In vitro propagation of embryonic stem cells

Abstract

The present invention relates to the use of leukaemia inhibitory factor (LIF) in the isolation and propagation of embryonic stem cells in vitro.

1 . A method for the isolation of embryonic stem (ES) cells from animal embryos in vitro which method comprises deriving and maintaining said embryos in culture medium containing an effective amount of leukaemia inhibitory factor (LIF) for a time and under conditions sufficient for the development of said ES cells.  
     
     
         2 . The method according to  claim 1  wherein the culture medium is free of feeder cells.  
     
     
         3 . The method according to claims  1  or  2  wherein the animal embryos are derived from humans, mice, birds, sheep, pigs, cattle, goats or fish.  
     
     
         4 . The method according to  claim 3  wherein the animal embryos are derived from mice.  
     
     
         5 . The method according to claims  1  or  2  wherein the culture medium is Eagle's medium or modifications thereof or equivalents thereto.  
     
     
         6 . The method according to any one of the preceding claims wherein the LIF is recombinant LIF.  
     
     
         7 . The method according to  claim 6  wherein the LIF is recombinant human or murine LIF.  
     
     
         8 . The method according to  claim 7  wherein LIF is added to the culture medium at a concentration of from 10 to 1,000,000 units/ml.  
     
     
         9 . The method according to  claim 8  wherein the LIF is added to the culture medium at a concentration of from 100 to 100,000 units/ml.  
     
     
         10 . The method according to  claim 9  wherein the LIF is added to the culture medium at a concentration of from 500 to 10,000 units/ml.  
     
     
         11 . A method according to any one of the preceding claims wherein the effective time is from 1 day to 20 weeks.  
     
     
         12 . The method according to  claim 11  wherein the effective time is from 1 to 8 weeks.  
     
     
         13 . A method for maintaining animal embryonic stem (ES) cells in vitro while retaining their pluripotential phenotype which process comprises culturing said cells in a culture medium containing an effective amount of leukaemia inhibitory factor (LIF) under conditions sufficient to maintain said cells.  
     
     
         14 . The method according to  claim 13  wherein the culture medium is free of feeder cells.  
     
     
         15 . The method according to  claim 13  or  14  wherein the animal ES cells are derived from humans, mice, birds, sheep, pigs, cattle, goats or fish.  
     
     
         16 . The method according to  claim 15  wherein the animal ES cells are derived from mice.  
     
     
         17 . The method according to  claim 13  or  14  wherein the culture medium comprises Eagle's medium or modifications thereof or equivalents thereto.  
     
     
         18 . The method according to any one of  claims 13  to  17  wherein the LIF is recombinant LIF.  
     
     
         19 . The method according to  claim 18  wherein the LIF is recombinant murine or human LIF.  
     
     
         20 . The method according to  claim 19  wherein the recombinant LIF is added to the culture medium at a concentration of from 10 to 1,000,000 units/ml.  
     
     
         21 . The method according to  claim 20  wherein the recombinant LIF is added to the culture medium at a concentration of from 100 to 100,000 units/ml.  
     
     
         22 . The method according to  claim 21  wherein LIF is added to the culture medium at a concentration of from 500 to 10,000 units/ml.  
     
     
         23 . Embryonic stem (ES) cells derived from animal embryos in vitro isolated by deriving and maintaining said embryos in culture medium, said culture medium containing an effective amount of leukaemia inhibitory factor (LIF) for a time and under conditions sufficient for the development of said ES cells.  
     
     
         24 . The ES cells according to  claim 23  wherein the culture medium is free of feeder cells.  
     
     
         25 . The ES cells according to  claim 23  or  24  derived from human, mouse, bird, sheep, pig, cattle, goat or fish embryos.  
     
     
         26 . The ES cells according to  claim 25  derived from mouse embryos.  
     
     
         27 . A chimaeric animal or transgenic progeny thereof generated using ES cells which have been isolated from animal embryos according to  claim 1 .  
     
     
         28 . A chimaeric animal or transgenic progeny thereof generated using animal ES cells which have been maintained in vitro according to the method of  claim 13 .  
     
     
         29 . The chimaeric animal or transgenic progeny thereof according to  claim 27  or  28  wherein said animal is a mouse.  
     
     
         30 . The chimaeric animal or transgenic progeny thereof according to claims  27  or  28  or  29  wherein the ES cells contain additional genetic material inserted therein.