Fertilization and Early Embryogenesis
(T320) DERIVATION, MAINTENANCE AND CHARACTERIZATION OF BOVINE ES-LIKE CELLS.
Vassiliev, Ivan1, 2, McConnell, Helen 1, 2, Verma, Paul1, 2, 1 Monash University, Clayton, Victoria, Australia2 CRC for Innovative Dairy Products, Melbourne, Victoria, Australia
ABSTRACT- The derivation and maintenance of ES cell lines from domestic species are important for a number of reasons. The most obvious are to facilitate manipulation of the genotype to produce animals for pharmaceutical or agricultural benefit, and to provide donor cells with an epigenetic profile similar to that of embryonic cells, aimed at improving cloning efficiency.Here we report on our attempts to obtain, maintain and characterize bovine ES-like cells. Three types of embryonal material from in vitro produced (IVP) blastocysts were investigated for isolation of bovine ES-like cells: 1) immunosurgically isolated ICM component of blastocysts, 2) blastocyst outgrowths from whole intact blastocysts and 3) blastocyst outgrowths developed from mechanically disrupted blastocysts. Embryonal outgrowths or parts thereof which demonstrated morphological criteria characteristic for ES cells were mechanically passaged onto fresh feeders. The resulting ES-like cell colonies were examined for presence of pluripotent markers using immunohistochemistry and RT-PCR. Bovine ES-like cells were successfully isolated and maintained in vitro for 6 passages. These cells retained morphology characteristic for bES cells: small cytoplasmic/nuclear ratio, nuclei with multiple nucleoli, and multiple lipid inclusions in cytoplasm. The colonies grew in monolayers, as islands of ES cells surrounded by trophectoderm (TE) cells. Immunohistochemical detection of SSEA-1 and SSEA-4 demonstrated expression of these markers in bovine ES-like cells but not in TE cells. Bovine ES cells and TE cells were microdissected and mRNAs were isolated for analysis. The expression of Oct-4, Rex-1 and SSEA-1 examined by RT-PCR using bovine specific primers was similarly detected in bES cells but not in TE cells. On spontaneous differentiation, these cells were able to form a variety of cell types including beating muscle cells, with the cells displaying a propensity to differentiate in a manner reminiscent of human ES cells. Further these bES cells were successfully transfected with a construct containing mouse Oct4-EGFP sequence, indicating that factors, regulating bovine Oct4 expression, could recognize mouse regulatory sequences of this gene.
KEY WORDS: bovive ES cells, bovine Oct-4, bovine Rex1, bovine SSEA-1