Pearce, Daniel (2001) Intracellular analysis of stem and progenitor cells. (PhD thesis), Kingston University, .
Abstract
A population of rare pluripotential stem cells with extensive proliferative and self-renewal capabilities sustains haemopoiesis throughout life. Such cells, capable of differentiating into any haemopoietic lineage are required for gene therapy, ex-vivo expansion and stem cell transplantation strategies. It is currently not possible to positively identify these cells; their presence can only be retrospectively assessed through elaborate, time consuming culture techniques and animal repopulating studies. Stem cells can be isolated through negative selection, a complex and very expensive procedure. The CD34 antigen is routinely used as a surrogate marker of primitive haemopoietic cells and identifies most of the committed progenitors of the bone marrow as well as some of the more immature pluripotential cells. AC133, discovered in 1997, is reportedly expressed on a subset of CD34[sup]+stem/progenitor cells, suggesting an alternative for CD34. The present study first assessed the potential of one or both molecules (CD34/AC133) for progenitor cell acquisition in order to determine the status of AC133 as a unique stem cell antigen. Results suggested that selection of cells on the basis of AC133 excluded the more mature committed cells of the CD34[sup]+ population (especially erythroid). Consequently, the AC133[sup]+ population is more highly enriched (than the whole CD34[sup]+ cell population) for cells of a high proliferative potential and long-term culture initiating ability. The CD34 molecule can be very quickly upregulated. Upregulation is rapid (1 minute), independent of transcription/translation, and is unaffected by inhibitors of protein synthesis. Upregulation is of a fully formed glycosylated molecule and is probably from preformed intracellular stores. This research developed methodology for simultaneously fluorescent antibody-labelling internal and external stores of CD34. This was applied to investigating the internal expression of CD34, attempting to determine its significance in the hierarchy of haemopoietic cells, and this was achieved via flow cytometry, simultaneously examining the expression of internal CD34, external CD34 and one other established haemopoietic cell marker (CD38, AC133, Thy-1, CD164, CD117 and CD7). The results indicated that the CD34 molecule translocates from internal stores to membrane surface during external CD34[sup]+ haemopoietic stem/progenitor cell maturation and development. In addition, internal CD34 expression identifies a novel subset of primitive haemopoietic external CD34 negative (CD34[sup]NEG) cells, which may contain cells that precede external CD34[sup]+ cells in the hierarchy of haemopoiesis. Specific fluorescent antibody labelling was then confirmed by laser confocal microscope examination of labelled cells. The technique of simultaneously labelling 2 antigens for the confocal microscope was established, and allowed the cellular localisation of internal and external CD34 to be confirmed. A possible clinical application of this technology (internal CD34 labelling) to the monitoring of mobilisation protocols was further investigated by analysing daily peripheral blood samples, taken from patients undergoing G-CSF mobilisation therapy. Results suggested that cells with a more primitive phenotype (internal CD34[sup]+, external CD34[sup]NEG) than external CD34[sup]+ cells are released in significant numbers during the early stages of mobilisation, and are missed by the conventional harvest date. Such cells may have an improved transplant potential. This Ph.D project has established the significance of internal CD34 expression a possible application and has identified a possible application for this technology.
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