The combined application of the two procedures of embryonic stem cell isolation and the transfer of nuclei from such cells would provide a unique method for the dissemination of genetic improvement through European herds and a source of exceptional embryos for sale on world markets. It was the objective of the project to gain new knowledge that will aid in the development of new procedures for the isolation of cell lines and embryo manipulation, as much as to develop the procedures themselves. This depends upon gaining an understanding of the mechanisms which determine whether a cell in the early embryo of a farm animal will retain the ability to contribute to all of the tissues of the conceptus or become committed to differentiation along a specific pathway.
Growth factors and their receptors in different cell lineages of bovine embryos; New cDNA libraries have been produced from bovine placenta, liver and ovary. By using these, new genes have been identified, including those for two receptor molecules required for growth factors to influence the pattern of cell division (full clone for gp130 and a partial clone for the LIF receptor). Clones have been identified for bovine Oct4 and two pseudogenes. Oct4 is a marker of totipotency in mice. and experiments are in progress to define their patterns of expression in cattle embryos.
Intracellular factors that regulate transcription and cell division; Studies of the characteristic cell cycle in mouse embryonic stem cells may facilitate the isolation of such cells from other species. The G1 phase of the cycle is virtually absent from mouse stem cells and this characterisation is being used in assessments of cultured pig cells. Specific kinases have been identified (cyclin E/CDK2 and cyclin D/CDK4) which are present at only very low levels in mouse stem cells, but which increase dramatically as soon as differentiation is induced. These observations suggest that stem cells lack regulation of the G1 phase of the cycle and that this contributes to their tumorigenic nature. It is proposed that absence of these cyclins is a marker of stem cells and that an increase in their level is an early event in differentiation. In the future, regulatory elements from genes specific to totipotent cells may be used to promote the growth of such cells in culture.
Heat shock family of genes; Earlier studies have shown that isolation of mouse embryo stem cells may depend upon an epigenetic change which is promoted by applying a heat shock to the embryos immediately before culture. Attempts to understand the mechanism of this effect by analysing the production of heat shock protein induced by the treatment have made the surprising observation that no induction could be detected in treated embryos, although transcription was depressed in the expected manner.
Specific markers for undifferentiated cells; A definitive analysis has been completed to clarify the pattern of expression of several potential markers of totipotency in livestock embryos and a seminal manuscript describing these results has been published. Several laboratories collaborated to confirm that absence of lamin A/C and of cytokeratins provide useful preliminary indications of totipotency. Markers characteristic of different lineages have also been defined.
Methods for the assessment of the developmental potential of cells; A detailed assessment has been made of three approaches to characterisation of cells from the embryo: the ability to differentiate into teratocarcinomas in immune deficient mice; to contribute to all of the tissues of chimeric offspring and developmental potential after nuclear transfer. Whereas cells from embryos of cattle and pigs retain the ability to differentiate into many different tissues in teratocarcinomas, cultured cells derived from such embryos have lost that potential. Although these transfers did not establish stable cell lines, they show that ability to form teratocarcinomas in immune-deficient mice may be a good indicator of developmental potential of cultured cells. The ability of cultured pig cells to contribute to chimeras was assessed, although no chimeras were born in this project.
Nuclear transfer of cultured embryonic cells has shown a loss of developmental potential in sheep and cattle. When sheep day 9 embryos were cultured as though for the isolation of stem cells, live lambs were born following transfer of nuclei after 1, 2 or 3 culture passages. By contrast none were born following transfer from passage 6, 7 or 11. These observations show that there were changes in the cells during culture which reduced their developmental potential. In a subsequent collaborative project, live lambs were born following nuclear transfer from a cell line established from these embryos when a new method for nuclear transfer was introduced. Similar experiments have been started in cattle. When bovine or rabbit germ cells were used as donors of nuclei pregnancies were established in both species, but no fetuses developed to term.
Culture of cells for ES cell isolation; Cells from embryos and from fetal gonads have been isolated and cultured. Cells with many of the characteristics of mouse ES cells have been derived from pig embryos and maintained for several passages. Although pig embryonic cells differentiated into epitheliod cells within one or two passages the addition of Differentiation Inhibitory Factor increased the rate of cell division significantly, suggesting that it does have a physiological role in the regulation of cell fate, even if is not sufficient to prevent differentiation. Removal of retinoic acid from the culture increased the period during which totipotent cells were present and inhibition of mesoderm formation from embryonic ectoderm was achieved by modifying SSEA-1 to inhibit the binding of bFGF.
In studies of committed cells, ovine heamatopoetic populations were established from both bone marrow and marrow stromal cells. Pluripotent cells were maintained for upto 6 months. These cells were found to be negative for lamin A/C and for cytokeratin 8/18, both of which are believed to be markers of pluripotency.
MAJOR SCIENTIFIC BREAKTHROUGHS:
Normal development to term following nuclear transfer from cultured sheep embryonic cells.
An understanding of the unique pattern of the cell cycle in mouse stem cells, in particular the low level of cyclins E/CDK2 and D/CDK4.
The characterisation of molecular markers of totipotency.
Cloning of bovine genes associated with totipotency in early embryos, including OCT4.
Prolonged maintenance of undifferentiated cells in culture from embryos, gonads and bone marrow of livestock species.
Funding SchemeCSC - Cost-sharing contracts
3584 CT Utrecht