and/or cholangiocytes.
  • the TGF- ⁇ antagonist is selected from SB431542 (Sigma-Aldrich S4317-5MG), SB 525334 (Sigma-Aldrich S8822-5MG), A83-01 (Tocris, 2929).
  • the aggregated cell maturation media comprises one or more factors which promote maturation, further lineage specification and/or expansion, optionally:
  • the aggregated cell maturation media comprises one or more factors which promote maturation, further lineage specification and/or expansion, optionally:
  • cell aggregates are generated from a monolayer of the cell population comprising hepatocyte and cholangiocyte progenitors by enzymatic treatment and/or manual dissociation.
  • the cell population comprising hepatocyte and/or cholangiocyte progenitors which have been cultured in maturation/specification media comprising HGF, OSM and DEX, optionally prior to cell aggregation are co-cultured with endothelial cells, optionally CD34+ positive endothelial cells.
  • endothelial cells are derived from embryonic ESC, preferably human.
  • the endothelial cells are mature endothelial cells optionally human, and/or derived from mature endothelial cells.
  • CD34+ endothelial cells can for example be generated as described in Example 8 from hESCs. 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 time the CD34 + cells (also CD31 + and KDR + ) can be isolated by FAGS. The sorted CD34 + cells can be further cultured for example for 6 days in endothelial cell growth media, optionally EMG2 media, and then used for the generation of chimeric aggregates.
  • the cell population comprising hepatocyte and/or cholangiocyte progenitors which have been cultured in maturation/specification media comprising HGF, OSM and DEX are co-cultured with CD34+ positive endothelial cells to form chimeric aggregates, optionally using an aggregation vessel (e.g. a vessel that promotes aggregation of a single cell type or mixed cell types) such as AggrewellsTM until chimeric aggregation is achieved, for example for about 1 day, about 2 days or about 3 days when using Aggrewells.
  • Aggregation can also be performed using a method described herein or known in the art.
  • the endothelial cells can be added to the vessels prior to the hepatic cell population to coat the bottom of the well.
  • the hepatic cell population can be added as a single cell suspension, for example day 25/26 hepatoblasts can be added on top of the endothelial cells and the mixture cultured in the Aggrewells.
  • the chimeric hepatic/endothelial aggregates can be subsequently removed from the Aggrewells and cultured. As shown in FIG. 12 b , the aggregates cultured together with the endothelial cells contained endothelial cells and were larger than those cultured alone.
  • the chimeric hepatic/endothelial aggregates cultured for an additional 12 days expressed substantially higher levels of CYP3A4 message than the hepatic aggregates generated without the endothelial cells ( FIG. 12 d ).
  • endothelial cells may promote maturation of the hPSC-derived hepatic cells.
  • the hepatic/endothelial chimeric aggregates are cultured for at least or about 6 days, at least or about 8 days, at least or about 10 days, at least or about 12 days, or until a desired or preselected level of CYP3A4 message is attained.
  • the hepatic endothelial chimeric aggregates are cultured in a gelatinous matrix, optionally a collagen comprising matrix, optionally a gel.
  • the collagen is collagen I or IV.
  • the gelatinous matrix comprises Matrigel, laminin, fibronectin, extracted ECM (e.g. extra cellular matrix from liver tissue) and/or combinations thereof.
  • the aggregates cultured in a collagen comprising matrix are cultured in the presence of cAMP, PD0325901 and XAV939.
  • the cells are treated with a notch agonist.
  • Addition of a notch agonist at such stages promotes cholangiocyte maturation.
  • inducing cAMP signaling is omitted.
  • the disclosure also provides a method of inducing maturation of cholangiocyte progenitors into cholangiocytes, the inducing maturation, further lineage specification and/or expansion comprising:
  • the method of producing functional cholangiocytes from pluripotent stem cells such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs):
  • pluripotent stem cells such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs):
  • the aggregates are cultured (or co-cultured when comprising Notch signaling donor cells) in a gelatinous matrix, optionally a collagen comprising matrix, optionally a gel.
  • a gelatinous matrix optionally a collagen comprising matrix, optionally a gel.
  • the collagen is collagen I or IV.
  • the gelatinous matrix comprises Matrigel, laminin, fibronectin, extracted ECM (e.g. extra cellular matrix from liver tissue) and/or combinations thereof.
  • Notch antagonist such as gamma-secretase inhibitor (GSI) L695,458 (Tocris #2627) DAPT (Sigma_Aldrich D5942) LY 411575 (Stemgent 04-0054) and L-685458) is demonstrated herein to inhibit cholangiocyte development and cells produced retain the characteristics of hepatocytes.
  • GSI gamma-secretase inhibitor
  • DAPT Sigma_Aldrich D5942
  • LY 411575 Stegent 04-0054
  • L-685458 is demonstrated herein to inhibit cholangiocyte development and cells produced retain the characteristics of hepatocytes.
  • the method of producing 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)
  • PSCs pluripotent stem cells
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • the Notch agonist can for example be any notch ligand bound to a surface such as a cell, plastic, ECM or bead.
  • the notch ligand is notch ligand delta Jagged-1 (EUROGENTEC 188-204), Jagged/peptide (abcam, ab94375).
  • Recombinant Human Pref-1/DLK-1/FA1 R&D 1144-PR.
  • inducing maturation, and further lineage specification and/or expansion comprises contacting a cell population comprising cholangiocyte progenitors with a notch signaling donor such as OP9, OP9delta, and/or OP9 Jagged1 cells and optionally in the presence of EGF, TGFbeta1, HGF and EGF, and/or HGF, TGFbeta1 and EGF, for at least or about 5 to about 10, about 14 or more days, for example 90 days, optionally for at least or about 5 to at least or about 60 days, at least or about 30 days, at least or about 25 days, 2 at least or about 1 days and/or at least or about 14 days, to induce the maturation of cholangiocyte progenitors into functional cholangiocytes.
  • a notch signaling donor such as OP9, OP9delta, and/or OP9 Jagged1 cells
  • the structures produced can be maintained in culture for over 60 days.
  • the cell population comprising cholangiocyte progenitors is contacted with a notch signaling donor (notch agonist) such as OP9, OP9delta, and/or OP9 Jagged1 cells and optionally in the presence of EGF, TGFbeta1, HGF and EGF, and/or HGF, TGFbeta1 and EGF, for at least 5 days and optionally up to any day between 5 and 90, or 5 and 60 days.
  • a notch signaling donor such as OP9, OP9delta, and/or OP9 Jagged1 cells
  • contacting a cell population comprising cholangiocyte progenitors with a notch signaling donor comprises co-culturing the cell population comprising cholangiocyte progenitors with a notch signaling donor such as OP9, OP9delta, and/or OP9 Jagged1 cells and optionally in maturatoin media comprising EGF, TGFbeta1, HGF and EGF, and/or HGF, TGFbeta1 and EGF, for at least or about 5 to at least or about 90 days, optionally for at least or about 5 to at least or about 60 days, at least or about 30 days, at least or about 25 days, 2 at least or about 1 days and/or at least or about 14 days to induce the maturation of cholangiocyte progenitors into a cholangiocytes, optionally functional cholangiocytes.
  • a notch signaling donor such as OP9, OP9delta, and/or OP9 Jagged1 cells and optionally in maturatoin media comprising
  • inducing maturation, and optionally further lineage specification and/or expansion comprises co-culturing the cell population comprising cholangiocyte progenitors with OP9, OP9delta, and/or OP9 Jagged1 cells and optionally in the presence of EGF, TGFbeta1, HGF and EGF, and/or HGF, TGFbeta1 and EGF, for at least 5, 8, 9, 10, 11, 12, 13 or 14 days or more, 90 days, optionally for at least or about 5 to at least or about 60 days, at least or about 30 days, at least or about 25 days, 2 at least or about 1 days and/or at least or about 14 days.
  • activator of Notch signaling refers to as used herein any molecule or cell that activates Notch signaling in a hepatocyte and/or cholangiocyte and includes, but is not limited to, notch signaling donors such as OP9 cells, a line of bone marrow-derived mouse stromal cells and notch ligands.
  • notch signaling donors such as OP9 cells, a line of bone marrow-derived mouse stromal cells and notch ligands.
  • OP-9 cells endogenously express and have been engineered to overexpress one or more notch ligands.
  • OP-9-Jagged1 are engineered to overexpress recombinant/exogenous Jagged 1 notch ligand and OP9-delta1 are engineered to overexpress recombinant delta1 notch ligand.
  • the OP9 notch signaling donor is selected from OP9, OP9-Jagged1 or OP-delta1 cells.
  • OP9 cells express notch ligand delta. As they express notch ligands they thereby can act as activators of Notch signaling.
  • molecules and/or cells expressing Jagged-1 (EUROGENTEC 188-204), Jagged1 peptide (abcam, ab94375) as well as recombinant Human Pref-1/DLK-1/FA1 (R&D 1144-PR).
  • the “notch agonist” can for example be bound to a surface such as a cell, plastic, ECM or bead.
  • the cell population comprising cholangiocyte progenitors is co-cultured with OP9, OP9delta and/or OP9Jagged1 cells in the presence of 10 to 20 ng/ml, optionally about 20 ng/ml HGF and/or 25 to 75 ng/ml, optionally about 50 ng/ml EGF induce the differentiation of at least one cholangiocyte progenitor into a functional cholangiocyte.
  • the hepatoblast cells e.g. stage day 25 or 26 as shown in FIGS. 1 a and 14 a can be aggregated (also referred to as 3D aggregates) as described in step g), step g) comprising inducing maturation, further lineage specification and/or expansion of hepatocyte and cholangiocyte progenitors of the cell population into hepatocytes and/or cholangiocytes, the inducing maturation, further lineage specification and/or expansion comprising generating aggregates of the cell population.
  • 3D aggregates comprise hepatoblast cells that can be matured/differentiated to hepatocytes or further specified to cholangiocytes.
  • further lineage specification is obtained by co-culturing the aggregates with a Notch signaling donor such as OP9, OP9delta and/or OP9 Jagged1 cells, optionally as chimeric aggregates comprising hepatoblast cells and Notch signaling donor cells.
  • a Notch signaling donor such as OP9, OP9delta and/or OP9 Jagged1 cells
  • the hepatoblast cell population comprising cholangiocyte progenitors is co-cultured with OP9, OP9delta and/or OP9Jagged1 cells, optionally as chimeric aggregates, in a matrix/gel comprising Matrigel and/or collagen.
  • 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.
  • the collagen comprises collagen I and/or collagen IV.
  • the matrix/gel comprises from about 0 to about 5 mg/mL collagen I, optionally about 1.0 mg/mL, about 2 mg/mL, about 3.0 mg/mL, or about 4.0 mg/mL collagen I.
  • the matrix/gel comprises from 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 collagen I
  • cyst structures are obtainable wherein the co-culture comprises a Matrigel composition of at least 30% or more. If increased branched structures are desired, the Matrigel concentration can be decreased to for example about 20%.
  • CFTR expressing cholangiocyte branched and cyst structures can be produced using a method described herein.
  • the CFTR is functional as shown using swelling assays.
  • the cholangiocytes produced and/or isolated are CFTR expressing cholangiocytes.
  • nodal agonist as used herein means any molecule that activates nodal signal transduction such as “nodal” (for example human nodal such as Gene ID: 4338) or “activin” in a hepatocyte lineage cell.
  • activin refers to “Activin A” (for example Gene ID: 3624), for example human activin, as well as active conjugates and fragments thereof, optionally including naturally occurring active conjugates and fragments, that can for example activate nodal signal transduction as well as active conjugates and fragments thereof, including naturally occurring active conjugates and fragments.
  • concentration of activin can for example range 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.
  • the activin concentration 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.
  • HGF refers to hepatocyte growth factor (Gene ID: 3082), for example human HGF, as well as active conjugates and fragments thereof, including naturally occurring active conjugates and fragments.
  • concentration of HGF can for example range 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.
  • the HGF concentration 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.
  • TGFbeta as used herein means any one 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.
  • TGFb1 promotes cholangiocyte branching when hepatoblasts are co-cultured with OP9.
  • TGFb2 and TGFb3 have also been tested and promote branching structures under similar conditions.
  • TGFbeta1 refers to transforming growth factor beta 1, for example human TGFbeta1 Gene ID 7040) as well as active conjugates and fragments thereof including naturally occurring active conjugates and fragments.
  • concentration of TGFbeta1 for cholangiocyte specification can for example range from about 5 ng/ml to about 10 ng/ml.
  • a wnt/beta-catenin agonist as used herein means any molecule that activates wnt/beta-catenin receptor signaling in a hepatocyte and includes for example Wnt3a and as well as GSK3 selective inhibitors such as CHIR99021 (StemoleculeTM CHIR99021 Stemgent), 6-bromo-Indirubin-3′-Oxime (BIO) (Cayman Chemical (cat:13123)), or StemoleculeTM BIO from Stemgent (cat:04003).
  • CHIR99021 is a selective inhibitor of GSK3.
  • the GSK3 selective inhibitors contemplated are for example selective inhibitors for GSK-3 ⁇ / ⁇ in the Wnt signaling pathway.
  • Wnt/beta receptor signaling in a hepatocyte can be determined by for example by measuring increases in Axin2 gene expression for example by qPCR and/or measuring beta catenin phosphorylation, for example using Cignal TCF/LEF reporter from Qiagen (Cignal TCF/LEF Reporter (luc) Kit: CCS-018L).
  • Wnt3a refers to wingless-type MMTV integration site family, member 3A factor (e.g. Gene ID: 89780), for example human Wnt3a, as well as active conjugates and fragments thereof, including naturally occurring active conjugates and fragments.
  • concentration of Wnt3a can for example range 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.
  • the Wnt3a concentration 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.
  • agonist means an activator, for example, of a pathway or signaling molecule.
  • a nodal agonist means a molecule that selectively activates nodal signaling.
  • a TGF beta antagonist is a molecule that selectively inhibits TGFbeta signaling, for example by measuring phosphorylation of Smad.
  • A83-01 is a more potent inhibitor of smad2 than SB431542.
  • selective inhibitor means the inhibitor inhibits the selective entity or pathway at least 1.5 ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ or 10 ⁇ more efficiently than a related molecule.
  • a GSK-3 selective inhibitor inhibits GSK-3 in the wnt pathway at least 1.5 ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ or 10 ⁇ more efficiently than it is inhibited by for example LiCl or at least 1.5 ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ or 10 ⁇ more efficiently than it inhibits other kinases, other GSKs and/or GSK3 in other pathways.
  • CHIR 99021 has been shown in in vitro kinase assays to specifically inhibit GSK3B with an IC50 of about 5 nM and GSK3a with an IC 50 of 10 nM with little effect on other kinases.
  • a selective inhibitor can be exhibit an IC50 that is at least 1.5 ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ or 10 ⁇ lower than other for example, 2 other, 3 other etc. unrelated kinases.
  • selective activator means an activator that activates the selective entity or pathway at least 1.5 ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ or 10 ⁇ more efficiently than a related molecule.
  • active fragments is a polypeptide having amino acid sequence which is smaller in size than, but substantially homologous to the polypeptide it is a fragment of, and where 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% effective biological action as compared to the full length polypeptide of which it is a fragment of or optionally has greater than 100%, for example 1.5-fold, 2-fold, 3-fold, 4-fold or greater than 4-fold effective biological action as compared to the polypeptide from which it is a fragment of.
  • active conjugates means a polypeptide (or other molecule” that is conjugated to a tag such as a fluorescent tag or stabilizing entity for example for improving stability under extended storage, heat, enzymes, low pH, stirring etc. that does not at all or substantially interfere with the activity of the active portion of the molecule.
  • the conjugate can have about at least 50%, or 60% or 70% or at 80% or 90% or 100% or greater than 100%, for example 1.5-fold, 2-fold, 3-fold, 4-fold or greater than 4-fold effective biological action (e.g. receptor activating activity) as compared to the unconjugated polypeptide or other molecule.
  • active fragments and conjugates refers to fragments and conjugates of a molecule that retain the ability to activate the cognate receptor of the molecule.
  • active fragments and conjugates are at least 60%, 70%, 80%, 90% or 95% as active as the full length and/or unconjugated molecule.
  • Variants such as conservative mutant variants and activating mutant variants for each of the polypeptides can also be used.
  • the term “Dex” as used herein refers to dexamethasone (Dex).
  • the concentration of Dex can for example range 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.
  • the Dex concentration 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.
  • OSM oncostatin M.
  • concentration of OSM can for example range 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.
  • the OSM concentration 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.
  • some embodiments of the present disclosure comprise activating the cAMP pathway within the aggregates to induce hepatocyte and/or cholangiocyte maturation.
  • cAMP pathway refers to the adenyl cyclase pathway, a G protein-coupled receptor-triggered signaling cascade used in cell communication.
  • the cAMP pathway is optionally the human cAMP pathway.
  • the term “activating the cAMP pathway” refers to inducing the pathway to convert ATP into cAMP e.g increase levels of cAMP.
  • activated GPCRs cause a conformational change in the attached G protein complex, which results in the G alpha subunit exchanging GDP for GTP and separation from the beta and gamma subunits.
  • the G alpha subunits in turn, activate adenylyl cyclase, which converts ATP into cAMP.
  • the cAMP pathway can also be activated downstream by directly activating adenylyl cyclase or PKA.
  • Molecules that activate the cAMP pathway include but are not limited to cAMP, cAMP analogs such as 8-bromoadenosine-3′,5′-cyclic monophosphate (8-Br-cAMP), dibutyryl-cAMP, Adenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS) and/or 8-Bromoadenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer (Sp-8-Br-cAMPS)).
  • 8-Br-cAMP, dibutyryl-cAMP and Sp-cAMPS are examples of cell permeable analogs of cAMP.
  • cAMP analogs that activate cAMP signaling are also known in the art and can be used.
  • Other compounds that activate the cAMP pathway include, but are not limited to, cholera toxin, forskolin, caffeine, theophylline and pertussis toxin.
  • cAMP agonists include, but are not limited to, cholera toxin, forskolin, caffeine, theophylline and pertussis toxin.
  • Sp-8-Br-cAMP Biolog: Cat. No.: B 002 CAS No.: [127634-20-2]
  • 8-Br-cAMP and forskolin (FSK) Sigma:66575-29-9 showing that these compounds can be interchanged.
  • the cAMP pathway is optionally activated by contacting the hepatic aggregates with 0.5 to 50 mM of a cell permeable cAMP analog such as 8-Br-cAMP, optionally 1-40, 1-30, 1-20, 5-15, 8-12 or about 10 mM 8-Br-cAMP.
  • the hepatic aggregates are optionally contacted with the cell permeable cAMP analog for example 8-Br-cAMP for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
  • the cell population comprising hepatocyte and/or cholangiocyte progenitors and/or the aggregates, is cultured in cell culture medium comprising HGF, dexamethasone and oncostatin M.
  • the aggregates are cultured in cell culture medium comprising Iscove's Modified Dulbecco's Medium (IMDM) supplemented with B27, ascorbic acid, glutamine, MTG, HGF, dexamethasone and oncostatin M.
  • IMDM Iscove's Modified Dulbecco's Medium
  • the cells are cultured in cell culture medium comprising HGF, dexamethasone and oncostatin M, optionally Iscove's Modified Dulbecco's Medium (IMDM) supplemented with B27, ascorbic acid, glutamine, MTG, HGF, dexamethasone and oncostatin M prior to aggregation and/or during aggregation.
  • IMDM Iscove's Modified Dulbecco's Medium
  • the cell culture medium is optionally also supplemented with Rho-kinase inhibitor and BSA.
  • the cell population comprising hepatocyte and/or cholangiocyte progenitors can be cultured in a maturation media comprising HGF, DEX and OSM for 10, 11, 12, 13 or 14 days and/or the aggregates can be cultured in a maturation media comprising HGF, DEX and OSM for 6, 7, 8, 9, 10 days.
  • the inducing maturation, further lineage specification and/or expansion step further comprises activating the cAMP pathway within the aggregates to induce the maturation of at least one hepatocyte or cholangiocyte progenitor into a functional hepatocyte and/or cholangiocyte cell.
  • activating the cAMP pathway comprises contacting the aggregates with a cAMP analog and/or cAMP agonist for example with a cAMP analog or cAMP agonist described above.
  • a maturation media comprising a cAMP analog and/or cAMP agonist and DEX and optionally HGF is added to the aggregates subsequent to culturing in the maturation media comprising HGF, DEX and OSM, for example for about 10, 11, 12, 13 or 14 days.
  • aggregates are cultured in cell culture medium comprising HGF, Dex and OSM until the cAMP pathway is activated.
  • the aggregates are cultured medium containing a cAMP analog and/or cAMP agonist and Dex.
  • OSM is removed from the medium when a cAMP analog and/or cAMP agonist is added.
  • HGF is also removed from the medium when a cAMP analog and/or cAMP agonist is added.
  • the amount of HGF in the medium is reduced following the addition of a cAMP analog and/or cAMP agonist (for example 10 ng/ml HGF is reduced from 20 ng/ml HGF).
  • aggregates are cultured in HGF, Dex and OSM for about 6, 7, 8, 9, 10, 11 or 12 days at which point the cAMP analog and/or cAMP agonist is added.
  • OSM and optionally HGF are removed when the cAMP analog and/or cAMP agonist is added.
  • the concentration of HGF in the media is reduced (for example from about 20 ng/ml to about 10 ng/ml).
  • the methods induce 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 nodal agonist treated induced endodermal cells.
  • Maturation for example can be detected by determining the level of mature hepatocyte markers.
  • CYP1A2, CYP2B6, CYP2D6, CYP3A4, CYP7A1, CYP2C9, ALB, CPS1, G6P, TAT, TDO1, NAT2, UGT1A1 and/or ASGPR1 are mature hepatocyte or functional hepatocyte markers whose expression can be detected for example by RT-PCR. Differentiation can also be detected using antibodies that recognize mature hepatocyte cells, for example an antibody that detects ASGPR-1.
  • the endodermal cell population is differentiated from pluripotent stem cells (PSCs) such as an embryonic stem cells (ESCs) or an induced pluripotent stem cells (iPSCs).
  • PSCs pluripotent stem cells
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • the pluripotent stem cell is from a mammal, such as a human.
  • the pluripotent stem cell is a human ESC (hESC) or a human iPSC (hiPSC).
  • iPSC induced pluripotent stem cell
  • a pluripotent stem cell artificially derived (e.g., induced or by complete reversal) from a non-pluripotent cell, typically an adult somatic cell, for example, by inducing expression of one or more genes (including POU4F1/OCT4 (Gene ID; 5460) in combination with, but not restricted to, SOX2 (Gene ID; 6657), KLF4 (Gene ID; 9314), cMYC (Gene ID; 4609), NANOG (Gene ID; 79923), LIN28/LIN28A (Gene ID; 79727)).
  • POU4F1/OCT4 Gene ID; 5460
  • SOX2 Gene ID; 6657
  • KLF4 Gene ID; 9314
  • cMYC Gene ID; 4609
  • NANOG Gene ID; 79923
  • LIN28/LIN28A Gene ID; 79727)
  • the method comprises steps for obtaining the endodermal cell population.
  • steps for obtaining the endodermal cell population For example, methods are provided herein for inducing a definitive endoderm in a pluripotent stem cell such as an ESC or an iPSC.
  • obtaining the endodermal cell population comprises forming embryoid bodies from the pluripotent stem cell culture.
  • EBs are formed by any method known in the art, for example the method described in Nostro, M. C. et al. 18 , wherein EBs are formed from small aggregates in culture for 24 hours in low levels of a BMP4 agonist.
  • obtaining the endodermal cell population comprises obtaining and/or growing the pluripotent stem cell culture in a monolayer.
  • the EBs and/or monolayer cells are subsequently contacted with high concentrations of activin A to induce definitive endoderm.
  • the EBs and/or monolayer are exposed to 80 to 120 ng/ml or 90 to 110 ng/ml activin, optionally about 100 ng/ml activin A for about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 14 days.
  • the EBs and/or monolayer cells are contacted with a wnt/beta-catenin agonist such as Wnt3a or a GSK-3 selective inhibitor such as CHIR-99021, 6-bromo-Indirubin-3′-Oxime (BIO), or StemoleculeTM BIO in addition to activin A.
  • a wnt/beta-catenin agonist such as Wnt3a
  • a GSK-3 selective inhibitor such as CHIR-99021, 6-bromo-Indirubin-3′-Oxime (BIO), or StemoleculeTM BIO
  • BIO 6-bromo-Indirubin-3′-Oxime
  • StemoleculeTM BIO StemoleculeTM BIO
  • the EBs and/or the monolayer cells are exposed to from 10 to 40 ng/ml Wnt3a, or 20 to 30 ng/ml Wnt3A, optionally about 25 ng/ml Wnt3a for about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.
  • the EBs and/or the monolayer cells are exposed to from about 0.03 ⁇ M to about 30 ⁇ M CHIR-99021, or from about 0.1 ⁇ M to about 3 ⁇ M, optionally about 0.3 ⁇ M to about 1 ⁇ M CHIR-99021.
  • the EBs are exposed to from about 0.1 ⁇ M to about 2 ⁇ M.
  • the monolayer cells are exposed to from about 1 ⁇ M to about 30 ⁇ M, for example from about 1 ⁇ M to about 3 ⁇ M CHIR-99021.
  • a person skilled in the art would be able to ascertain equivalently useful amounts of other GSK-3 inhibitors.
  • the EBs and/or monolayer cells are first contacted with 80 to 120 ng/ml activin, or 90 to 110 ng/ml activin, optionally about 100 ng/ml activin A for about 1, 2, 3, 4, 5, 6, 7, 8, 9 or about 10 days prior to being contacted with 80 to 120 ng/ml activin, or 90 to 110 ng/ml activin, optionally about 100 ng/ml activin A and 10 to 40 ng/ml Wnt3a, or 20 to 30 ng/ml Wnt3A, optionally about 25 ng/ml Wnt3a for about 1, 2, 3, 4, 5, 6, 7, 8, 9 or about 10 days to produce an induced endodermal cell population.
  • the induced endodermal cell population is cultured with a nodal agonist such as ActA for at least 36, 38, 42, 44, 46, 48, 50, 52, 56, 58 or 60 hours or for about 1 to about 4 days to produce the extended nodal agonist treated induced endodermal population.
  • a nodal agonist such as ActA for at least 36, 38, 42, 44, 46, 48, 50, 52, 56, 58 or 60 hours or for about 1 to about 4 days to produce the extended nodal agonist treated induced endodermal population.
  • the base culture media for inducing definitive endoderm is any media known in the art for inducing definitive endoderm, optionally neural base media or StemPro34.
  • the cell culture medium is supplemented with activin A, glutamine, ascorbic acid, MTG, bFGF and BMP4.
  • the cell culture medium is further supplemented with a wnt/beta-catenin agonist such as Wnt3a or a GSK-3 selective inhibitor such as CHIR-99021.
  • the definite endoderm or induced endodermal 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, greater than 50%, 60%, 70%, 80%, 85%, 90% or 95% of the endodermal cell population expresses CXCR4, CKIT and EPCAM following activin induction. In another embodiment, greater than 50%, 60%, 70%, 80%, 85%, 90% or 95% of the endodermal cell population expresses SOX17 and/or FOXA2 following activin induction.
  • the method further comprises enriching and/or isolating functional hepatocytes and/or cholangiocytes to optionally generate an isolated population of functional hepatocytes and/or cholangiocytes.
  • the isolating step comprises contacting the population of cells with a specific agent that binds functional hepatocytes and/or cholangiocytes.
  • isolated population refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells.
  • an isolated population is a substantially pure population of cells as compared to the heterogeneous population from which the cells were isolated or enriched from.
  • the cells can for example be single cell suspensions, monolayers and/or aggregates.
  • the isolated population can also comprise a notch ligand expressing cells such as OP9, OP9delta and/or OP9Jagged1 cells.
  • the isolated population can also comprise endothelial cells.
  • the isolated population, optionally in dissociated cell suspension and/or aggregates can be used for example in screening applications, disease modeling applications and/or transplanting applications comprising for example scaffold etc.
  • substantially pure 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, with respect to the cells making up a total cell population.
  • substantially pure population of functional hepatocytes and/or cholangiocytes refers to a population of cells that contain fewer than about 30%, fewer than about 20%, more preferably fewer than about 15%, 10%, 8%, 7%, most preferably fewer than about 5%, 4%, 3%, 2%, 1%, or less than 1%, of cells that are not functional hepatocytes and/or cholangiocytes or their progeny as defined by the terms herein.
  • the present invention encompasses methods to expand a population of functional hepatocytes and/or cholangiocytes, wherein the expanded population of functional hepatocytes and/or cholangiocytes is a substantially pure population of functional hepatocytes and/or cholangiocytes.
  • enriching or “enriched” are used interchangeably herein and mean that the yield (fraction) of cells of one type is increased 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% over the fraction of cells of that type in the starting culture or preparation. Enriching and partially purifying can be used interchangeably.
  • the population of cells can be enriched using different methods such as methods based on markers such as cell surface markers (e.g. FACS sorting etc).
  • hepatocytes and/or cholangiocytes can be isolated using the methods described herein.
  • a further aspect of the application includes a population of cells produced according to a method described herein.
  • the population of cells is an enriched, purified or isolated cell population of hepatoblasts, hepatocytes and/or cholangiocytes, optionally mature and/or functional hepatocytes and/or cholangiocytes, for example produced according to a method described herein, and expressing for example markers of mature and/or functional cells.
  • the enriched, purified or isolated are optionally single cell suspensions, aggregates, chimeric aggregates, and/or structures, including branched structures and/or cysts.
  • the mature and/or functional hepatocytes lack expression of AFP and/or fetal CYP3A7.
  • the mature and/or functional cholangiocyte cells express MDR transporter gene, aquaporin, CFTR and/or a mutant thereof.
  • the cell population is a hepatoblast cell population, optionally expressing Notch2.
  • the isolated, purified and/or enriched population is in vitro produced.
  • the population of cells are comprised in a composition with a suitable diluent.
  • a suitable dilument includes for example a suitable culture medium, or freezing medium containing for example serum, a serum substitute or serum supplement and/or a suitable cryoprotectant such as dimethyl sulphoxide (DMSO), glycerol methylcellulose or polyvinyl pyrrolidone.
  • a further aspect comprises a culture medium supplement composition comprising optionally a FGF and/or a BMP4 agonist which can be used as a supplement for a cell culture base medium.
  • the supplement can also include other components discussed herein such as activin A, Wnt3A, a GSK-3 selective inhibitor such as CHIR-99021, HGF, dexamethasone, Oncostatin M, ascorbic acid, glutamine and B27 supplement.
  • the functional hepatocyte and/or cholangiocyte is derived from an iPS of a subject affected with a liver and/or biliary disease.
  • the disease is a monogenic disease e.g. cystic fibrosis, Alagille syndrome, progressive familial intrahepatic cholestasis (PFIC types 1, 2 and 3).
  • the disease is cystic fibrosis.
  • the subject carries a mutation, for example in the cystic fibrosis gene, for example deltaF508, 997 CFTR del and/ C1 CFTR mutation.
  • the methods described can generate heptoblast populations from iPSCs generated/derived from a cystic fibrosis patient.
  • the disease is a complex biliary disease, optionally primary sclerosing cholangitis or biliary atresia.
  • Another aspect includes an implantable construct or extracorporal bioartificial liver device (BAL) comprising a population of cells described herein, prepared according to a method described herein.
  • BAL extracorporal bioartificial liver device
  • the functional hepatocytes and/or cholangiocytes described herein and their derivatives are can be used in one or more applications.
  • the methods can be used to produce a population of hepatic lineage cells from iPSCs derived from or obtained from a subject affected by a liver and/or biliary disease.
  • Another aspect is a method for generating a liver and/or biliary disease cell model comprising:
  • the disease is in an embodiment the disease is a monogenic disease e.g. cystic fibrosis, Alagille syndrome, progressive familial intrahepatic cholestasis (PFIC types 1, 2 and 3).
  • the disease is complex biliary disease, optionally primary sclerosing cholangitis or biliary atresia.
  • Another aspect is a method for generating a cystic fibrosis cell model comprising:
  • the functional hepatocyte and/or cholangiocyte cells can be used for predictive drug toxicology, drug screening and drug discovery.
  • an assay comprising: contacting a functional hepatocyte and/or cholangiocyte population generated using a method described herein with a test compound, and measuring: 1) cell expansion, 2) maturation of hepatocyte cells and/or cholangiocyte specification, 3) one or more hepatoblast, hepatocyte and/or cholangiocyte properties; and/or 4) restoration and/or amelioration of one or more liver and/or biliary disease cell model deficiencies and compared to a wildtype cell population and/or other control tested in the absence of the test compound.
  • the method further comprises measuring one or more hepatoblast, hepatocyte and/or cholangiocyte properties, including for example as measured in Example 9.
  • the one or more cholangiocyte properties comprises:
  • CFTR activity can for example be assessed by measuring cyst swelling, for example using a cAMP agonist such as forskolin.
  • Example 9 provides an example of a method that can be employed to measure CFTR activity.
  • Another aspect includes a functional CFTR assay comprising:
  • test agent can be compared to and/or tested in the presence of CFTR channel potentiator such as VX-770.
  • VX-770 is an FDA approved drug (also known as Kalydeco) which is used for a patients carrying a particular CF mutation, G551 D.
  • the cells described can also be used for cell transplantation.
  • mixed population of cells, enriched and/or isolated functional hepatocytes and/or cholangiocytes can be introduced into a subject in need thereof, for example for treating liver disease.
  • an aspect includes obtaining cells and/or preparing isolated hepatocytes and/or cholangiocytes optionally functional hepatocytes and/or cholangiocytes according to a method described herein, and administering said cells to a subject in need thereof, for example a subject with liver and/or biliary disease.
  • Yusa et al (55) described correcting a known gene defect in iPSC-derived hepatocytes and retransplanting them.
  • the corrected cells were re-transplanted back into mice and showed functionality that was previously absent in the diseased state. Yusa et al found the most suitable transposon for their purposes to be piggyBac, a moth-derived DNA transposon, which can transpose efficiently in mammalian cells including human embryonic stem (ES) cells.
  • ES human embryonic stem
  • the mobile element enables the removal of transgenes flanked by piggyBac inverted repeats without leaving any residual sequences.
  • the iPSC-3-G5-A7 generated had the corrected A1AT, an intact genome compared to the parental fibroblast and expressed normal A1AT protein when differentiated to hepatocyte-like cells.
  • a transposon is optionally used.
  • Other method of introducing an expression construct include lentiviral, adenoviral based methods. Efficient systems for the transfer of genes into cells both in vitro and in vivo are vectors based on viruses, including Herpes Simplex Virus, Adenovirus, Adeno-associated virus (AAV) and Lentiviruses. Alternative approaches for gene delivery in humans include the use of naked, plasmid DNA as well as liposome-DNA complexes (Ulrich et al., 1996; Gao and Huang, 1995). It should be understood that more than one transgene could be expressed by the delivered vector construct. Alternatively, separate vectors, each expressing one or more different transgenes, can also be delivered to the cell.
  • Cotransfection (DNA and marker on separate molecules) are optionally employed (see e.g. U.S. Pat. No. 5,928,914 and U.S. Pat. No. 5,817,492).
  • a marker such as Green Fluorescent Protein marker or a derivative
  • the vector itself preferably a viral vector.
  • TALENs transcription activator-like effector nucleases
  • CRISPR-Cas9 transcription activator-like effector nucleases
  • a further method includes ZFN (Zinc finger nuclease), optionally combined with TALENs (transcription activator like effector nuclease for gene editing and correction of a mutated gene. See for example Gaj et al (58).
  • the method comprises obtaining cells, optionally blood cells, from a patient affected by a liver and/or biliary disease, genome editing and/or inserting a construct encoding a functional and/or therapeutic protein; and either before or after inserting the construct, inducing hepatoblasts, hepatocytes and/or cholangiocytes according to a method described herein.
  • the population of cells administered is an about day 25/26 population of cells.
  • the cells are specified to a hepatocyte or cholagiocyte fate.
  • Example 9 demonstrates that CFTR functional cholangiocytes can be produced in vitro and/or in vivo.
  • a method of transplanting or treating a subject in need thereof with hepatocytes generated according to a method described herein includes use of the cells generated according to a method described herein for treating a subject in need thereof of example a subject with liver disease and/or a biliary disease.
  • a therapeutically effective amount is administered.
  • the cells obtained are derived from autologous cells, for example iPSCs generated from blood and/or skin cells from a subject.
  • the PSC can for example be iPSCs obtained from a biopsy, blood cells, skin cells, hair follicles and/or fibroblasts.
  • hepatocytes and/or cholagiocytes generated using a method described herein are contacted with a test agent in a toxicity screen.
  • CYPs are the 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.
  • drugs may increase or decrease the activity of various CYP isozymes, either by inducing a CYP isozyme (CYP induction) or by directly inhibiting the activity of a given CYP (CYP inhibition). Changes in CYP enzyme activity may 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 within the body to toxic levels.
  • CYP inhibition screens can be conducted. As it is demonstrated that hepatocytes produced using a method described herein are shown to express CYP1A2, CYP2B6, CYP3A4, CYP2B6, CYP2C9, CYP2D6, and/or CYP7A1, screens for inhibition of one or more these isozymes for example using LC-MS/MS or fluorescent assays, can be conducted. CYP IC 50 and/or K i can be determined.
  • induction of CYP enzymes can be assessed.
  • some compounds induce CYP enzymes resulting in increased metabolism of co-administered drugs that are substrates for the induced CYP enzymes.
  • co-administered drugs can hence lose efficacy.
  • CYP enzymes such as CYP1A2, CYP2B6, CYP2C and CYP3A4 are susceptible to induction. Catalytic activity and mRNA levels of the CYPs can be measured relative to controls with the result being expressed as a fold induction.
  • drug metabolites can be assessed, e.g. the metabolite spectrum of a drug can be determined.
  • different concentrations of the test agent are added to cells obtained using a method described herein, and the cells evaluated for survival, CYP 450 isozyme activity, CYP 450 isozyme mRNA level, and/or metabolite profile.
  • the methods can be used for example to screen drugs generally or to assess a patient's specific toxicity to a drug.
  • the functional hepatocytes and/or cholangiocytes are used in tissue engineering.
  • access to purified populations of functional hepatocytes and/or cholangiocytes allows generation of engineered constructs with defined numbers of functional hepatocytes and/or cholangiocytes.
  • access to purified populations of functional hepatocytes and/or cholangiocytes allows generation of bioartifical liver devices
  • the liver is the main source of plasma proteins, including albumin, components of the complement system and clotting 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 blood bound to transport proteins, mainly albumin (middle chain fatty acids, tryptophan and metabolites of it, endogenous benzodiazepines and other neuro-active substances, mercaptans, toxic bile acids, bilirubin, heavy metals and endogenous vasodilators).
  • albumin middle chain fatty acids, tryptophan and metabolites of it, endogenous benzodiazepines and other neuro-active substances, mercaptans, toxic bile acids, bilirubin, heavy metals and endogenous vasodilators.
  • Plasma exchange techniques utilize for example highly selective membranes and albumin dialysis to increase the clearance of albumin-bound toxins along with water soluble toxins.
  • Obtaining functional hepatocytes and/or hepatocytes that produce albumin can be used with BAL. For example, if functional hepatocytes are obtained, it may not be necessary to perform albumin dialysis.
  • ES/iPS derived hepatocytes that are able to generate albumin protein, which is a major protein secreted from liver can also be used with BAL and albumin dialysis.
  • the production of albumin from a human source is for example important in BAL.
  • Albumin transports hormones, fatty acids and other compounds including toxic agents.
  • the benefit of albumin dialysis is that toxic compounds binding Albumin can be eliminated from the blood stream.
  • Human serum albumin which is clinically used for liver and kidney disease is only obtained at present from donated blood. Generating albumin secreating cells and/or together with higher hepatic function activity, would be an advantage for establishing a BAL system.
  • the methods are applied to patient specific disease hiPSCs and used for example to model liver disease.
  • liver or other cells from a patient with liver disease can be isolated, treated to obtain hiPSCs which can then be cultured and induced to differentiate to functional hepatocyte and/or cholangiocyte cells. These cells can be used to assess characteristics of the disease, such as the genes involved in the disease or the response to patients' immune cells.
  • normal cells and patient specific disease hiPSCs can be induced to functional hepatocytes and/or cholangiocytes and compared.
  • genetic, epigenetic and proteomic analyses of pancreatic progenitors and beta cells from normal and patient specific hiPSCs can be conducted. Such detailed analyses 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.
  • subject includes all members of the animal kingdom including mammals, and suitably refers to humans.
  • treat as applied to an isolated cell, include subjecting the cell to any kind of process or condition or performing any kind of manipulation or procedure on the cell.
  • the terms refer to providing medical or surgical attention, care, or management to an individual.
  • treatment refers to an approach aimed at obtaining beneficial or desired results, including clinical results and includes medical procedures and applications including for example pharmaceutical interventions, surgery, radiotherapy and naturopathic interventions as well as test treatments.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • administering introducing
  • transplanting are used interchangeably in the context of delivering cells (e.g. functional hepatocytes and/or cholangiocytes) into a subject, by a method or route which results in at least partial localization of the introduced cells at a desired site.
  • the cells can be implanted directly to the liver, or alternatively be administered by any appropriate route which results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable.
  • a further aspect includes a kit.
  • the kit can comprise one or more of the agonists, antagonists, maturation factors etc e.g. described above, one or more medias, vessels for growing cells and the like, which can be used in a method described herein and/or cells expanded and/or prepared according to a method described herein.
  • the kit comprises instructions for use according to a method herein.
  • the kit comprises a population of cells produced herein, optionally with instructions, one or more of the agonists, antagonists, maturation factors etc e.g. described above, including for example one or more medias, vessels for growing cells and the like.
  • FIG. 1 a The strategy used to generate hepatic cells from hESCs using embryoid bodies (EBs) is shown in FIG. 1 a . Similar to protocols using monolayer cultures 16 , it involves specific steps that recapitulate the critical stages of liver development in the early embryo. EBs are formed from small aggregates by culture for 24 hours in low levels of BMP4, as previously described 18 . The EBs are subsequently exposed to high concentrations of activin A (hereafter referred to as activin) for five days to induce definitive endoderm, a population defined by expression of the surface markers CXCR4, CKIT and EPCAM and the transcription factors SOX17 and FOXA2. As shown in FIG.
  • activin activin A
  • the induction of highly enriched endoderm is an important first step in the efficient and reproducible generation of hepatocyte-like cells from hPSCs. Induction levels of for example of at least 90% CXCR4+CKIT+ and 80% SOX17+ cells were found to result in optimal hepatic lineage development.
  • the FGF10/BMP4 step was included as it was found to increase albumin expression compared to bFGF/BMP4 alone in the differentiation cultures ( FIG. 2 b ). With these induction conditions, substantial numbers of albumin positive cells consistently developed in the cultures between days 12 and 24 of differentiation.
  • the extended activin treatment maintained the CXCR4 + CKIT + population until day 8 of culture ( FIG. 3 c ) and resulted in higher levels of expression of genes indicative of hepatic progenitor (hepatoblast) development, including HEX, AFP, ALB and HNF4 ⁇ at day 26 of culture ( FIG. 3 d ).
  • Cultures generated from non-treated CXCR4 + CKIT + endoderm contained contaminating mesoderm 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 ( FIG. 3 d,e ).
  • Aggregates were generated from the monolayer by a combination of enzymatic treatment and manual dissociation and then cultured in the presence of HGF, Dexamethasone (Dex) and Oncostatin M (OSM) for six days ( FIG. 4 a ). Aggregation did impact differentiation and led to an increase in the expression of a number of genes associated with liver function including ALB (albumin), CPS1 (Carbamonyl-phosphatase synthase 1), TAT (Tyrosine aminotransferase), G6P (Glucose 6 phosphatase) and TDO (Tryptophan 2,3-dioxygenase) ( FIG. 4 b ).
  • ALB albumin
  • CPS1 Carmonyl-phosphatase synthase 1
  • TAT Teyrosine aminotransferase
  • G6P Glucose 6 phosphatase
  • TDO Tryptophan 2,3-dioxygenase
  • the levels were similar to (ALB) or higher than (TDO) the levels found in adult liver ( FIG. 4 b ). Aggregation also increased the expression of several cytochrome P450 genes including CYP7A1, CYP3A7 and CYP3A4. The levels of CYP3A4 were similar to that in found in primary hepatocytes but well below that in adult liver ( FIG. 4 c ). Expression of other P450 genes including CYP1A2 and CYP2B6 as well as the Phase II enzyme UGT1A1 were not induced to any significant level.
  • the cell surface marker asialo-glycoprotein receptor-1 (ASGPR-1) is found on mature hepatocytes and has been shown to mark maturing cells in hESC-differentiation cultures. Aggregation resulted in a dramatic increase in the proportion of ASGPR-1 + cells detected in the culture, consistently yielding populations that contain greater than 50% positive cells ( FIG. 4 d ). Immunostaining showed that ASGPR-1 and E-cadherin was detected on albumin + day 32 aggregate cells.
  • cAMP Signaling Induces Maturation of hESC-Derived Hepatocyte-Like Cells.
  • cAMP signaling was investigated. Studies using hepatic cell lines have shown that activation of this pathway can induce hepatic gene expression, in part through the induction of the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1- ⁇ ), a co-activator that functions together with HNF4 ⁇ to regulate the expression of many genes involved in hepatocyte function 25-28 .
  • POC1- ⁇ peroxisome proliferator-activated receptor gamma coactivator 1-alpha
  • 8-bromoadenosine-3′5′′-cyclic monophosphate 8-bromoadenosine-3′5′′-cyclic monophosphate
  • 8-Br-cAMP 8-bromoadenosine-3′5′′-cyclic monophosphate
  • ICG Indocyanine green
  • albumin ARB
  • HNF4 ⁇ protein The levels of albumin secreted by hESC-derived monolayer and aggregate populations, as well as by HepG2 cells, Huh7 cells and cryopreserved hepatocytes (PH, lot OSI) was detected using an ELISA assay.
  • Albumin secretion was detectable at day 20 in low levels in monolayer cultures but was dramatically increased (about 5 fold) in day 32 aggregated cultures. Only low levels were detected in HepG2, Huh7 and PH cells.
  • ICG indocyanine green
  • cAMP Signaling Increases Metabolic Enzyme Activity in hESC-Derived Hepatocytes.
  • cAMP signaling also induced changes in the expression pattern of key Phase I cytochrome P450 genes, notably a reduction in the levels of expression of the fetal gene CYP3A7, and a significant increase in expression of the adult genes CYP3A4 (2.5-fold), CYP1A2 (18-fold) and CYP2B6 (4.7-fold) ( FIG. 6 a ).
  • UGT1A1 an important Phase II enzyme, was also significantly induced (11-fold) by 8-Br-cAMP ( FIG. 6 a ).
  • the induced levels of CYP3A4 and CYP1A2 were significantly higher than those found in primary hepatocytes whereas the levels of CYP2B6 were similar in the two populations.
  • UGT1A1 expression in the hESC-derived population did not reach the levels found in the primary hepatocytes. Without being bound by theory, given that expression of CYP1A2 is restricted to the liver and only detected after birth 31 , these findings suggest that cAMP signaling promotes differentiation beyond the fetal stage of development.
  • the ability to metabolize isozyme-selective marker drugs was measured by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the 8-Br-cAMP-treated cells O-deethylated the CYP1A2-selective substrate phenacetin at levels as high as primary cultured hepatocytes ( FIG. 6 e ).
  • Non-treated cells did not show detectable activity.
  • CYP2B6 activity as measured by the hydroxylation of bupropion was also detected in the 8-Br-cAMP-treated cells, at levels comparable to those found in primary hepatocytes ( FIG. 60 .
  • Analyses of phase II metabolic enzymes including the arylamine N-acetyltransferases NAT1 and/or NAT2 ( FIG. 6 g ) and UDP-glucuronosyltransferase (UGT) ( FIG. 6 h ) revealed activity higher than that of primary cultured hepatocytes, indicating that cAMP signaling induced the up-regulation of expression of a broad range of enzymes, consistent with maturation of the population. Together, these observations indicate that cAMP signaling promotes maturation of the hESC-derived hepatocyte-like cells in the 3-D aggregates.
  • CYP1A2 and CYP3A4 were also evaluated. 8-Br-cAMP-treated cells were able to metabolize the CYP1A2-selective substrate phenacetin. Induction of the cells with lansoprazole for 72 hours resulted in a 3.4-fold increase in this activity. The non-treated (8-Br-cAMP) cells had low levels of activity that were not inducible. Two independent primary hepatocyte samples showed lower or comparable levels of basal metabolic activity, but did display higher levels of induction (18- and 9-fold). CYP3A4 activity was measured by the ability of the cells to metabolize testosterone to 6 ⁇ -hydroxyl testosterone.
  • 8-Br-Camp treated cells displayed this activity.
  • Addition of the CYP3A4 inducer rifampicin increased the activity 2.2-fold, indicating that this enzyme was also inducible in the hESC-derived cells.
  • the primary hepatocytes showed low but significant levels of CYP3A4 induction.
  • iPSC induced pluripotent cell
  • H9-derived cells were generated following two days of activin treatment with H9-derived cells, both H1 and 38-2 cells required four days of additional activin signaling. With this treatment, it was possible to generate cultures consisting of 90%, 85% and 70% ALB + cells from the H9, H1 and 38-2 cell lines, respectively ( FIG. 7 c ). H9-derived cells at day 26 of differentiation showed a cobblestone morphology very similar to that of cultured hepatocytes ( FIG. 7 d ).
  • H9-derived cells possessed that lansoprazole-inducible CYP1A2 activity.
  • H9 and iPSC-derived cells also showed CYP3A4 activity that was inducible with rifampicin. Inducible CYP1A2 activity was not detectable in the iPSC-derived cells, possibly reflecting suboptimal differentiation of this population.
  • a microarray analysis was carried out to compare the global expression profile of the different populations.
  • a total of 23038 filtered transcripts were used in the final analysis.
  • a two-way unsupervised hierarchical cluster analysis revealed that the three groups appear as distinct populations.
  • the three cAMP-induced populations were the most similar to one another, whereas the three primary hepatocyte populations showed the most divergent expression patterns.
  • a FDR corrected ANOVA (q ⁇ 0.05) was used to identify 784 transcripts that showed the most statistically significant variability across all three sample groups.
  • a hierarchically clustered visualization of these data identified clusters of highly expressed transcripts in each of the biological groups.
  • clusters consisted of 181 transcripts in the primary hepatocytes, 106 transcripts in the 8-Br-cAMP-induced cells and 80 transcripts in the non-treated cells.
  • Genes enriched in 8-Br-cAMP-induced cells included most of the key P450 enzymes, as well as gene ontogeny categories of those involved in many aspects of liver function including gluconeogenesis, glucose homeostasis and lipid metabolism.
  • the cluster expressed at highest levels in the primary hepatocytes consisted of immune system, inflammatory related and MHC genes.
  • the cluster detected in the non-induced hESC-derived cells did not contain any enriched gene ontology categories.
  • transcripts that encode proteins involved in key aspects of liver function were analyzed. These included a subset of Phase I and II drug metabolizing enzymes, transporters, coagulation factors, lipoproteins, nuclear receptors and transcription factors and general liver enzymes and other functional molecules. Analyses of these data revealed that many of the genes are expressed at comparable levels in the 8-Br-cAMP-treated hESC-derived cells and the primary hepatocytes. Select genes in each category are expressed at significantly higher levels in the 8-Br-cAMP treated cells compared to the untreated cells or the primary hepatocytes.
  • Phase I enzymes CYP1A2 and CYP3A4 include the Phase I enzymes CYP1A2 and CYP3A4, confirming the qPCR and functional studies, the Phase II enzyme SULT2A1, the transporter SLCO1B1, the general liver enzymes TAT, G6P and TDO (responsible for tyrosine metabolism, gluconeogenesis and tryptophan metabolism, respectively), the surface receptor ASGPR-1, and ALB. Since cellular uptake of ICG in hepatocytes is regulated by the organic anion transporters SLCO1B1 and SLCO1B3 and the Na + -independent transporter SLC10A1 29 , induction of their expression is consistent with the findings that cAMP treated aggregates showed higher levels of ICG uptake.
  • hPSC-derived hepatocytes For hPSC-derived hepatocytes to be useful for drug metabolism analyses and for transplantation for the treatment of liver disease, the cells must be relatively mature and display many characteristics of adult hepatocytes including measurable levels of key Phase I and Phase II drug-metabolizing enzymes.
  • a number of different studies have shown that it is possible to generate immature hepatic lineage cells from both hESCs and hiPSCs using staged protocols designed to recapitulate critical developmental steps in the embryo. The success of these studies reflects the fact that the pathways controlling the early stages of differentiation are reasonably well defined. In contrast, the factors and cellular interactions that control hepatocyte maturation are poorly understood, and as a consequence only a few studies have reported the development of metabolically functional cells.
  • Duan et al 12 showed that it was possible to derive hepatic cells from H9 hESCs that displayed levels of CYP1A2, CYP3A4, CYP2C9 and CYP2D6 enzyme activities comparable to those found in primary hepatocytes.
  • Duan et al used serum in their methods which comprises numerous factors some of which vary between batches of serum. The factors responsible were undefined. While these findings indicate that relatively mature hESC-derived hepatocytes can be generated, the study did not provide any details on the pathways that promote maturation nor did it demonstrate that the strategy is broadly applicable to other hPSC lines. As shown in FIG. 6 of Duan et al, they measured metabolism drugs in hepatocyte from human ES cells (H9).
  • Phenacetin induces and provides an assessment of CYP1A2 activity
  • Midazolam induces and provides an assessment of CYP3A4, and CYP2B6 and CYP2D6, respectively.
  • CYP1A2 and CYP2B6 almost equivalent levels of enzyme activity (CYP1A2 and CYP2B6) compared to primary hepatocytes was seen.
  • qPCR analysis the level of expression of CYP1A2 and CYP2B6 in H9 cells was 5-8 fold higher than those found in HES2 cell line.
  • the methods described herein result in cells that more closely resemble primary hepatocytes in terms of CYP enzymes CYP1A2 and CYP2B6. Similarly almost the same or comparable level of CYP2D6 expression is seen in cells generated using the present methods compared to primary hepatocyte.
  • the additional activin culture step may also play a role in appropriately patterning the endoderm to a ventral foregut fate, as this pathway is known to play role in the anterior-posterior patterning of the gut tube 35,36 . It was previously demonstrated that sustained activin/nodal signaling also impacted pancreatic development from hESCs 18 .
  • the maturation stage of the protocol involves 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 the maturation of mouse and human primary fetal hepatocytes 24,37,38 .
  • Recently, Miki et al. reported that 3D culture in perfusion bioreactors can improve the differentiation of hESC-derived hepatocytes indicating that a 3D environment may be important for maturation of the cells. The magnitude of these differences was, however, difficult to interpret, as comparisons were not made to fetal and adult liver control.
  • the second step of the maturation strategy is optionally the activation of the cAMP pathway within the 3D aggregates through the addition of the cell permeable and more slowly hydrolyzed cAMP analogue 8-Br-cAMP.
  • Specific genes within the liver including PGC1- ⁇ , TAT and G6P, contain CREB elements in their promoter regions and as a consequence are direct targets of cAMP signaling 25, 39, 40 . Given this, these target genes were induced in 2D monolayers as well as in the 3D aggregates.
  • cAMP signaling extends beyond the induction of target genes as activation of the pathway induced changes in gene expression patterns associated with different aspects of hepatocyte function including drug metabolism, mitochondrial biogenesis, lipid synthesis and glucose metabolism.
  • cAMP signaling promotes maturation of hepatoblasts.
  • sustained cAMP signaling was not required to maintain the elevated levels of expression further supports the interpretation that the effect is one of maturation and not simply induction and maintenance of expression of specific genes.
  • CYP1A2 CYP3A4 and UGT1A1 which were detected at levels as high as or higher than those found in primary human hepatocytes.
  • the transcript levels were indicative of function, as the HES2-derived cells displayed levels of functional enzyme comparable to that in primary hepatocytes. Similar patterns of induction were observed in hepatocyte-like cells from two hESC lines and one hiPSC line indicating that this maturation strategy is broadly applicable.
  • Endocrine hormones such as insulin and glucagon can influence cAMP levels in the adult liver that have acute effects on glucose metabolism as well as chronic effects via regulating gene expression.
  • cAMP levels are upregulated resulting in the rapid induction of PGC1- ⁇ and genes involved in gluconeogenesis, ensuring an energy supply 41, 42 .
  • expression of PGC1- ⁇ is dramatically upregulated 1 day after birth in the mouse liver 43 . This upregulation is thought to rapidly promote maturation of the neonatal hepatocytes.
  • the inventors have, for the first time, defined steps that promote the maturation of hepatic 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 end point as it demonstrates that these advances will enable the routine production of hPSC-derived hepatocyte-like cells for drug metabolism analyses in the pharmaceutical industry.
  • the cAMP-induced cells also provide an ideal candidate population for the development of bio-artificial liver devices and ultimately for transplantation for cell replacement therapy for the treatment of liver disease.
  • HPSCs were maintained on irradiated mouse embryonic feeder cells in hESC media consisting of DEME/F12 (50:50; Gibco) supplemented with 20% Knock-out serum replacement (KSR) as described previously 44 .
  • KSR Knock-out serum replacement
  • hESCs Prior to the generation of embryoid bodies (EBs), hESCs were passaged onto MatrigelTM-coated plates for 1 day to deplete the population of feeder cells. At this stage, the hESCs were dissociated by 0.25% Trypsin-EDTA to generate small cluster as previously described 44,45 and then cultured in serum free differentiation (SFD) media in the presence of BMP4 (3 ng/ml) for 24 hours (day 0 to day 1).
  • SFD serum free differentiation
  • induction medium A that consisted of StemPRO-34® supplemented with glutamine (2 mM), ascorbic acid (50 ⁇ g/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) for 3 days.
  • induction medium A consisted of StemPRO-34® supplemented with glutamine (2 mM), ascorbic acid (50 ⁇ g/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) for 3 days.
  • EBs were harvested and re-cultured in StemPRO-34® supplemented with bFGF (10 ng/ml), activin A (100 ng/ml), Wnt3a (25 ng/ml) and BMP4 (0.25 ng/ml) (medium B).
  • EBs were harvested at day 6, dissociated to single cells and the cells cultured for 2 days on MatrigelTM-coated 12 well plates at a concentration of 4 ⁇ 10 5 cells in media B without Wnt3A and with activin A at a concentration of 50 ng/ml.
  • hepatic specification media that consisted of Iscove's Modified Dulbecco's Medium (IMDM) supplement with 1% vol/vol 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). Media was changed every 2 days from day 8 to day 14. To promote maturation of HES2-derived hepatic cells, they were cultured in maturation media A for 12 days.
  • IMDM Iscove's Modified Dulbecco's Medium
  • Maturation media A consisted of IMDM with 1% vol/vol B27 supplement, ascorbic acid, Glutamine, MTG, Hepatocyte growth factor (HGF) (20 ng/ml), Dexamethasone (Dex) (40 ng/ml) and Oncostatin M (20 ng/ml). Aggregates were generated from the population at day 26 of culture. To generate aggregates the cells were dissociated with collagenase and TrypleLE and then cultured in six well ultra-low cluster dishes at a concentration of 6 ⁇ 10 5 cells per well in maturation medium A supplemented with Rho-kinase inhibitor and 0.1% BSA. Aggregates were maintained under these conditions until day 32, with media changes every 3 days.
  • the media was changed to maturation medium B that consisted of Hepatocyte culture medium (NCM) (Lonza: CC-4182) without EGF. 10 mM 8-Br-cAMP (Biolab: B007) was added at this stage. Media was changed every 3 days.
  • NCM Hepatocyte culture medium
  • 8-Br-cAMP Biolab: B007
  • Media was changed every 3 days.
  • the concentration of bFGF was increased to 40 ng/ml and the base media was switched from IMDM to H16 DMEM for culture from days 8 to 14 and then to H16 DMEM plus 25% Ham's F12 from days 14 to 20.
  • IMDM was replaced with H21 DMEM plus 25% Ham's F12 and 0.1% BSA for the maturation media A used from days 20 to 32. All cytokines were human and purchased from R&D Systems, unless stated otherwise. EB and monolayer cultures were maintained in a 5% CO2, 5% 02, 90% N2 environment. Aggregation cultures were maintained in a 5% CO2 ambient air environment.
  • Flow cytometric analyses were performed as described previously 45 .
  • staining was carried out in PBS with 10% FCS.
  • staining was performed on cells fixed with 4% paraformaldehyde (Electron Microscopy Science, Hatfield, Pa., USA) in PBS. Cells were permeabilized with 90% ice-cold methanol for 20 minutes for Sox17 and FoxA2 staining 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).
  • Immunostaining was carried out as described previously 45 .
  • Cells were fixed in the culture wells with 4% PFA at 37° C. for 15 minutes, washed three times in DPBS (with CaCl 2 and MgCl 2 )+0.1% BSA, and then permeabilized in wash buffer with 0.2% Triton-X100 for 20 minutes. Following an additional 3 washes in DPBS (with CaCl 2 and MgCl 2 )+0.1% BSA, the cells were blocked with protein block solution (DAKO; X0909) for 20 minutes at room temperature.
  • DAKO protein block solution
  • albumin and alpha-fetoprotein positive cells were stained for 1 hour at room temperature with either a goat anti-ALB antibody (Bethyl) or a rabbit anti-AFP antibody (DAKO). Concentrations of isotype controls were matched to primary antibodies. To visualize the signal, the cells were subsequently incubated for 1 hour at room temperature with either a donkey anti-goat Alexa 488 antibody (Invitrogen) or a donkey anti-rabbit-Cy3 antibody (Jackson lmmunoresearch). For Sox17 staining, the cells were fixed, permeabilized and blocked as described above. The cells were incubated with goat-anti-SOX17(R&D) over night at 4° C.
  • a goat anti-ALB antibody Bethyl
  • DAKO rabbit anti-AFP antibody
  • the signal was visualized by incubation with donkey anti-goat Alexa 488 (Invitrogen).
  • ASGPR-1 staining aggregates were cultured on MatrigelTM-coated cover glass for 1 day. Following attachment and spreading, the cells were fixed with 4% PFA at 37° C. for 15 minutes and then permeabilized with cold 100% methanol for 10 minutes. The cells were washed and blocked as above. The fixed cells were incubated with goat anti-ASGPR-1 (Santa Cruz) overnight at 4° C. and then with the rabbit anti ALB (DAKO) for 1 hour at room temperature. The signals were visualized by incubation with donkey anti-goat Alexa 488 antibodies and donkey anti-rabbit CY3 antibodies.
  • TATA box binding protein TATA box binding protein
  • ICG indocyanine green
  • Hepatic aggregates were incubated in HCM containing either the CYP1A2 substrate phenacetin (200 ⁇ M), the CYP2B6 substrate bupropion (900 ⁇ M), the NAT1/2 substrate sulfamethazine (SMZ) (500 ⁇ M), or the total UGT substrate 4-methylumbeliferone (4-MU) (200 ⁇ M) for either 24 or 48 hours.
  • HCM containing either the CYP1A2 substrate phenacetin (200 ⁇ M), the CYP2B6 substrate bupropion (900 ⁇ M), the NAT1/2 substrate sulfamethazine (SMZ) (500 ⁇ M), or the total UGT substrate 4-methylumbeliferone (4-MU) (200 ⁇ M) for either 24 or 48 hours.
  • SMZ NAT1/2 substrate sulfamethazine
  • 4-MU 4-methylumbeliferone
  • 4-MU glucuronide was measured by high-performance liquid chromatography coupled with tandem mass spectrometry as described previously 47 .
  • Cryopreserved hepatocytes were thawed and plated on collagen culture dishes at a density of 1 ⁇ 10 4 cells per well for either 24 or 48 hours.
  • Supernatant was harvested following either 24 or 48 hours of culture and activities for CYP1A2, CYP2B6, NAT1/2 and total UGT measured.
  • RNA samples were run on Affymetrix Human Gene ST v1.0 chips following standard Affymetrix guidelines at the University Health Network Genomics Centre. Briefly, 300 ng of total RNA starting material for each sample was used as input to the Ambion WT Expression Kit. 2.7 ⁇ g of amplified cDNA was then fragmented, labeled and hybridized to Affymetrix Human Gene ST v1.0 chips for 18 hours (45° C. at 60 RPM). Arrays were washed using a GeneChip Fluidics Station P450 fluidic station and scanned with an Affymetrix GeneChip Scanner 7 G. After scanning, each chip was checked and found to pass Affymetrix quality control guidelines.
  • Raw CEL files were imported into Genespring software (Agilent, v11.5.1) and probe level data was summarized using the ExonRMA16 algorithm based on the HuGene-1 — 0-st0v1_na31_hg19 — 2010-09-03 build. Furthermore, each gene was normalized to the median value across all samples under consideration. All statistics were performed on log 2 transformed data. In total, 28869 transcripts are represented on this array.
  • transcripts were filtered to remove those that were consistently in the lower 20th percentile of measured expression across all of the 3 sample groups.
  • An unsupervised hierarchical clustering analysis with a Pearson centered distance metric under average linkage rules was used to address overall similarity and differences between the samples and groups.
  • Directed statistical analysis between the 3 sample groups was performed using an ANOVA with a Benjamini and Hochberg False Discovery Rate (FDR, q ⁇ 0.05) 48 .
  • FDR Benjamini and Hochberg False Discovery Rate
  • a gene ontology (GO) analysis was performed using a corrected Benjamini and Yuketieli hypergeometric test at the q ⁇ 0.1 significance level 49 .
  • Two a priori defined sets of specific transcripts were examined in more detail: transcripts related to specific liver related activity of interest; and transcripts found to be expressed and liver specific based on publicly available information from the HOMER database 50 .
  • CHIR99021 is a selective inhibitor of GSK3 that has been reported to mimic the canonical Wnt signal pathway. CHIR99021 was tested as a replacement of wnt3a (e.g. added in combination with activin) in inducing Embryoid bodies and monolayer induction for definitive endoderm cells from hPSCs. As see in FIG. 8 a ) and b ), the proportion of CKIT+CXCR4+ and CXCR4+EPCAM+ cells induced using CHIR99021 to replace Wnt3a is greater than 95% of the population and is comparable to what is seen with Activin/wnt3a induction.
  • FIGS. 8 ( a ) and ( b ) demonstrate that CHIR99021 can induce definitive endoderm cells.
  • FIG. 8 ( a ) is a flow cytometric analysis showing the proportion of CXCR4+, CKIT+ and EPCAM+ cells in day six activin/CHIR 99021 of Embryoid body induction with activin/wnt3a.
  • FIG. 8 ( b ) is a flow cytometric analysis of showing the proportion of CXCR4+, CKIT+ and EPCAM+ cells in day six activin/CHIR 99021.
  • FIGS. 8( c ) and ( d ) show day seven of monolayer induction with (c) activin/wnt3a or (d) activin/CHIR 99021.
  • CHIR99021 (0.3 NM) was replaced from Wnt3a for endoderm induction.
  • HPSCs were maintained on irradiated mouse embryonic feeder cells in hESC media consisting of DEME/F12 (50:50: Gibco) supplemented with 20% Knock-out serum replacement (KSR) as described previously (Kennedy et al., 2007).
  • KSR Knock-out serum replacement
  • hESCs Prior to the induction of endoderm in monolayer culture, hESCs were passaged onto a Matrigel coated surface (typically 12 well plates) for 1 day. At day 0, the cells were cultured in a RPMI based medium supplemented with glutamine (2 mM), MTG (4.5 ⁇ 10-4 M: Sigma), activin A (100 ng/ml), CHIR99021 (0.3 NM) or Wnt3a (25 ng/ml).
  • Transplanted hESC-derived hepatoblasts engraft and generate cells that express hepatocyte differentiation markers.
  • FIGS. 8 ( e ), ( f ) and ( g ) demonstrates engraftment of ES derived liver cells prepared using the method described in Example 1 except that the GSK3 inhibitor CHIR99021 was used to make the transplanted cells as described in Example 2.
  • FIGS. 8 ( e ), ( f ) and ( g ) (h) Ectopic liver tissue in NSG Mice.
  • FIG. 8( e ) is a demonstrative photomicrograph of H&E staining of the intestinal mesentery area, showing a cluster of hESC-derived hepatocyte (arrowhead) 2 months after transplant. Magnification was 5 ⁇ . Intestine (arrow), engrafted cells (arrowhead).
  • FIG. 8 ( f ) shows high magnification (10 ⁇ ) photomicrographs of H&E stained section from FIG. 8 ( e ).
  • FIG. 8 ( g ) (h) Immunohistochemical staining shows the presence of hESC-derived cells in the intestinal mesentery area two months after transplant. Double staining for human Albumin (Alexa 488: green) (showing as an arrow) and CK19 (Cy3: red) (showing as an arrowhead) shows that the transplanted cells have the potential to differentiate into the hepatocyte and cholangiocyte lineages. HESC-derived hepatocyte-like cells were observed as albumin positive cells (Arrow), whereas cholangiocyte-like cells expressed CK19 and were found in duct like structures (Arrowhead).
  • mice Six week-old NSG mice were obtained from The Jackson Laboratories (Bar Harbor, Me., USA) and housed at UHN animal facility. Aggregates (day 27) consisting of hESC-derived hepatocyte progenitors (hepatoblasts) and hESC-derived CD34+ endothelial cells were suspended 50 ⁇ l Matrigel (BD bioscience) and kept on the ice until transplantation. Recipient mice were anesthetized with 1-3% isoflurane and laparotomized. The intestinal mesentery areas were exposed and the cells mixture with Matrigel was positioned on the mesentery area and covered with a absorbable hemostat agent, Surgicel (Ethicon 360, USA). Two months following transplantation, the, mice were sacrificed and evaluated for presence of hESC-derived cells by histological analyses.
  • hepatoblasts hepatoblasts
  • CD34+ endothelial cells were suspended 50 ⁇ l Matrigel (BD bioscience) and kept on the ice until transplantation
  • a small molecule related to Wnt/ ⁇ -catenin pathway can expand hepatic progenitor cells.
  • Day 27 hepatic progenitor cells (H9) were dissociated and plated on 96 well Matrigel coated dish at the density of 1 ⁇ 10 4 cell per well. Cells were treated with different concentrations of CHIR99021 (0.3 ⁇ M, 1 ⁇ M and 3 ⁇ M) and cultured for 9 days. Increases in the ratio of hepatic progenitor cells was examined by the counting the cell number compared to day 27 cell number without treatment ( FIG. 9 a ).
  • CHIR99021 (0.3 ⁇ M) replaced Wnt3a during endoderm induction.
  • HPSCs were maintained on irradiated mouse embryonic feeder cells in hESC media consisting of DEME/F12 (50:50: Gibco) supplemented with 20% Knock-out serum replacement (KSR) as described previously (Kennedy et al., 2007).
  • KSR Knock-out serum replacement
  • hESCs were passaged onto 12 wells Matrigel coated plate for 1 day. At day 0, the cells were cultured in RPMI based medium that supplemented with glutamine (2 mM), MTG (4.5 ⁇ 10 ⁇ 4 M: Sigma), activin A (100 ng/ml), CHIR99021 (0.3 ⁇ M) or Wnt3a (25 ng/ml).
  • the cell aggregates were cultured in HGF, Dex and OSM until day 32 at which point cAMP was added. OSM was removed when cAMP analog and/or cAMP agonist is added. In some experiments, HGF was also removed from the cultures when cAMP analog and/or cAMP agonist is added. In other experiments, the addition of 10 ng/ml HGF (reduced from 20 ng/ml) when cAMP was added was shown to promote survival of the aggregates.
  • OSM has an inhibitory effect on the induction of expression of Phase 1 CYP enzymes, in particular CYP 3A4.
  • H9-derived day 27 hepatic progenitors were co-cultured with OP 9 cells (Notch signaling donor) in the presence of HGF 20 ng/ml and EGF 50 ng/ml.
  • the H9-derived day 27 hepatic progenitors were derived as in Example 1.
  • the albumin positive cells were completely diminished and turned into CK19 positive cells with an organized branching appearance ( FIG. 10 a,b ).
  • an H&E section of the co-cultured cells either in the presence or absence of GSI showed that in the presence of GSI, chimeric aggregation was maintained.
  • cells were arranged in an epithelial duct like structure containing lumen ( FIG. 10 a ).
  • cholangiocyte-like cells forming a bile-like structure can be induced from H9-derived hepatic progenitor cells through the activation of Notch signaling (for example by co-culturing with OP9, OP9delta and/or OP9Jagged1 cells).
  • Notch signaling for example by co-culturing with OP9, OP9delta and/or OP9Jagged1 cells.
  • CFTR a marker of functional cholangiocytes
  • IMDM IMDM, 1% vol/vol B27 supplement, glutamine (2 mM), ascorbic acid (50 ⁇ g/ml; Sigma), MTG (4.5 ⁇ 10 ⁇ 4 M; Sigma)
  • Hepatocyte growth factor (20 ng/ml) (20 ng/ml), Dexamethasone (Dex) (40 ng/ml) and Oncostatin M (20 ng/ml).
  • H16/Ham's F12 (75%/25%), 1% vol/vol B27 supplement, glutamine (2 mM), ascorbic acid (50 ⁇ g/ml; Sigma), MTG (4.5 ⁇ 10 ⁇ 4 M; Sigma)
  • Hepatocyte growth factor (20 ng/ml) (20 ng/ml), Dexamethasone (Dex) (40 ng/ml) and Oncostatin M (20 ng/ml).
  • H21/Ham's F12 (75%/25%), 1% vol/vol B27 supplement, glutamine (2 mM), ascorbic acid (50 ⁇ g/ml; Sigma), MTG (4.5 ⁇ 10 ⁇ 4 M; Sigma)
  • Hepatocyte growth factor (20 ng/ml) (20 ng/ml), Dexamethasone (Dex) (40 ng/ml) and Oncostatin M (20 ng/ml).
  • IMDM or H21/Ham's F12 (75%/25%), 1% vol/vol B27 supplement, glutamine (2 mM), ascorbic acid (50 ⁇ g/ml; Sigma), MTG (4.5 ⁇ 10 ⁇ 4 M; Sigma), Rho-kinase inhibitor (10 ⁇ M) and 0.1% BSA.
  • Hepatocyte growth factor (20 ng/ml) (20 ng/ml), Dexamethasone (Dex) (40 ng/ml) and Oncostatin M (20 ng/ml).
  • HCM Hepatocyte culture medium
  • EGF EGF
  • 10 mM 8-Br-cAMP Biolab: B007
  • small molecule related to the inhibition of Wnt/beta.catenin signal XAV 939: 1 ⁇ M
  • MEK/Erk signal PD032590: 1 ⁇ M
  • H21/Ham's F12 (75%/25%), 1% vol/vol B27 supplement, glutamine (2 mM), ascorbic acid (50 ⁇ g/ml; Sigma), MTG (4.5 ⁇ 10 ⁇ 4 M; Sigma),
  • HGF Hepatocyte growth factor
  • EGF Epidermal Growth factor
  • endothelial cells play an important role in liver development, this lineage was assessed for its influence on the growth and/or maturation of the hESC-derived hepatic cells.
  • CD34+ endothelial cells were generated from hESCs.
  • the HES2 hESC line was used which is engineered to express the red fluorescence protein (RFP) cDNA from the ROSA locus to enable us to track the endothelial cells.
  • Endothelial cells were generated by induction with a combination of BMP4, bFGF and VEGF for 6 days at which time the CD34 + cells (also CD31 + and KDR + ) were isolated by FACS.
  • the sorted CD34 + cells were cultured for 6 days in EGM2 endothelial cell growth media and then used for the generation of chimeric aggregates using AggrewellsTM.
  • the endothelial cells were added to the Aggrewells 2 days prior to the hepatic cells to allow them to coat the bottom of the well ( FIG. 12 a ).
  • a single cell suspension of day 25 hepatoblasts was added on top of the endothelial cells and the mixture cultured in the Aggrewells for 48 hours.
  • the aggregates were subsequently removed from the Aggrewells and cultured for an additional 6 days, at which time they were harvested and analyzed. As shown in FIG.
  • the aggregates cultured together with the endothelial cells contained RFP + cells and were larger than those cultured alone.
  • Flow cytometric analysis revealed that the RFP + cells represented greater than 30% of the population ( FIG. 12 c ), indicating that significant numbers had integrated with hepatic cells in the aggregates.
  • qRT-PCR analyses showed that the chimeric aggregates cultured for an additional 12 days expressed substantially higher levels of CYP3A4 message than the hepatic aggregates without the endothelial cells ( FIG. 12 d ). Importantly, these levels were achieved without the addition of cAMP, suggesting that endothelial cells can promote maturation of the hPSC-derived hepatic cells.
  • the combination of 3D aggregation, cAMP and PD/XAV did promote significant differentiation of the human pluripotent stem cell-derived hepatocytes ( FIG. 9 ), ( FIG. 9 d and e ) The cells did retain some expression of AFP and fetal CYP3A7 indicating they may not be fully mature.
  • the chimeric endothelial/hepatic aggregates were treated with the combination of cAMP, PD and XAV. These aggregates were maintained either in liquid culture or in collagen gels to provide a source of extracellular matrix proteins. As shown in FIG.
  • pluripotent stem cells pluripotent stem cells
  • PSCs pluripotent stem cells
  • staged differentiation protocols that promote the generation of cells albeit in low efficiencies and lacking metabolic function, that display some characteristics of mature hepatocytes, including the expression of functional P450 enzymes.
  • iPSCs patient specific induced pluripotent stem cells
  • biliary tract disorders involving the biliary tract are common causes of chronic liver disease that result in significant morbidity and often require whole organ transplantation for definitive management.
  • the underlying mechanisms of monogenic biliary diseases such as cystic fibrosis liver and Alagille syndrome remain incompletely understood, and more complex biliary diseases such as primary sclerosing cholangitis and biliary atresia lack appropriate models for understanding their pathophysiology or for screening novel pharmacological agents.
  • the ability to generate functional cholangiocytes from hPSCs would fulfill these unmet needs.
  • cholangiocytes develop early in fetal life and derive from a bipotential progenitor known as the hepatoblast that also gives rise to the hepatocyte lineage.
  • hepatoblast a bipotential progenitor known as the hepatoblast that also gives rise to the hepatocyte lineage.
  • Targeting studies in the mouse have shown that specification of the cholangiocyte lineage from the hepatoblast is a Notch dependent event that is mediated by the interaction of Notch 2 expressed by the progenitors and Jagged-1 present on the developing portal mesenchyme.
  • hPSC-derived cholangiocytes could be induced to form epithelialized cystic structures that express markers found in mature bile ducts including the cystic fibrosis transmembrane conductance regulator (CFTR).
  • CFTR function in these structures was demonstrated through the regulation of cyst swelling following stimulation of the cAMP pathway with forskolin.
  • Cysts generated from cystic fibrosis patient iPSCs showed a deficiency in the forskolin-induced swelling assay that could be rescued by the addition of CFTR correctors.
  • the progenitor cells downregulate Tbx3 and maintain and/or upregulate the expression of a combination of genes that are normally expressed in the hepatic and/or cholangiocyte lineages including albumin (ALB), alpha fetoprotein (AFP) cytokeratin 19 (CK19), Sox9, NHF6 ⁇ and NOTCH2.
  • ALB albumin
  • AFP alpha fetoprotein
  • CK19 alpha fetoprotein
  • Sox9 NHF6 ⁇ and NOTCH2.
  • RT-qPCR analyses of the bFGF/BMP4 treated hESC-derived endoderm population revealed a transient upregulation of TBX3 expression at day 13 of differentiation, identifying this time as the stage of hepatoblast specification ( FIG. 14 c ).
  • Immunostaining revealed that the majority of the cells in the day 13 population were TBX3 + , indicating that hepatoblast specification was efficient.
  • the onset of SOX9 and HNF6B expression overlapped with that of TBX3 ( FIG. 14 c ). However, unlike TBX3, the expression of these genes continued to increase until day 25, the final day of the analyses. Expression of ALB and AFP was upregulated at day 19 and also increased at day 25.
  • CK19 showed a biphasic pattern, with peak levels of expression detected at days 13 and 25.
  • Immunofluorescent staining and flow cytometric analyses revealed that the majority of the cells at day 25 of differentiation were ALB + , AFP + and CK19 + . Together these findings strongly suggest that the cells within the day 25 population are representative of the expanded hepatoblast stage of development, the equivalent of the liver bud in vivo. Notch 2 but not Notch1 expression was also upregulated at day 25, further supporting the interpretation that this population contains hepatoblasts capable of signaling through this pathway.
  • Notch Signaling Promotes Cholangiocyte Development from the hPSC-Derived Hepatoblast-Like Population.
  • FIG. 15 a and flow cytometric analyses confirmed the immunostaining findings and demonstrated a complete absence of ALB + cells following co-culture with OP9.
  • gamma secretase inhibitor an antagonist of the Notch pathway, blocked the downregulation of ALB expression, reduced the proportion of CK19 + cells in the cultures and inhibited the development of the branched structures indicating that these effects were mediated by Notch signaling ( FIG. 15 a ).
  • Notch signaling FIG. 15 a .
  • Expression of the Notch targets HES1, HES5 and HEY1 was upregulated following nine days of culture on OP9.
  • FIG. 15 b This increase in expression was block by the addition of the ⁇ -secretase inhibitor demonstrating that co-culture with OP9 effectively activated the Notch pathway ( FIG. 15 b ).
  • FIG. 22 Aggregates derived from both lines generated branched structures consisting of CK19 + ALB ⁇ cells following co-culture with the OP9 stromal cells. As observed with the H9-derived populations, the downregulation of ALB expression and the development of these structures were NOTCH dependent events.
  • ZO-1 Zonula occuludens 1
  • the tight junction marker was also expressed and was found to be restricted to the apical side of the structures, suggesting that the cells had acquired apicobasal polarity, a feature of mature epithelial ducts.
  • the cells in the ducts also expressed the Cystic fibrosis transmembrane conductance regulator (CFTR), a transmembrane channel that is first expressed in the adult biliary tract.
  • CFTR Cystic fibrosis transmembrane conductance regulator
  • the CFTR protein was detected predominantly on the apical side of the duct-like structure.
  • Western blot analyses confirmed the presence of the protein in the population generated from the chimeric aggregates ( FIG. 23 d ).
  • the cells within the ducts were RFP + demonstrating that they were of human origin, derived from the HES2-RFP cells ( FIG. 17 c,d ). Additionally the cells expressed CK19 and CFTR, indicating that they displayed characteristics of cholangiocytes. As observed with the structures generated in vitro, CFTR expression segregated to apical side of the duct. Teratomas were not observed in any of the transplanted animals
  • rhodamine123 a tracer dye used to measure the functional activity of the MDR1 transporter that is present in normal bile duct cells.
  • the cystic structures derived from either H9 hESCs or the iPSCs transported dye to the luminal space, indicative of active transporter activity.
  • an inhibitor of the MDR transporter, rhodamine did not accumulate in the lumen of the structures confirming that the movement of the dye reflected active transport likely via the MDR transporter protein.
  • cyst formation from iPSCs generated from two different cystic fibrosis patients carrying the common F508 deletion (e.g. deltaF508).
  • Both hiPSC lines generated hepatoblast populations with kinetics similar to those observed for wild type hPSCs ( FIG. 24 a,b ).
  • cyst formation from the patient iPSCs was clearly impaired as only branched structures were observed in the gels following two weeks of culture ( FIG. 19 a ). Cyst formation from the patient cells could be induced by the addition of forskolin for the first week of the two-week culture period (change) ( FIG. 19 a,b ).
  • cysts that developed from the patient iPSC we not completely hollow, but rather contained branched ductal structures ( FIG. 19 c ).
  • a higher frequency of hollow cysts, typical of those that developed from normal iPSCs were detected following longer periods of culture, suggesting that maturation of the mutant cells was delayed.
  • Addition of the CFTR inhibitors to cultures of normal iPSC-derived cholangiocytes also delayed cyst formation, indicating that the generation of these structures was dependent, to some degree, on a functional CFTR ( FIG. 19 b ).
  • CFTR CFTR-associated fibroblasts
  • the majority of CFTR in normal cells is the larger mature form identified by the upper band (C) in lane HBE in FIG. 20 a .
  • the patient derived cells contained much less CFTR protein and the majority was the smaller immature form. Addition of the correctors dramatically increased the proportion of mature protein in the patient cells.
  • cysts generated from patient Cl increased by approximately 2.18+/ ⁇ 0.52 fold where as those from patient 997 increased by 1.64+/ ⁇ 0.08 folds 24 hours following induction with forskolin/IBMX and VX770.
  • a system for the directed differentiation of hPSCs into functional cholangiocyte-like cells that self-organize into duct-like structures in vitro and in vivo is described.
  • Notch pathway is important for inducing cholangiocyte fate decision in vivo, including Jagged 1 interaction with portal mesenchyme cells and Notch 2 on hepatocytes.
  • Notch signaling provided by OP9 cells successfully manipulated the fate decision in not only monolayer, but also three dimensional gel cultures. Reversed effect was observed when Notch signaling was affected in addition to G secretase inhibitor. Previous reports have shown that cholangiocyte-like duct structures generated, albeit in low efficiency, from human ES cells resulted in showing the function with polarity and rhodamine 123 uptake
  • Notch signaling provided by OP9 promoted the engraftments from human ES derived cholangiocyte-like cells and cells formed the RFP-positive duct-like structures in mouse mammary fat pad. These structures were not observed in the absence of OP9. Taken together, OP9 co-culture system efficiently provides notch signaling to induce cholangiocyte-like cells from human PSCs derived-hepatoblasts both in vitro and in vivo.
  • Hepatocyte maturation from hPSCs is shown to be enhanced by three dimensional culture environments herein. Similarly, maturation of cholangiocyte lineage cells was also promoted by three dimensional gel culture system. When hepatoblast aggregates stimulated by Notch signaling via OP9 cells, gene expression associated with cholangiocyte lineage and maturation was significantly increased. Furthermore functional activity as a cholangiocyte was detected in vitro.
  • the human iPS-derived cholangiocyte-like duct structure demonstrated functional CFTR activity. These cells can be used for drug screening in a patient-specific manner.
  • patient-specific cholangiocyte-like duct structures can be obtained efficiently and used to validate existing or new therapeutic drugs n other severe biliary diseases such as the monogenic conditions progressive familial intrahepatic cholestasis (PFIC types 1, 2 and 3), and Alagille syndrome, and the more common and complex biliary diseases, biliary atresia and primary sclerosing cholenagitis.
  • PFIC types 1, 2 and 3 progressive familial intrahepatic cholestasis
  • Alagille syndrome the more common and complex biliary diseases, biliary atresia and primary sclerosing cholenagitis.
  • accession numbers provided herein including for example accession numbers and/or biomarker sequences (e.g. protein and/or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely.

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