Download Lineage specification, commitment and self

Lineage Specification, Commitment and Self-Renewal in Embryos and Embryonic
Stem Cells.
Maurice A. Canham, Fella Hammachi, Gillian M. Morrison and Joshua M. Brickman
Centre Development in Stem Cell Biology, Institute for Stem Cell Research, University of
Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JQ
Embryonic Stem (ES) cells are in vitro cell lines that are karyotypically normal and capable
of differentiating into all the lineages of the foetus and adult. So what is an ES cell culture?
Is it a uniform cell type or a heterogeneous mixture of cells with different potentials? I will
present several lines of evidence that ES cultures are heterogeneous and that cells with in
these cultures can shift identity within certain parameters. However, what keeps them ES
cells? The ES cell specific transcription factor Oct4 is a key to answering this question.
We have shown that Oct4 can inhibit commitment in multiple lineages, allowing ES cells to
progress in and out of immediate early states of differentiation, but always remaining
uncommitted. The means by which Oct4 does this is not the suppression of differentiation
down specific lineages, but by maintaining a positive program to block commitment. This
program is conserved in evolution and derived from mechanism(s) essential for embryonic
development. This general mechanism may act on a variety of cell types and thus would be
particularly effective at blocking differentiation in heterogeneous ES cell cultures. We have
recently found a specific example of this phenomona in the early stages of differentiation in
the extra-embryonic primitive endoderm lineage. We generated a fluorescent reporter ES
cell line that is expressed in a sub-population of normal undifferentiated ES cell cultures.
This cell line exploits an early marker of the endoderm lineage, Hex. We have replaced the
first exon of Hex with a cDNA encoding a tagged version of the Hex protein upstream of
an internal ribosomal entry site (IRES) driving the expression of the highly sensitive
Yellow Fluorescent Protein, Venus. These cells express both wild type levels of Hex RNA
and Venus in cells as they begin to form endoderm. Interestingly, Venus is also expressed
in undifferentiated ES cell cultures, but never in the entire population of cells. As ES cells
can easily differentiate towards primitive endoderm, we considered the possibility that
these populations might represent an immediate early stage of primitive endoderm
differentiation. Experiments using defined agonists and antagonists of the MAP kinase
pathway alongside molecular analysis confirm this hypothesis. Venus positive cells express
higher levels of the primitive endoderm marker, GATA6 and less of the ES cell
pluripotency gene Nanog. However, while these cells are shifted in the direction of
primitive endoderm, they are still not committed. Clonal analysis indicates that a Venus
positive cell can readily revert to Venus negative ES cells. At all times these Hex positive
and negative populations expresses equivalent levels of the master ES cell regulator, Oct4.
These observations support our contention that ES cell cultures represent a heterogeneous
mixture of cell types maintained in an uncommitted state. Thus when cultured under
conditions that promote ES cell self-renewal, the ability of these different cells to progress
further in differentiation towards their defined lineages may be suppressed by the master
ES cell regulator Oct4.