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We succeeded in the derivation and maintenance of pluripotent embryonic stem (ES) cells from equine and bovine blastocysts. These cells expressed markers that are characteristics of mouse ES cells, namely, alkaline phosphatase, stage-specific embryonic antigen 1, STAT 3 and Oct 4. We confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells as a source of nuclei for nuclear transfer and we generated cloned cattle with a higher frequency of pregnancies to term than has been achieved with somatic cells. On the other hand, we established human fetal membrane derived stem cell lines by the colonial cloning techniques using MEMalpha culture medium containing 10 ng/ml of EGF, 10 ng/ml of LIF and 10% fetal bovine serum (FBS). These cells appeared to maintain normal karyotype in vitro and expressed markers characteristics of stem cells. Furthermore, these cells contributed to the formation of chimeric murine embryoid bodies and gave rise to all three germ layers in vitro. Results from animal ES cells and human fetal membrane derived stem cells clearly demonstrate that these cells might be used for providing different types of cells for regenerative medicine as well as used for targeted genetic manipulation of the genome.

Mouse embryonic stem (ES) cells can be maintained indefinitely in the presence of leukemia inhibitory factor (LIF) and they express markers of self-renewal and pluripotency, which include the transcription factor Oct 4, STAT-3, stage-specific embryonic antigen (SSEA)-1, and alkaline phosphatase (AP). Upon removal of LIF, from the culture medium they cease to express markers such as Oct 4, rapidly losing the capacity for self-renewal and differentiating into a variety of cell types. Gene targeting is feasible in murine ES cells because these cells can be maintained in an undifferentiated state long enough to allow selection of properly targeted cell colonies with a high frequency of homologous recombination. Furthermore, blastocysts cloned from cultured murine ES cells develop to term at an efficiency (10-30%) that is three to ten times higher than blastocysts cloned from the nuclei of differentiated somatic cells. It seems likely that ES cells require less extensive reprogramming than do somatic cells, perhaps because in ES cells, many genes that are essential for early development are already active and thus do not require reactivation. Recently, we succeeded in isolating immortalized equine and bovine ES cells with a normal karyotype, that exhibit features similar to those of murine ES cells and express Oct 4, STAT-3, SSEA-1 and AP. We further confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells, as a source of nuclei for nuclear transfer (NT) and we generated cloned cattle with a higher frequency of pregnancies to term than has been achieved with differentiated somatic cells. Moreover, bovine ES cells that expressed enhanced green fluorescent protein (EGFP) were incorporated into both the inner cell mass (ICM) and the trophectdermal cells of developing blastocysts. These findings should facilitate targeted genetic manipulation of the genome and should allow production of cloned cattle in a single step after modification, as appropriate, of the genome.