|Rexroad Jr, Caird|
Submitted to: Journal of Molecular Reproduction and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 7, 1997
Publication Date: N/A
Interpretive Summary: Embryonic stem cells are powerful tools for understanding how genes regulate physiological processes. Stem cells for cows have been difficult to produce because the factors that cause the cells of embryos to become the tissues of fetuses are poorly understood. A technique was developed to study the cells of the early embryo in culture so that various factors that cause cells to become part of specific tissues, especially the germ line, can be studied in closely controlled conditions. The outcome is expected to be the production of embryonic stem cells which will be used by embryologists to study gene function and will be used by biotechnologists to produce genetically modified animals.
Technical Abstract: A culture system was devised to study the differentiation of isolated bovine blastomeres. Blastomeres from embryos produced by in vitro maturation, fertilization and culture of oocytes (IVMFC) from slaughterhouse ovaries were cultured in 24-well culture plate on feeder layers in medium BCM (blastomere culture medium: equal parts of tissue culture medium 199 and low glucose Dulbecco's Modified Eagle's medium (DMEM) ) with 10% fetal calf serum (FCS). Ovine embryonic fibroblasts (OEF) and STO cells were superior to bovine (BEF) and mouse embryonic fibroblasts (MEF) as mitotically inactivated feeder cells. Over 5 studies in which 4 blastomeres from an embryo were added to each culture well, an average of 1 colony per well was observed to form from the blastomeres. The colonies continued to grow throughout the 10 day culture period and most colonies resembled trophectoderm in their cellular characteristic, although some cultures resembled endoderm. When the number of blastomeres cultured in each well was varied over the range of 2 to 8 the number of colonies formed was proportional to the number of blastomeres added. Blastomeres from Day 5 and Day 6 embryos produced fewer colonies than did those from Day 4 embryos perhaps as a result of differentiation and tighter blastomere adhesion resulting in damage during separation of blastomeres. The absence of serum did not alter the number of colonies formed. A number of growth factors, including LIF, OM, PDGFa & FGF4, has no effect on colony formation & differentiation beyond that provided by the feeder layer or serum when present. Blastomeres were not culturable in the absence of feeder layers.