Maturation of mammalian hepatocytes

Abstract

Directed differentiation and maturation of mammalian hepatocytes, such as human hepatocytes. The hepatocyte obtained show a phenotype which is more similar to that of primary hepatocytes than previously shown. In particular, exposure of mammalian hepatocytes, such as human hepatocytes, to at least one maturation factor selected from the group consisting of Src kinase inhibitors, vitamin D including precursors, metabolites and analogs thereof, hypoxia inducing compounds, sphingosine and sphingosine derivatives, activators of peroxisome proliferator-activated receptors (PPARs), platelet-activating factor (PAF), PKC inhibitors, and combinations thereof.

1 . A method for producing mammalian hepatocytes, the method comprising:
 culturing mammalian hepatic progenitor cells under differentiation conditions to obtain hepatocytes, and   exposing said hepatocytes to at least one hypoxia inducing compound selected from the group consisting of RAR-related orphan receptor alpha (ROR-alpha) ligands, CoCl2, and NaN3, optionally in combination with at least one maturation factor selected from the group consisting of Src kinase inhibitor, vitamin D including precursors, metabolites and analogs thereof, sphingosine and sphingosine derivatives, activators of peroxisome proliferator-activated receptors (PPARs), platelet-activating factor (PAF), and PKC inhibitors.   
     
     
         2 . The method according to  claim 1 , further comprising initially culturing cells of the definitive endoderm (DE) under differentiation conditions to obtain said hepatic progenitor cells, or further comprising initially culturing mammalian pluripotent stem (PS) cells under differentiation conditions to obtain cells of the definitive endoderm (DE cells) and further culturing the obtained cells under differentiation conditions to obtain said hepatic progenitor cells. 
     
     
         3 . The method according to  claim 1 , wherein said mammalian cells are human cells. 
     
     
         4 . The method according to  claim 1 , wherein said mammalian hepatocytes are exposed to at least one RAR-related orphan receptor alpha (ROR-alpha) ligand. 
     
     
         5 . The method according to  claim 1 , wherein said mammalian hepatocytes are exposed to at least one RAR-related orphan receptor alpha (ROR-alpha) ligand selected from the group consisting of CGP52608, a CGP52608 analog, melatonin, melatonin analogs, cholesterol, cholesterol derivatives, and combinations thereof. 
     
     
         6 . The method according to  claim 1 , wherein said mammalian hepatocytes are exposed to CGP52608 or a CGP52608 analog. 
     
     
         7 . The method according to  claim 1 , wherein said mammalian hepatocytes are exposed to at least one Src kinase inhibitor, preferably selected from the group consisting of PP1, PP2, 1-NA PP1, 1-NM-PP1, Src Inhibitor-1 (Src-I1), Src Kinase Inhibitor I, Src Kinase Inhibitor II, A-419529, A-770041, AZM 475271, bosutinib, CGP77675, Damnacanthal, dasatinib, dasatinib monohydrate, ER 27319 maleate, Fingolimod (FTY720), Geldanamycin, Herbimycin A, KB SRC 4, KX2-391, KX1-004, Lavendustin A, Lavendustin C, LCK inhibitor 2, Lyn peptide inhibitor, MLR-1023, MNS, N-Acetyl-O-phosphono-Tyr-Glu Dipentylamide, N-Acetyl-O-phosphono-Tyr-Glu-Glu-Ile-Glu, NVP-BHG712, PD 166285, PD173952, PD 180970, Piceatannol, pp60 c-src, quercetin, radicicol from Diheterospora chlamydosporia solid, saracatinib, SU 6656, TC-S 7003, TG 100572, WH-4-023, ZM 306416, and combinations thereof; more preferably selected from PP1 and PP2. 
     
     
         8 . The method according to  claim 1 , wherein said mammalian hepatocytes are exposed to at least one vitamin D, vitamin D precursor, vitamin D metabolite or vitamin D analog. 
     
     
         9 . The method according to  claim 8 , wherein said mammalian hepatocytes are exposed to at least one vitamin D3, vitamin D3 precursor, vitamin D3 metabolite or vitamin D3 analog, preferably selected from the group consisting of cholecalciferol, calcifediol, calcitriol, and combinations thereof. 
     
     
         10 . The method according to  claim 1 , wherein said mammalian hepatocytes are exposed to at least one sphingosine, such as D-erythro-sphingosine, or sphingosine derivative, such as sphingosine-1-phosphate or sphingolipid. 
     
     
         11 . The method according to  claim 10 , wherein said sphingolipid is a ceramide or a ceramide analog, such as L-erythro MAPP or D-erythro MAPP. 
     
     
         12 . The method according to  claim 1 , wherein said mammalian hepatocytes are exposed to at least one activator of peroxisome proliferator-activated receptors (PPARs), preferably selected from the group consisting of thiazolidinediones, free fatty acids (FFAs), eicosanoids including eicosanoid precursors and eicosanoid analog, and combinations thereof. 
     
     
         13 . The method according to  claim 1 , wherein said mammalian hepatocytes are exposed to at least one platelet-activating factor (PAF). 
     
     
         14 . The method according to  claim 1 , wherein said mammalian hepatocytes are exposed to at least one protein kinase C (PKC) inhibitor.