Graham, Susan M.
Isolation and characterisation of CML and normal quiescent haemopoietic stem cells.
PhD thesis, University of Glasgow.
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The work contained in this thesis was aimed at isolation of quiescent stem cells from both normal and CML samples and characterisation of the properties of these rare populations.
To isolate these specific cell populations, enrichment of the stem cell fraction was achieved by selection methods based on the stem cell marker CD34. Positive or negative CD34 selection yielded up to 99% pure CD34+ cell populations. A fluorescent activated cell-sorting (FACS) strategy was developed to allow isolation of quiescent and cycling stem cells. Using Hoechst (HST) and Pyronin Y (Py) to stain the DNA and RNA respectively and gating on the viable, Propidium lodide- (PI)-, CD34+ population, it was possible to isolate viable stem cells in G0, G1 and S/G2/M phases of the cell cycle.
Success of the sorting strategy was demonstrated by PCR analysis of differential cell cycle gene expression in the sorted populations. Further development of FACS methodology allowed the division history and stem cell status, as defined by CD34 expression, to be monitored under a range of conditions.
Using this experimental strategy, it was shown that a number of CML stem cells were able to survive exposure to concentrations of ST1 in excess of those achievable in vivo, and that the surviving cells were undivided or quiescent.
In an effort to further characterise quiescent stem cells, the HST/Py FACS strategy was used to isolate quiescent and cycling stem cell populations from both normal and CML samples, which were then processed for microarray studies.
The results have shown a differential gene expression profile between normal G0 and cycling cells that fully validates the sorting strategy. The most significant different gene expression was in a set of chemokine genes, which were up-regulated in both normal and CML G0 cells compared to cycling cells. This has not been previously reported and represents a novel finding. Additional data analysis is ongoing and may, in time, yield therapeutic targets for CML disease eradication.
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