Derivation of pluripotential embryonic cell lines from domestic animals

Abstract

A method of selecting and growing pluripotential embryonic stem cells isolated from an ungulate species blastocysts of embryos that develop by way of an embryonic disc is disclosed. The method comprises growing blastocysts in tissue culture growth medium which includes both heat-inactivated new born calf serum and heat-inactivated fetal calf serum; disaggregating the blastocysts either after spontaneous hatching or after mechanical removal of the zone pellucida; growing stem cell colonies from the disaggregated cells in issue culture growth medium; selecting stem cell colonies by morphological characteristics; and growing the selected stem cells in tissue culture growth medium. The cells are round cells, tightly packed with large nuclei in relation to cytoplasm, and fairly prominent nucleoli. They grow in tightly adherent coloedes and as the colonies get larger the cells tend to flatten out in the center of the colony. The outer, less flattened cells of a larger colony or all the cells of a smaller colony without central flattening are readily disaggregated by trypsinization into small spherical cells which have a bright phase contrast appearance, and if observed after a short time of incubation at 37 C. they show lobular pseudopodia

1 . Pluripotential embryonic stem cells isolated from in vitro treatment of non-human and non-rodent blastocysts.  
     
     
         2 . Stem cells according to  claim 1  from ungulate species.  
     
     
         3 . Stem cells according to  claim 1  from bovine species.  
     
     
         4 . Stem cells according to  claim 1  from porcine species.  
     
     
         5 . A blastocyst to which has been introduced one or more stem cells according to any of  claims 1  to  4 .  
     
     
         6 . An embryonic cell to which has been introduced by nuclear transfer a nucleus of a stem cell according to any of  claims 1  to  4 .  
     
     
         7 . A chimeric animal which is the progeny of a blastocyst according to  claim 5  or an embryonic cell according to  claim 6 .  
     
     
         8 . A method of obtaining pluripotential embryonic stem cells according to any of  claims 1  to  4 , comprising growing blastocysts in tissue culture growth medium which includes both new born calf serum and foetal calf serum (both sera having been heat inactivated before use); causing disaggregation of the blastocysts either after spontaneous hatching or after mechanical removal of the zona pellucida; growing the disaggregated cells in tissue culture growth medium; selecting stem cell colonies by morphological characteristics; and growing the selected stem cells in tissue culture growth medium; wherein the morphologically selected cells grow in distinctive flat polarised epithelial colonies which tend to spread to form monolayers and the cells are round, have relatively large clear nuclei, have prominent nucleoli and relatively little cytoplasm.  
     
     
         9 . A method according to  claim 8 , further including passaging the selected stem cells by trypsinisation onto fresh tissue culture growth medium at intervals to prevent differentiation of the cells and to maintain a cell line in culture.  
     
     
         10 . A method which comprises introducing into a blastocyst one or rare stem cells made according to the method of  claim 8 .  
     
     
         11 . A method which comprises introducing by nuclear transfer into an embryonic cell a nucleus of a stem cell made according to the method of  claim 8 .  
     
     
         12 . A method which comprises introduction to the uterus of a pseudo-pregnant foster mother animal, a viable embryo obtained using a technique involving any of the methods of  claim 8  to  11 , so as to produce progeny in the form of a chimeric animal.