Methods for generating hepatocytes and cholangiocytes from pluripotent stem cells

Abstract

Methods for producing hepatocyte and/or cholangiocyte lineage cells from pluripotent stem cells, 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 FGF agonist and a BMP4 agonist and/or active conjugates and/or fragments thereof; (b) inducing maturation, and optionally further lineage specification and/or expansion of the hepatocyte and/or cholangiocyte progenitors of the cell population to obtain a population comprising hepatocyte lineage cells such as hepatoblasts, hepatocytes and/or cholangiocytes, the inducing maturation step comprising generating aggregates of the cell population. Optionally, the method also comprises activating the cAMP pathway within the aggregates and forming co-aggregates.

1 . A method of producing hepatocyte and/or cholangiocyte lineage cells 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 FGF agonist and a BMP4 agonist and/or active conjugates and/or fragments thereof;   (b) inducing maturation, and optionally further lineage specification and/or expansion of the hepatocyte and/or cholangiocyte progenitors of the cell population to obtain a population comprising hepatocyte lineage cells such as hepatoblasts, hepatocytes and/or cholangiocytes, the inducing maturation step comprising generating aggregates of the cell population.   
     
     
         2 . The method of  claim 1 , wherein 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. 
     
     
         3 . The method of  claim 1 , wherein maturation, further lineage specification and/or expansion can comprise one or more additional steps, optionally wherein 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. 
     
     
         4 . A method of producing hepatocytes and/or cholangiocytes from a pluripotent stem cell population, the method comprising:
 a) contacting the pluripotent stem cells cultured as a monolayer, with an induction media comprising nodal agonist such as ActA and optionally a wnt/beta-catenin agonist such as i) Wnt3a and/or ii) a GSK-3 selective inhibitor such as CHIR-99021, to provide an induced endodermal cell population;   b) contacting the induced endodermal cell population with a nodal agonist to provide an extended nodal agonist treated induced endodermal cell population;   c) specifying the extended nodal agonist treated induced endodermal cell population according to  claim 1  step a);   d) optionally contacting the cell population comprising hepatocyte and/or cholangiocyte progenitors with a maturation media comprising HGF, dexamethasone and/or Oncostatin M and/or active conjugates and/or fragments thereof; and   e) inducing maturation, and optionally further lineage specification and/or expansion of hepatocyte and cholangiocyte progenitors of the cell population into hepatocytes and/or cholangiocytes, the inducing maturation step comprising generating aggregates of the cell population.   
     
     
         5 . A method of producing hepatocytes and/or cholangiocytes from a pluripotent stem cell population, the method comprising:
 a) forming embryoid bodies (EBs) of the pluripotent stem cells, optionally by contacting the pluripotent stem cells with a BMP4 agonist;   b) contacting the EBs with an induction media comprising a nodal agonist such as ActA and optionally a wnt/beta-catenin agonist such as i) Wnt3a and/or ii) a GSK-3 selective inhibitor such as CHIR-99021, to provide an induced endodermal cell population;   c) dissociating the induced endodermal cell population to provide a dissociated induced endodermal cell population;   d) contacting the dissociated induced endodermal cell population with a nodal agonist to provide an extended nodal agonist treated induced endodermal cell population;   e) specifying the extended nodal agonist treated induced endodermal cell population according to  claim 1  step a);   f) optionally contacting the cell population comprising hepatocyte and/or cholangiocyte progenitors with a maturation media comprising HGF, dexamethasone and/or Oncostatin M and/or active conjugates and/or fragments thereof;   g) inducing maturation, and optionally further lineage specification and/or expansion of hepatocyte and cholangiocyte progenitors of the cell population into an expanded hepatoblast population and/or hepatocytes and/or a cholangiocytes, the inducing maturation, comprising generating aggregates of the cell population.   
     
     
         6 . The method of  claim 1 , wherein 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. 
     
     
         7 . The method of  claim 1 , wherein 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, optionally wherein the FGF is bFGF, FGF10, FGF2 or FGF4 or combinations thereof. 
     
     
         8 . The method of  claim 1 , wherein the aggregates are generated from a cell population comprising at least 70%, 80%, 85%, or 90% albumin positive cells. 
     
     
         9 . The method of  claim 1 , wherein 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, optionally wherein, activating the cAMP pathway comprises contacting the aggregates with a cAMP analog such as 8-bromoadensoine-3′5″-cyclic monophosphate. 
     
     
         10 . A method of producing hepatocytes and/or cholangiocytes from pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), the method comprising:
 a) contacting the pluripotent stem cells cultured as a monolayer or formed into embryoid bodies, with an induction media comprising nodal agonist such as ActA and optionally a wnt/beta-catenin agonist such as i) Wnt3a and/or ii) a GSK-3 selective inhibitor such as CHIR-99021 to provide an induced endodermal cell population;   b) contacting the induced endodermal cell population with a nodal agonist to provide an extended nodal agonist treated induced endodermal cell population; and   c) specifying the extended nodal agonist treated induced endodermal cell population according to  claim 1  step a); and   d) inducing maturation, and optionally further lineage specification and/or expansion of hepatocyte and/or cholangiocyte progenitors into hepatoblasts, hepatocytes and/or cholangiocytes, the inducing maturation, and optionally further lineage specification and/or expansion comprising:   (i) culturing the cell population comprising hepatocyte and/or cholangiocyte progenitors with a maturation media comprising HGF, OSM and DEX;   (ii) generating aggregates of the cell population, optionally when the cell population comprises at least 70%, 80%, 85%, or 90% albumin positive cells or after about 20 to about 40 days of culture for example after about 24 to about 28 days of culture;   (iii) culturing the aggregated cells in an aggregated cell maturation media: and   (iv) optionally activating the cAMP pathway in the aggregated cells, optionally within about 1 to about 10 days of aggregation, for example within 6 days of aggregation, optionally after about 27 to about 36 days of culture.   
     
     
         11 . The method of  claim 10 , wherein the aggregated cell maturation media comprises one or more factors which promote maturation, further lineage specification and/or expansion, optionally:
 a. a wnt agonist such as CIHR 99021, optionally in combination with a TGFbeta antagonist such as SB431542 which promotes expansion of an albumin+/HNF4+ progenitor population; or   b. a Wnt antagonist such as XAV939 and/or a Mek/Erk antagonist, for example PD0325901 which is added during the cAMP activation step, which enhances expression of CYP enzymes and promotes maturation of hepatocyte precursors;   
     
     
         12 . The method of  claim 10  wherein the aggregated cell maturation media comprises one or more factors which promote maturation, further lineage specification and/or expansion, optionally:
 a. a Notch agonist which promotes cholangiocyte lineage specification; 
 b. a Notch antagonist such as gamma-secretase inhibitor (GSI) L695,458 which promotes hepatocyte lineage specification. 
 
     
     
         13 . The method of  claim 11  wherein 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. 
     
     
         14 . The method of  claim 12 , wherein the maturation media and/or the aggregate specification media comprises a notch agonist, cAMP activation is omitted. 
     
     
         15 . A method of producing hepatocytes and/or cholangiocytes from pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), comprises:
 a) contacting the pluripotent stem cells cultured as a monolayer or formed into embryoid bodies, with an induction media comprising a nodal agonist such as ActA and optionally a wnt/beta-catenin agonist such as i) Wnt3a and/or ii) a GSK-3 selective inhibitor such as CHIR-99021, optionally for about 4 to about 8 days, to provide an induced endodermal cell population;   b) contacting the induced endodermal cell population with a nodal agonist, optionally for about 1, 2, 3, or about 4 days, to provide an extended nodal agonist treated induced endodermal cell population;   c) specifying the extended nodal agonist treated induced endodermal cell population according to  claim 1  step a), optionally for about 4 to about 10 days, to obtain a cell population comprising hepatocyte and/or cholangiocyte progenitors, and   d) inducing maturation, and optionally further lineage specification and/or expansion of the hepatocyte or cholangiocyte progenitors into hepatocytes and/or cholangiocytes, the inducing maturation, and optionally further lineage specification and/or expansion comprising:   (i) culturing the cell population comprising hepatocyte and/or cholangiocyte progenitors with a maturation media comprising HGF, Dex and/or OSM, optionally for about 10 to 14 days;   (ii) generating aggregates of the cell population, optionally when the cell population comprises at least 70%, 80% 85%, or 90% albumin positive cells or after about 20 to about 40 days for example after about 24 to about 28 days of culture;   (iii) culturing the aggregates in maturation medium comprising Dex for about 1 to 10 days;   iv)
 a) culturing aggregates in a hepatocyte maturation medium comprising Dex and optionally a cAMP agonist, optionally a cAMP analog, for about 6 days to about 10 days, optionally adding the cAMP agonist within about 1 to about 10 days of the generating aggregates step, for example within 6 days of the generating aggregates step, optionally after about 27 to about 36 days of culture; or 
 b) culturing the aggregates in a cholangiocyte maturation/specification medium comprising a notch agonist, optionally a notch signal donor such as OP-9 cells, and optionally a cAMP agonist, optionally EGF, TGFb1, HGF and EGF, and/or EGF, TGFb1 and HGF for about 5 days to about 90 days, optionally adding the notch agonist within about 1 to about 10 days of the generating aggregates step, for example within 6 days of the generating aggregates step, optionally after about 20 to 40 days of culture and/or coculturing the aggregates when the notch agonist is a notch signaling donor cell. 
   
     
     
         16 . The method of  claim 1 , wherein the hepatocytes produced are functional hepatocytes. 
     
     
         17 . The method of  claim 16 , wherein the functional hepatocyte comprises increased expression and/or activity of at least one gene or protein selected from the group consisting of ALB, CPS1, G6P, TDO, CYP7A1, CYP3A7, CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2D6, NAT2 and UGT1A1 compared to the cells of the cell population comprising hepatocyte and/or cholangiocyte progenitors. 
     
     
         18 . The method of  claim 1 , wherein at least 40, 50, 60, 70, 80 or 90% of the hepatocytes, optionally functional hepatocytes, are ASGPR-1+ cells. 
     
     
         19 . The method of  claim 1 , wherein the cholangiocyte fate is specified by treating aggregates of the cell population during the inducing maturation, further lineage specification and/or expansion step with a notch agonist. 
     
     
         20 . The method of  claim 19  wherein the notch agonist comprises a notch ligand bound to a surface such as a cell, plastic, ECM or bead. 
     
     
         21 . The method of  claim 20 , wherein the notch agonist comprises a notch signaling donor such as OP9, OP9delta and/or OP9Jagged1 cells. 
     
     
         22 . The method of  claim 19  wherein, the notch agonist comprises a notch ligand selected from notch ligand delta, Jagged-1 Jagged1 peptide, or Pref-1/DLK-1/FA1. 
     
     
         23 . The method of  claim 19  wherein the method comprises contacting a cell population comprising cholangiocyte progenitors with a notch agonist such as a notch signaling donor such as OP9, OP9delta and/or OP9Jagged1 cells, optionally in the presence of EGF, TGFbeta1, HGF and EGF, and/or HGF, TGFbeta1 and EGF for at least or about 5 to about 14 days, to induce further lineage specification and/or maturation of the cholangiocyte progenitors into cholangiocytes, optionally functional cholangiocytes. 
     
     
         24 . The method of  claim 1 , wherein the population of cholangiocytes produced is a population of functional cholangiocytes optionally wherein 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, optionally wherein at least 40%, 50%, 60%, 70%, 80% or 90% of the population of cholangiocytes are CK19+ cholangiocytes and/or CFTR+ cholangiocytes. 
     
     
         25 . The method of  claim 1 , wherein 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). The pluripotent stem cells are optionally human ESCs (hESCs) or human iPSCs (hiPSCs). 
     
     
         26 . The method of  claim 19 , wherein inducing maturation, and optionally further lineage specification and/or expansion comprises co-culturing the cell population comprising cholangiocyte progenitors with a notch signalling donor such as OP9, OP9delta and/or OP9Jagged1 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, to induce the maturation of at least one hepatic progenitor into a functional cholangiocyte. 
     
     
         27 . A method for generating a functional hepatocyte and/or cholangiocyte comprising:
 a. producing a population of cells comprising hepatoblasts, optionally day 25 stage hepatoblasts; hepatocytes and/or cholangiocytes, according to the method of  claim 1 ; and   b.
 i. co-culturing the population of cells, optionally wherein the population of cells comprises hepatoblasts, with:
 1. endothelial cells, optionally CD34+ endothelial cells, optionally embryonic CD34+ endothelial cells, optionally cultured in chimeric aggregates optionally wherein the aggregates are embedded in a gel/matrix to produce a population of cells comprising functional hepatocytes, or 
 2. a notch agonist, optionally notch signaling donor cells, optionally OP9 cells, OP9-Jagged 1 cells and/or OP9delta1 cells, optionally in chimeric aggregates, optionally wherein the aggregates are embedded in a gel/matrix and cultured with EGF, TGFb1, EGF and HGF, or EGF, TGFb1 and HGF to produce a population of cells comprising functional cholangiocytes, and/or 
 
 ii. introducing the population of cells from a) or b i), or a functional hepatocyte and/or cholangiocyte enriched or isolated population therefrom into a subject. 
   
     
     
         28 . The method of  claim 27  wherein the population of cells is co-cultured with: i) endothelial cells for at least about 4 days, about 6 days, about 8 days, about 10 days, about 12 days or more days, up to for example 15 days, 20 days, 30 days, 40 days, 50 days, 60 days or 90 days, or ii) a notch agonist, optionally OP9 cells, OP9-Jagged 1 cells and/or OP9delta1 cells for at least about 4 days, about 6 days, about 8 days, about 10 days, about 12 days or more days, up to for example about 90 days or about 60 days. 
     
     
         29 . The method of  claim 1  wherein the aggregates are embedded in a gel/matrix and 3D cultured. 
     
     
         30 . The method of  claim 1  further comprising enriching or isolating a hepatocyte and/or cholangiocyte lineage population of cells, optionally a functional hepatocyte population of cells. 
     
     
         31 . The method of  claim 30  comprising at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or up to about 95% hepatocytes and/or cholangiocytes, optionally functional hepatocytes. 
     
     
         32 . A population of cells produced according to  claim 1 . 
     
     
         33 . Use of the population of cells of  claim 32  for drug discovery, drug metabolism analysis, the development of bioartificial liver devices and/or as cell replacement therapy for the treatment of liver and/or biliary disease. 
     
     
         34 . A kit comprising 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 or for use in a method described herein, and optionally instructions for use according to a method herein.