Isolation and culture of bovine embryonic stem cells.

Isolation and culture of primary embryonic stem (ES) cell colonies are the first critical step towards establishment of stable ES cell lines. Here we introduce a novel method designated as "Separate and Seed" that contributes remarkably to efficient derivation of bovine primary ES cell colonies from blastocysts. The bovine ES cell colonies can self-renew to passage 10 with the growth factors bFGF and BIO. The bovine ES cells exhibit morphology typical of ES cells and express pluripotent molecular markers including Oct4, Nanog, SSEA1, SSEA4, and alkaline phosphatase (AP). These pluripotent markers may be used for the characterization of authentic bovine ES cell lines. Although continued efforts are required for improving long-term culture of bovine ES cells, this novel "Separate and Seed" method plus the growth factors bFGF and BIO provides an initial effective step that may eventually lead to the derivation of authentic bovine ES cells.