Pdx1-expressing dorsal and ventral foregut endoderm

Abstract

Disclosed herein are cell cultures comprising dorsal and/or ventral PDX1-positive foregut endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified dorsal and/or ventral PDX1-positive foregut endoderm cells as well as methods for enriching, isolating and purifying dorsal and/or ventral PDX1-positive foregut endoderm cells from other cell types. Methods of identifying differentiation factors capable of promoting the differentiation of dorsal and/or ventral PDX1-positive foregut endoderm cells, are also disclosed.

1 . A cell culture comprising human cells wherein at least 10% of said human cells are pancreatic-duodenal homoebox factor-1 (PDX1) positive, endoderm cells that express at least one marker selected from the group consisting of SOX9, HNF6, and PROX1, and which express the PDX1 protein. 
     
     
         2 . The cell culture of  claim 1 , wherein at least 40% of said human cells are PDX1-positive endoderm cells. 
     
     
         3 . The cell culture of  claim 1 , wherein at least 50% of said human cells are PDX1-positive endoderm cells. 
     
     
         4 . The cell culture of  claim 1 , wherein at least 60% of said human cells are PDX1-positive endoderm cells. 
     
     
         5 . The cell culture of  claim 1 , wherein at least 75% of said human cells are PDX1-positive endoderm cells. 
     
     
         6 . The cell culture of  claim 1 , wherein human feeder cells are present in said culture, and wherein at least 10% of human cells other than said human feeder cells are PDX1-positive endoderm cells. 
     
     
         7 . The cell culture of  claim 1 , wherein the expression of PDX1 is greater than the expression of a marker selected from the group consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in said PDX1-positive endoderm cells, wherein expression is determined by quantitative PCR. 
     
     
         8 . The cell culture of  claim 1 , wherein said cell culture is substantially free of cells selected from the group consisting of visceral endodermal cells, parietal endodermal cells and neural cells. 
     
     
         9 . The cell culture of  claim 1  further comprising a retinoid. 
     
     
         10 . The cell culture of  claim 9 , wherein said retinoid is retinoic acid (RA). 
     
     
         11 . The cell culture of  claim 10  further comprising B27. 
     
     
         12 . The cell culture of  claim 1 , wherein said human cells are non-recombinant. 
     
     
         13 . The cell culture of  claim 1 , wherein said endoderm cells are multipotent cells that can further differentiate to cells derived from the foregut. 
     
     
         14 . A cell population comprising cells wherein at least 90% of said cells are human PDX1-positive, endoderm cells that express at least one marker selected from the group consisting of SOX9, HNF6, and PROX1, and which express the PDX1 protein. 
     
     
         15 . The cell population of  claim 14 , wherein at least 98% of said cells are PDX1-positive endoderm cells. 
     
     
         16 . The cell population of  claim 14 , wherein the expression of PDX1 is greater than the expression of a marker selected from the group consisting of AFP, SOX7, SOX1, ZIC1 and NFM in said PDX1-positive endoderm cells, wherein expression is determined by quantitative PCR. 
     
     
         17 . The cell population of  claim 14 , wherein said human cells are non-recombinant. 
     
     
         18 . The cell population of  claim 14 , wherein said endoderm cells are multipotent cells that can further differentiate to cells derived from the foregut. 
     
     
         19 . A method of producing PDX1-positive endoderm cells, said method comprising:
 (a) culturing a population of pluripotent human cells in the presence of exogenous TGFβ superfamily growth factor;   (b) culturing the cells from step (a) in the presence of an exogenous FGF-family growth factor; and   (c) culturing the cells from step (b) in the presence of a retinoid, thereby producing multipotent PDX1-positive endoderm cells.   
     
     
         20 . The method of  claim 19 , wherein said TGFβ superfamily growth factor comprises activin A. 
     
     
         21 . The method of  claim 19 , wherein the FGF-family growth factor comprises FGF-7. 
     
     
         22 . The method of  claim 19 , wherein said retinoid is retinoic acid (RA). 
     
     
         23 . The method of  claim 22 , wherein RA is provided in a concentration ranging from 0.01 μM to about 50 μM. 
     
     
         24 . The method of  claim 19 , wherein at least 25% of said cell population expresses PDX1. 
     
     
         25 . A method of screening growth factors capable of promoting the differentiation of PDX1-negative definitive endoderm cells to PDX1-positive definitive endoderm cells, said method comprising:
 (a) culturing a population of PDX1-negative definitive endoderm cells;   (b) determining the expression of PDX1 in said population;   (c) providing one or more candidate growth factors to said population;   (d) determining the expression of PDX1 in said population after addition of the one or more candidate growth factors;   (e) comparing expression of PDX1 at step (b) with expression of PDX1 at step (d);   (f) evaluating whether expression of PDX1 at step (d) is increased as compared to expression of PDX1 at step (b) thereby determining that the candidate differentiation factor is capable of promoting the differentiation of PDX1-negative definitive endoderm cells to PDX1-positive definitive endoderm cells.