Appeal No. 1995-1977
Application 07/669,403
Claims 1 and 8 are representative of the subject matter on appeal, and
read as follows:
1. Pluripotential embryonic stem cells isolated from an ungulate species
blastocysts of embryos that develop by way of an embryonic disc.
8. A method of selecting and growing pluripotential embryonic stem cells
according to claim 1, comprising the steps of:
growing blastocysts in tissue culture growth medium which includes both
heat-inactivated new born calf serum and heat-inactivated fetal calf serum;
disaggregated the blastocysts either after spontaneous hatching or after
mechanical removal of the zone pellucida;
growing stem cell colonies from 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 are capable of culture in a
tissue culture dish to exhibit the following morphological features:
a) they are round cells, tightly packed with large nuclei in relation
to cytoplasm, and fairly prominent nucleoli;
b) they grow in tightly adherent colonies, and as the colonies get
larger the cells tend to flatten out in the centre of the colony, with the colony
having an outer rim of cells of the form described in a), and
c) on trypsinization of the such a colony it may be seen that 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° show lobular pseudopodia.
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