Method of obtaining chicken embryonic stem cells

Abstract

The present invention concerns a method of culturing embryonic stem (ES) cells of avian, comprising the steps of: a) suspending ES cells originating from the blastoderm disk of fertilized un-incubated avian egg(s) in a basal culture medium supplemented with: insulin-like growth factor-1 (IGF-1) and ciliary neurotrophic factor (CNTF); and animal serum; and optionally, at least one growth factors selected in the group comprising interleukin 6 (II-6), interleukin 6 receptor (II-6R), stem cell factor (SCF), fibroblast growth factor (FGF), leukaemia inhibitory factor (LIF), interleukin 11 (II-11), oncostatin and cardiotrophin; b) seeding the suspension of ES cells obtained in step a) on a layer of feeder cells and further culturing the ES cells for at least between 2 to 10 passages; c) optionally removing at least one growth factors selected SCF, FGF, II-6, II-6R, LIF, oncostatin and cardiotrophin and II-11 from the culture medium; d) further culturing the ES cells in the medium of step c) on a layer of feeder cells.

1 . A method of culturing embryonic stem (ES) cells of avian, comprising the steps of:
 a) suspending ES cells originating from the blastoderm disk of fertilized un-incubated avian egg(s) in a basal culture medium supplemented with:
 insulin-like growth factor-1 (IGF-1) and ciliary neurotrophic factor (CNTF); and 
 animal serum; and 
 optionally, at least one growth factors selected in the group comprising interleukin 6 (Il-6), interleukin 6 receptor (Il-6R), stem cell factor (SCF), fibroblast growth factor (FGF), leukaemia inhibitory factor (LIF), interleukin 11 (Il-11), oncostatin and cardiotrophin; 
   b) seeding the suspension of ES cells obtained in step a) on a layer of feeder cells and further culturing the ES cells for at least between 2 to 10 passages;   c) optionally removing at least one growth factors selected SCF, FGF, Il-6, Il-6R, LIF, oncostatin and cardiotrophin and Il-11 from the culture medium;   d) further culturing the ES cells in the medium of step c) on a layer of feeder cells.   
     
     
         2 . The method of culturing embryonic stem (ES) cells of avian according to  claim 1 , comprising the steps of:
 a) suspending ES cells originating from the blastoderm disk of fertilized un-incubated avian egg(s) in a basal culture medium supplemented with insulin-like growth factor-1 (IGF-1), ciliary neurotrophic factor (CNTF), interleukin 6 (Il-6), interleukin 6 receptor (Il-6R), stem cell factor (SCF) and fibroblast growth factor (FGF) and animal serum;   b) seeding the suspension of ES cells obtained in step a) on a layer of feeder cells and further culturing the ES cells for at least between 2 to 10 passages;   c) optionally removing at least one growth factor selected from the group comprising SCF, FGF, Il-6 and Il-6R from the culture medium;   d) further culturing the ES cells in the medium of step c) on a layer of feeder cells.   
     
     
         3 . The method of culturing embryonic stem (ES) cells of avian according to  claim 1 , comprising the steps of:
 a) suspending ES cells originating from the blastoderm disk of fertilized un-incubated avian egg(s) in a basal culture medium supplemented with insulin-like growth factor-1 (IGF-1), ciliary neurotrophic factor (CNTF), interleukin 6 (Il-6), interleukin 6 receptor (Il-6R), stem cell factor (SCF), fibroblast growth factor (FGF) and animal serum;   b) seeding the suspension of ES cells obtained in step a) on a layer of feeder cells and further culturing the ES cells for at least between 2 to 10 passages;   c) optionally removing at least one growth factor selected from the group comprising SCF and FGF from the culture medium;   d) further culturing the ES cells in the medium of step c) on a layer of feeder cells.   
     
     
         4 . The method according to  claim 1  wherein the avian is a chicken. 
     
     
         5 . The method according to  claim 1  wherein the avian is a duck. 
     
     
         6 - 33 . (canceled) 
     
     
         34 . A culture medium for avian embryonic stem cells comprising at least insulin-like growth factor-1 (IGF-1), ciliary neurotrophic factor (CNTF) and optionally at least one compound selected in the group comprising interleukin 6 (Il-6), interleukin 6 receptor (Il-6R), stem cell factor (SCF), fibroblast growth factor (FGF) wherein said medium is sufficient for the maintenance of said avian embryonic stem cells into culture for at least 7 days, preferably for at least 100 days. 
     
     
         35 . The culture medium of  claim 34  further comprising a layer of feeder cells. 
     
     
         36 - 39 . (canceled)