Regionalised endoderm cells and uses thereof

Abstract

The present invention relates to the generation of anterior definitive endoderm (ADE) cells from embryonic stem cells and the differentiation of such cells to, for example, pancreatic or liver cells. The invention also relates to cell lines, cell culture methods, cell markers and the like and their potential uses in a variety of applications.

1 .- 31 . (canceled) 
     
     
         32 . A method of identifying anterior definitive endoderm (ADE) cells derived from a population of ES cells, comprising detecting two or more ADE cell markers in a cell population derived from said ES cell population, in order to identify said ADE cells. 
     
     
         33 . The method according to  claim 32 , wherein said two or more markers are selected from and group consisting of Sst, Pyy, Ghrl, Slc38a5, Chga, xph5, Tmprss2, Clic6, 1110003E01Rik, Sstr2, 4732456N10Rik, Rab15, Rnase4, Slc6a4, Fxyd3, Slc12a2, 1700027A23Rik, Tm4st2, 0610040B21Rik (txndc12), Apoa1, 2410021P16Rik (acad10), 4832420M10, Fzd5, Gckr, Enpp5, Syt5, Cklfsf8 (Cmtm8), Robo1, Ctsh, Rarres1, Sec1412, Ripk4, Slc7a8, Ng23, Krt2-7, Sfrp5, Pga5, foxa1, foxa2, foxa3, Cerl, Hhex, Otx2, and Gatn3. 
     
     
         34 . The method according to  claim 33 , wherein the cell markers are cell surface expressed. 
     
     
         35 . The method according to  claim 34 , wherein the cell surface markers are selected from the group consisting of Sic38a5, Tmprss2, Clic6, Sstr2, Sic6a4, Fxyd3, Sic12a2, Tm4st2, Fzd5, Cklfsf8 (Cmtm8), Rarres1, Sic7a8, and Sfrp5. 
     
     
         36 . The method according to  claim 33 , further comprising detecting at least 3 of the makers. 
     
     
         37 . The method according to  claim 32 , further comprising obtaining a cell population comprising greater than 40% ADE cells. 
     
     
         38 . The method according to  claim 32 , further comprising differentiating said ADE cells in order to obtain differentiated cells in which greater than 40% of the cells differentiated in the cell culture are pancreatic cells. 
     
     
         39 . The method according to  claim 32 , further comprising differentiating said ADE cells in order to obtain differentiated cells in which greater than 10% are liver cells. 
     
     
         40 . The method according to  claim 38 , comprising purifying said differentiated cells such that the resulting differentiated cell population is essentially free of ES cells. 
     
     
         41 . A mixed cell population obtained directly from an ES cell culture, comprising at least 40% ADE cells. 
     
     
         42 . A mammalian ES cell line comprising a nucleic acid construct comprising a marker gene which has been inserted within an ADE specific gene locus of the genome such that the marker gene is under control of the ADE specific gene promoter. 
     
     
         43 . A method of reprogramming a somatic cell or somatic cell progenitor in order to generate an ADE cell, comprising:
 i) providing one or more ADE cell markers;   ii) providing a somatic cell or somatic cell progenitor; and   iii) transferring the ADE marker(s) or genes encoding said markers into the cell or cell progenitor, thereby reprogramming the cell or cell progenitor.   
     
     
         44 . A method of differentiating ES cells to ADE cells, comprising:
 (i) culturing the ES cells in a first culture medium, optionally on a feeder, in (a) the presence of serum or an extract thereof or (b) in a serum free or serum extract free medium;   (ii) replacing the first culture medium with a second serum free medium comprising activin or removing any serum or extract thereof and any feeder present in the first culture medium and adding activin; and   (iii) propagating the ES cells in the medium comprising activating, in order to obtain cells comprising ADE cells, with the proviso that the first and second medium are not SF03 medium.   
     
     
         45 . The method according to  claim 44 , wherein said first and/or second medium is N2B27. 
     
     
         46 . The method according to  claim 45 , wherein the first or second medium further comprises EGF and/or an agonist of a receptor of the TGF-β superfamily. 
     
     
         47 . The method according to  claim 45 , wherein the ES cells are grown in suspension. 
     
     
         48 . The method according to  claim 47 , wherein the first culture medium comprising serum is GMEM and the second medium comprising activin is N2B27. 
     
     
         49 . The method according to  claim 44 , wherein the initial cell density in first culture medium containing the serum or extract thereof is less than 1×10 5  cells/ml. 
     
     
         50 . The method according to  claim 44 , wherein the ES cells are grown in monolayer culture and the first medium comprises one or more of activin, Fgf4, Bmp4 and CHIR99021, and wherein the second medium optionally comprises EGF and/Fgf4. 
     
     
         51 . The method according to  claim 50 , wherein the second medium is SF03. 
     
     
         52 . The method according to  claim 44 , wherein the second medium comprises Bmp4 or EGF4. 
     
     
         53 . The method according to  claim 43 , wherein the one or more markers ADE cell markers is selected from the group consisting of Clic6, Sstr2, Rab15, Rnase4, Slc6a4, Fxyd3, Slc12a2, Tm4sf2, Apoa1, Fzd5, Gckr, Enpp5, Syt5, Cklfsf8, Robo1, Ctsh, Rarres1, Sec1412, Ripk4, Slc7a8, Ng23, Krt2-7m, Sfrp5, Pga5, Foxa1, 2, 3, Cerl, Hhex, Otx2, Gata3, Cish, Pik3cb, and Rfxdc1. 
     
     
         54 . The method according to  claim 53 , wherein the cell markers are cell surface expressed. 
     
     
         55 . The method according to  claim 54 , wherein the cell markers are selected from the group consisting of Sic38a5, Tmprss2, Clic6, Sstr2, Sic6a4, Fxyd3, Sic12a2, Tm4sf2, Fzd5, Cklfsf8 (Cmtm8), Rarres1, Sic7a8, and Sfrp5. 
     
     
         56 . The method according to  claim 53 , wherein at least 2 of the cell markers are used to generate the ADE cells. 
     
     
         57 . A method of treating a disease of the liver, pancreas, lung,  thymus , thyroid or parathyroid, comprising administering ADE cells prepared according to  claim 43  or  claim 44  to a subject in need thereof. 
     
     
         58 . The cell population according to  claim 57 , wherein the ADE cells are derived from humans, primates, rodents or birds. 
     
     
         59 . The cell population according to  claim 57 , wherein the ADE cells are derived from mammals, especially mice, rats and humans. 
     
     
         60 . The cell population according to  claim 57 , wherein the ES cells from which the ADE cells are derived are either wild-type or genetically modified ES cells.