Method for inducing human cholangiocyte differentiation

Abstract

The invention relates to a method for inducing human cholangiocyte differentiation of progenitor cells called hepatoblasts. More specifically, the invention relates to a method for differentiating hepatoblasts to cholangiocytes by culturing said hepatoblasts with a particular medium having interleukin-6 (IL-6) activity. The differentiation method can specifically induce cholangiocyte dilferentiation from hepatoblasts, and the human cholangiocytes differentiated according to the invention may be useful for drug discovery for treatment of cholangiopathies and bioengineered livers.

1 - 11 . (canceled) 
     
     
         12 . A population of human cholangiocytes produced by a method of
 (i) providing a population of human hepatoblasts (hHB); and   (ii) culturing the population of hHB in at least one cholangiocyte induction medium to specifically induce cholangiocyte differentiation and produce a population of cholangiocytes, wherein the step (ii) comprises the steps of:   (a) culturing the population of hHB in a first cholangiocyte induction medium, wherein said first cholangiocyte induction medium is a chemically defined medium (CDM) comprising both growth hormone (GH) and epidemal growth factor (EGF), without human interleukin-6 or a variant thereof, and without sodium taurocholate hydrate or sodium butyrate;   (b) further culturing the population of hHB produced in step (a) in a second cholangiocyte induction medium according to step (ii), wherein said second cholangiocyte induction medium is the CDM of step (ii) to which is added human interleukin-6 (IL-6) or a variant thereof having at least 90% amino acid identity to the human IL-6 and at least 90% of the activity of the human IL-6; and   (c) further culturing the population of hHB produced in step (b) in a third cholangiocyte induction medium, wherein said third cholangiocyte induction medium is a CDM of step (a) to which is added sodium taurocholate hydrate and optionally sodium butyrate,   wherein steps (a), (b) and (c) are performed sequentially (a) to (c),   wherein at least 60% of differentiated cells produced in step (ii) are cholangiocytes, and   wherein said population of cholangiocytes displays a normal phenotype.   
     
     
         13 . (canceled) 
     
     
         14 . A method of treating a patient with a cholangiopathy, comprising
 providing a population of human cholangiocytes produced by a method of
 (i) providing a population of human hepatoblasts (hHB); and 
 (ii) culturing the population of hHB in at least one cholangiocyte induction medium to specifically induce cholangiocyte differentiation and produce a population of cholangiocytes, wherein the step (ii) comprises the steps of: 
 (a) culturing the population of hHB in a first cholangiocyte induction medium, wherein said first cholangiocyte induction medium is a chemically defined medium (CDM) comprising both growth hormone (GH) and epidemal growth factor (EGF), without human interleukin-6 or a variant thereof, and without sodium taurocholate hydrate or sodium butyrate; 
 (b) further culturing the population of hHB produced in step (a) in a second cholangiocyte induction medium according to step (ii), wherein said second cholangiocyte induction medium is the CDM of step (ii) to which is added human interleukin-6 (IL-6) or a variant thereof having at least 90% amino acid identity to the human IL-6 and at least 90% of the activity of the human IL-6; and 
 (c) further culturing the population of hHB produced in step (b) in a third cholangiocyte induction medium, wherein said third cholangiocyte induction medium is a CDM of step (a) to which is added sodium taurocholate hydrate and optionally sodium butyrate, 
 wherein steps (a), (b) and (c) are performed sequentially (a) to (c), 
 wherein at least 60% of differentiated cells produced in step (ii) are cholangiocytes, and 
 wherein said cholangiocytes display a normal phenotype, and 
   administering to the patient a pharmaceutically effective amount of the cholangiocytes.   
     
     
         15 . A method of screening for a compound useful in the treatment of a cholangiopathy comprising the steps of:
 providing a population of human cholangiocytes produced by a method of
 (i) providing a population of human hepatoblasts (hHB); and 
 (ii) culturing the population of hHB in at least one cholangiocyte induction medium to specifically induce cholangiocyte differentiation and produce a population of cholangiocytes, wherein the step (ii) comprises the steps of: 
 (a) culturing the population of hHB in a first cholangiocyte induction medium, wherein said first cholangiocyte induction medium is a chemically defined medium (CDM) comprising both growth hormone (GH) and epidemal growth factor (EGF), without human interleukin-6 or a variant thereof, and without sodium taurocholate hydrate or sodium butyrate; 
 (b) further culturing the population of hHB produced in step (a) in a second cholangiocyte induction medium according to step (ii), wherein said second cholangiocyte induction medium is the CDM of step (ii) to which is added human interleukin-6 (IL-6) or a variant thereof having at least 90% amino acid identity to the human IL-6 and at least 90% of the activity of the human IL-6; and 
 (c) further culturing the population of hHB produced in step (b) in a third cholangiocyte induction medium, wherein said third cholangiocyte induction medium is a CDM of step (a) to which is added sodium taurocholate hydrate and optionally sodium butyrate, 
 wherein steps (a), (b) and (c) are performed sequentially (a) to (c), 
 wherein at least 60% of differentiated cells produced in step (ii) are cholangiocytes, and 
 wherein said cholangiocytes display a normal phenotype, 
   
       contacting the cholangiocytes produced with a test compound, and 
       determining the effect of the test compound on said cholangiocytes. 
     
     
         16 . The method of  claim 15 , wherein the chemically defined medium (CDM) comprises human IL-6. 
     
     
         17 . The method of  claim 16 , wherein the human IL-6 has an amino acid sequence as shown in Uniprot Accession number P05231 for a full length human IL-6 protein. 
     
     
         18 . The method of  claim 15 , wherein the variant has 100% of the activity of human IL-6. 
     
     
         19 . The method of  claim 15 , wherein at least 70% of differentiated cells are cholangiocytes. 
     
     
         20 . The method of  claim 15 , wherein at least 80% of differentiated cells are cholangiocytes. 
     
     
         21 . The method of  claim 14 , wherein in the providing step the chemically defined medium (CDM) comprises human IL-6. 
     
     
         22 . The method of  claim 21 , wherein the human IL-6 has an amino acid sequence as shown in Uniprot Accession number P05231 for a full length human IL-6 protein. 
     
     
         23 . The method of  claim 14 , wherein in the providing step the variant has 100% of the activity of human IL-6. 
     
     
         24 . The method of  claim 14 , wherein in the providing step at least 70% of differentiated cells are cholangiocytes. 
     
     
         25 . The method of  claim 14 , wherein in the providing step at least 80% of differentiated cells are cholangiocytes.