To assess the involvement of Notch signalling

To assess the involvement of Notch signalling on defined cell populations within the AGM microenvironment we established a novel system whereby we co-cultured isolated FLK1+ oxycodone hydrochloride on AGM-derived stromal cell lines. We used inducible expression of either NotchIC or dominant negative MAML as gain- and loss-of-function strategies respectively to manipulate Notch activity within this defined cell population at specific time points during differentiation. Potential problems associated with transgene silencing during differentiation have been avoided by targeting our inducible constructs into the permissive Rosa26 and Hprt loci. The Cre-mediated recombination strategy that we have used to express NotchIC allows us to compare directly NotchIC+ and NotchIC− cells from the same culture, avoiding clonal variability between ESC subclones (Schroeder et al., 2003a). Using this elegant system we have been able to dissect precisely the role of Notch signalling on the specification of haematopoietic cells from Flk1+ precursors within the AGM microenvironment, uncovering an inhibitory effect of Notch signalling on haematopoietic commitment.
Materials and methods
Results
Discussion We describe the co-culture of ESC-derived FLK1+ cells on AGM-stroma that provides a simple model to study the molecular pathways involved in haematopoietic development in the AGM microenvironment. We demonstrate that the AGM derived stromal cell line promotes the differentiation of FLK1+ mesoderm progenitor cells into haematopoietic lineages and we have used our system to assess the role of the Notch pathway at this point in the differentiation of a defined subpopulation of cells. AM14 stromal cells have a distinct Notch ligand profile compared to OP9 with the expression of Jagged 1 being significantly lower and the expression of Jagged 2 being higher than OP9. This indicates that the different microenvironments have distinct Notch ligand activity. Our goal in this study was to study the role of Notch signalling within the AGM-type microenvironment. We noted that Notch signalling is dynamic during the co-culture with the lowest level being associated with the time of haematopoietic progenitor production. Further reduction of this low level had no functional consequences with the haematopoietic differentiation of FLK1+ cells being unaffected by the overexpression of dominant negative MAML. In contrast, activation of the Notch pathway by NotchIC inhibited the subsequent production of haematopoietic progenitors from FLK1+ cells as assessed by CFU-C formation and the expression of haematopoietic cell surface markers and transcription factors. Our Cre-mediated recombination strategy, whereby NotchIC-expressing cells were marked with the hCD2 receptor, has been a powerful approach because it has enabled us to directly compare FLK1+ cells that express exogenous NotchIC with control, FLK1+ NotchIC− cells from the same culture. Thus we overcome some of the problems that have been associated with the interpretation of data derived from complex heterogeneous cell populations that has resulted in confusing and sometimes contradictory reports in the literature (Bigas and Espinosa, 2012). At this stage during haematopoietic differentiation the FLK1+ cell population includes progenitors for a number of mesodermal lineages including endothelial and cardiac cells and NotchIC could be acting via interactions between these cell types.
Conclusion In summary, using an inducible genetic system which has allowed us to compare NotchIC+ and NotchIC− cells from the same culture we have resolved some of the discrepancies in the literature about the role of this signalling pathway during haematopoietic differentiation. We conclude that within the AGM microenvironment the production of haematopoietic cells from FLK1+ progenitors is optimal when Notch signalling is below a specific threshold. The opportunity to modulate Notch signalling via its interaction with other signalling pathways such as Wnt and Hedgehog could lead to new strategies for regenerative medicine and the production of haematopoietic cells from pluripotent cells for therapy.