OBJECTIVE: Notch signalling has been implicated in haematopoietic stem cell self-renewal. Although several studies have tested the effect of activating or inhibiting the Notch signalling pathway in stem cells, no study has yet determined the functional differences associated with expressing Notch1. The aims of this study were to characterise the expression of human cell-surface Notch1 in cord blood (CB) CD34(+) cells and to study the function of Notch in CD34(+) cells in vitro. METHODS: A monoclonal antibody against the extracellular domain of Notch1 was developed, and Notch1 expression in CB CD34(+) cells was assessed by flow cytometry. CB CD34(+) cells were sorted on the basis of their Notch1 expression and cultured in serum-free media. Single sorted CD34(+) CD38(-) Notch1(+) /(-) cells were cultured for 8 wks on murine stroma monolayers and assayed for stem cell activity and lineage potential using a cobblestone area-forming cell (CAFC) assay. RESULTS: Cell-surface Notch1 expression was characterised in various primitive CD34(+) cell compartments including a small subpopulation of CD34(+) CD38(-) cells. We found the CD34(+) CD38(-) Notch1(+) population to be enriched for stem cell activity. Moreover, CD34(+) CD38(-) Notch1(+) , but not Notch1(-) cells, demonstrated multilineage potential. CONCLUSIONS: These data show that Notch1 is expressed on a functionally distinct subpopulation of CD34(+) cells that is highly enriched for stem cell activity and multilineage potential and could suggest that Notch1 could be used as a novel stem cell marker.
OBJECTIVE:Notch signalling has been implicated in haematopoietic stem cell self-renewal. Although several studies have tested the effect of activating or inhibiting the Notch signalling pathway in stem cells, no study has yet determined the functional differences associated with expressing Notch1. The aims of this study were to characterise the expression of human cell-surface Notch1 in cord blood (CB) CD34(+) cells and to study the function of Notch in CD34(+) cells in vitro. METHODS: A monoclonal antibody against the extracellular domain of Notch1 was developed, and Notch1 expression in CB CD34(+) cells was assessed by flow cytometry. CB CD34(+) cells were sorted on the basis of their Notch1 expression and cultured in serum-free media. Single sorted CD34(+) CD38(-) Notch1(+) /(-) cells were cultured for 8 wks on murine stroma monolayers and assayed for stem cell activity and lineage potential using a cobblestone area-forming cell (CAFC) assay. RESULTS: Cell-surface Notch1 expression was characterised in various primitive CD34(+) cell compartments including a small subpopulation of CD34(+) CD38(-) cells. We found the CD34(+) CD38(-) Notch1(+) population to be enriched for stem cell activity. Moreover, CD34(+) CD38(-) Notch1(+) , but not Notch1(-) cells, demonstrated multilineage potential. CONCLUSIONS: These data show that Notch1 is expressed on a functionally distinct subpopulation of CD34(+) cells that is highly enriched for stem cell activity and multilineage potential and could suggest that Notch1 could be used as a novel stem cell marker.