Fibronectin peptides and murine embryonic stem cells : an in vitro study

Murine embryonic stem cells are cells that are obtained from the inner cell mass of the embryo blastocyst at day 3.5. These cells have the novel capability of differentiating into almost any cell lineage in the mammal and this characteristic has resulted in them being referred to as pluripotent. Fibronectin is involved in a number of different functions including differentiation, growth, migration and the adhesion of cells. In addition it is well known to interact with the extracellular matrix. Fibronectin also has an essential role to play in early development of the embryo as studies have shown that knocking out either fibronectin or its primary receptor eg 51 integrin receptor is lethal to the developing embryo. The section of fibronectin identified as being essential for binding and activation of 51 integrin receptor has been identified as the 9th and 10th type III fibronectin domain. This peptide of fibronectin has subsequently been generated. In this study, the effect of this fibronectin peptide on murine embryonic stem cells was examined. In particular it was identified that the generation of 2-D orientated fibronectin peptide displays in no way enhances the cell attachment potential of the fibronectin peptides. The optimal conformation of the peptides for the attachment of embryonic stem cell colonies was identified as being the dimeric form of the peptide. The fact that these fibronectin peptides have no effect on the proliferation of embryonic stem cells was also determined. There was an apparent morphological change in embryonic stem cells grown on the fibronectin peptides. These embryonic stem cells were flatter and reminiscent of differentiated embryonic stem cells. This occurred even when the embryonic stem cells were cultured in the presence of LIF, the cytokine essential to maintain embryonic stem cells in a state of self renewal. Alkaline phosphatase staining confirmed that while there was a morphological change the embryonic stem cells maintained their undifferentiated state when grown on fibronectin peptides in the presence of LIF. Finally Quantitative Polymerase Chain Reaction was carried out in an attempt to ascertain why there appeared to be a morphological change in embryonic stem cells grown on fibronectin peptides as opposed to embryonic stem cells grown on gelatin controls. In the presence of LIF, there was a transient increase in the number of Oct-4, Brachyury and Nestin transcripts. Up regulation of these genes is known in some cases to come from a pro differentiation signal. However concomitant with these transcriptional up regulations, the levels of Nanog, the gene well known to resist and/or reverse changes in gene expression states in embryonic stem cells also increased upon growth on fibronectin peptides. It is possible that in the presence of LIF, Nanog increases to inhibit the differentiation inducing signals produced by interaction of the fibronectin peptides with the 51 integrin receptor. When grown in complete media containing foetal calf serum in the absence of LIF, there were no obvious transcriptional changes that gave an indication to which, if any, lineage the fibroenctin peptides may be instructing the embryonic stem cells to differentiate into. Upon using serum replacement media as opposed to foetal calf serum and allowing the embryonic stem cells to differentiate more obvious transcriptional changes were observed. ES cells grown on the fibronectin peptides appeared to be inhibited from increasing the levels of transcripts with known roles in cardiac and neuroectodermal differentiation. However there was a transient increase in the levels of FoxA2, a gene involved in the differentiation of embryonic stem cells into endodermal tissue. This increase was shortly followed by a significant decrease in the levels of transcripts when grown on the fibronectin peptides as opposed to gelatin controls. Importantly it appears that allowing the differentiation of embryonic stem cells to occur in a monolayer instead of the classically used embryoid bodies could potentially be used as an alternative model to study embryonic stem cell differentiation