The Role of Notch-1 in ex vivo Expansion of CD34(+) Cells

Abstract

Notch signalling has been shown to prevent maturation and enhance self-renewal of haematopoietic stem cells (HSCs). Effective manipulation of this pathway could therefore improve protocols for ex vivo expansion of HSCs, which rapidly lose their 'stem' potential when grown in vitro. In mammals, four Notch receptors have been identified, which through different receptor-ligand combinations may lead to different outcomes of Notch signal transduction. Therefore, it is important to distinguish via which Notch protein pathway activity is modulated. An antibody against the extracellular domain (ECN) of a certain Notch homologue would be a useful tool to study its surface expression at the physiological level in a defined population of live cells, allowing their isolation for further experiments. In this project, two antibodies against ECN of human Notch-1 (hECN1) were tested and compared. The first, 'in house' hECN1, was developed in the Buckle lab ten years ago, whereas the second, commercial hECN1, has been available recently from R&D Systems. Using both antibodies, it was shown for the first time that the Notch-1 receptor is expressed on the surface of CD34(+) progenitors from mobilised peripheral blood (MPB) and that CD34(+)'in house' hECN1(+) cells create a small subpopulation with a lower level of Notch-1 expression within the CD34(+)commercial hECN1(+) fraction. Due to significant differences observed in staining of CD34(+) cells with both antibodies, their specificity was verified. The commercial hECN1 antibody does not cross-react with other Notch homologues and recognises the canonical Notch-1 heterodimer. The 'in house' hECN1 antibody seems to detect an unprocessed Notch-1 protein, which does not undergo post-translational modifications. Comparison of CD34(+)Notch-1(+) and CD34(+)Notch-1(-) MPB cells using the commercial hECN1 antibody only showed for the first time a greater 'stem' potential of the former subpopulation. Initially CD34(+)Notch-1(+) cells demonstrated a slower proliferation rate, better maintenance of the CD34 phenotype and cell viability, a greater multi-lineage potential, therefore they appear to be more primitive than CD34(+)Notch-1(-) cells. Because Notch-1 expression oscillated during the in vitro culture, further studies are required, especially on cells which acquired the CD34(+)Notch-1(+)aq phenotype as they could increase the pool of cells available for successful ex vivo expansion. Particularly worth examination is the possible relationship between surface expression of the Notch-1 receptor and pathway activity, which can be lower or not present in CD34(+)Notch-1(+)aq cells.

Date of Award	1 Aug 2013
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Dean Jackson (Supervisor)