Methods of culturing embryonic stem cells and controlled differentiation

Abstract

The present invention provides a preparation of undifferentiated embryonic stem (ES) cells sustainable for a prolonged period in an undifferentiated state which will undergo stem cell renewal or somatic differentiation. Preferably the cells are capable of somatic differentiation in vitro and are inclined to differentiate away from an extraembryonic lineage. The present invention also provides method of culturing embryonic stem (ES) cells to improve stem cell maintenance and persistence in culture. The method also provides a culture of ES cells prepared by the method as well as differentiated cells derived from the embryonic cells resulting from directed differentiation procedures provided by the present invention.

1 . A preparation of undifferentiated embryonic stem (ES) cells maintained in an undifferentiated state and wherein said cells will undergo stem cell renewal or somatic differentiation.  
   
   
       2 . The preparation according to  claim 1  wherein said undifferentiated ES cells will differentiate into a somatic lineage upon introduction of a differentiating signal.  
   
   
       3 . A method of culturing undifferentiated ES cells, said method including: 
 obtaining a source of undifferentiated ES cells; and    culturing the ES cells in the presence of an indirect or direct antagonist of a BMP-mediated default pathway of extraembryonic endoderm differentiation.    
   
   
       4 . A method of producing a progenitor cell from an ES cell, said method comprising: 
 obtaining a source of undifferentiated ES cells; and    culturing the ES cells in the presence of an antagonist of a BMP mediated default pathway of extra embryonic endoderm differentiation for a period sufficient to differentiate the ES cell to a progenitor cell.    
   
   
       5 . The method according to  claim 4  wherein the source of undifferentiated ES cells is selected from the group including an embryo, a blastocyst, or a culture of undifferentiated orientated stem cells.  
   
   
       6 . The method according to  claim 5  wherein the ES cells are cultured in the presence of an indirect or direct antagonist of a BMP-2 mediated default pathway of extraembryonic endoderm differentiation.  
   
   
       7 . The method according to  claim 6  wherein the antagonist is a direct antagonist of the BMP-2 mediated default pathway selected from the group consisting of fetuin, noggin, chordin, gremlin, follistatin, cerberus, amnionless, DAN, or the ectodomain of BMPR1A (a BMP receptor protein) or ligand binding domains from other BMP receptors.  
   
   
       8 - 12 . (canceled)  
   
   
       13 . An undifferentiated ES cell prepared by a method according to  claim 3 .  
   
   
       14 . A progenitor cell prepared by the method according to  claim 4 .  
   
   
       15 . The undifferentiated ES cell or a progenitor cell according to any one of claims  1 ,  13  or  14  characterised by being unreactive with any one of the antibodies including PHM4 recognising MHC Class 1 surface molecules, anti-desmin, UJ13A reactive with polysialylated N-CAM, Cam 5.2 reactive with low molecular weight cytokeratins, AMF reactive with vimentin intermediate filaments, antibody to 160 kDa neurofilament protein, GCTM-2 reactive with a proteoglycan present on the surface of ES cells, TG42.1 reactive with a 25 kDa protein which copurifies with the proteoglycan recognised by GCTM-2 and is found on stem cells and other cell types, monoclonal antibody GCTM-5 reactive with an unknown molecule present on a small proportion of cells in spontaneously differentiating human EC cell cultures.  
   
   
       16 . The method according to  claim 6  wherein the antagonist is an indirect antagonist of the BMP-2 mediated default pathway selected from the group consisting of insulin, insulin analogue, or a cell derived insulin or insulin analogue-induced-factor.  
   
   
       17 . The method according to  claim 16  said method including: 
 obtaining a source of undifferentiated ES cells; and    culturing the ES cells in the presence of insulin or an insulin analogue.    
   
   
       18 . The method according to  claim 17  further including culturing the embryonic tem cells in the presence of a fibroblast feeder layer.  
   
   
       19 . The method according to  claim 18  including the steps of: 
 obtaining a source of undifferentiated ES cells;    culturing the ES cells on a fibroblast feeder cell layer; and    subjecting the cultured ES cells on the fibroblast feeder cell layer to an effective amount of insulin or an insulin analogue.    
   
   
       20 . The method according to  claim 17  wherein the insulin or insulin analogue is present in the range of 10 ng/ml to 10 μg/ml.  
   
   
       21 . The method according to  claim 16 , said method including: 
 obtaining a source of undifferentiated ES cells; and    culturing the ES cells in the presence of a cell derived insulin or insulin analogue induced factor.    
   
   
       22 . The method according to  claim 21  wherein the cell derived insulin or insulin analogue induced factor is derived from conditioned medium of another culture of ES cells exposed to insulin or insulin analogue.  
   
   
       23 . The method according to  claim 22  wherein the other culture of ES cells is exposed to insulin in the range of 10 ng/ml to 10 μg/ml.  
   
   
       24 . An undifferentiated ES cell or a progenitor cell prepared by the method according to any one of  claims 16  to  23 .  
   
   
       25 . A method of culturing undifferentiated ES cells, said method including: 
 obtaining a source of undifferentiated ES cells; and    culturing the ES cells in the presence of an indirect and direct antagonist of a BMP-mediated default pathway of extraembryonic endoderm differentiation.    
   
   
       26 . The method according to  claim 25  wherein the BMP mediated default pathway is a BMP-2 mediated default pathway.  
   
   
       27 . The method according to  claim 25  wherein the culturing of the ES cell in the presence of the direct antagonist of the BMP-2 mediated default pathway selected from the group consisting of fetuin, noggin, chordin, gremlin, follistatin, cerberus, amnionless, DAN, or the ectodomain of BMPR1A (a BMP receptor protein) or ligand binding domains from other BMP receptors.  
   
   
       28 . The method according to  claim 25  wherein the culturing of the ES cell in the presence of the indirect antagonist of the BMP-2 mediated default pathway selected from the group consisting of insulin, insulin analogue, or a cell derived insulin or insulin analogue-induced-factor.  
   
   
       29 . An undifferentiated ES cell or a progenitor cell prepared by the method according to any one of  claims 25  to  28 .  
   
   
       30 . A method of producing a somatic cell from an undifferentiated ES cell, said method including: 
 obtaining a progenitor cell comprising obtaining a source of undifferentiated ES cells;    culturing the ES cells in the presence of an antagonist of a BMP mediated default pathway of extra embryonic endoderm differentiation for a period sufficient to differentiate the ES cell to a progenitor cell;    obtaining a progenitor culture medium;    culturing the progenitor cell in the progenitor culture medium; and    obtaining a somatic cell from a lineage of the progenitor.    
   
   
       31 . The method according to  claim 30  wherein the progenitor culture medium is derived from a culture of neural progenitors which can differentiate into neural or glial cells.  
   
   
       32 . A somatic cell prepared by the method according to  claim 30 .  
   
   
       33 . A neural cell prepared by the method according to  claim 31   
   
   
       34 . A method according to  claim 30  wherein the ES cells are cultured in the absense of a feeder cell layer.  
   
   
       35 . A glial cell prepared by the method according to  claim 34 .  
   
   
       36 . A cell derived insulin or insulin analogue induced factor capable of maintaining ES cells in an undifferentiated state but capable of differentiation into a somatic lineage said factor derived from ES cells to insulin or insulin analogue.  
   
   
       37 . The factor according to  claim 36  wherein the insulin or insulin analogue is exposed to the ES cells in the range of 10 ng/ml to 10 μg/ml for a period of at least 5 days.  
   
   
       38 - 44 . (canceled)