Gene Expression Profiling in Quiescent Stem Cells from Normal and Chronic Myeloid Leukaemia Patients.

Earlier studies have shown that Ph+ quiescent cells exist in chronic myeloid leukaemia (CML) (Blood (1999)94:2056) and we have previously shown that these cells are primitive in that they express the stem cell marker CD34. We have also shown that quiescent CML stem cells are insensitive to the effects of imatinib (IM Novartis Pharma) (Blood (2002) 99:319) and may present a possible source for relapse. This quiescent population therefore represents a potentially significant clinical problem and thus studies aimed at developing methods for eradicating this population are timely. In an effort to identify molecular markers of this population that may allow it to be specifically targeted during therapy, we have set out to investigate the transcriptional differences between quiescent and cycling stem cells. To this end, we have used specific stem cell enrichment and sorting protocols. Leukapheresis products from CML patients (N=5) in chronic phase at diagnosis and mobilised peripheral blood from allogeneic donors (N=3), were selected for CD34⁺ cells. Hoechst 33342 and Pyronin Y were used to discriminate the quiescent (G0) cells identified as Hoechst^lo/Pyronin^lo from the cycling cells. In combination with propidium iodide for dead cell exclusion we were able to sort 4–9x10⁵ viable, quiescent stem cells and 4–11x10⁶ cycling cells, which were processed for microarrays. Affymetrix gene chips (U133A) were used for the analysis and the data obtained was analysed using GeneSpring.

Number of Genes Changed in Each Comparison

Initial analysis indicates that the greatest differences in gene expression are between the normal quiescent cells (G0) and normal dividing cells (Div) and between the normal quiescent cells and CML quiescent cells. A large percentage of the genes differentially expressed between the quiescent and cycling normal cells encode regulators of the cell cycle confirming the success of the sorting strategy for quiescent and cycling cells

A selection of Genes Up-Regulated in Normal Cycling Cells Compared to G0

In addition, many of the genes identified in our analysis are consistent with other published expression profiles for haemopoietic cells. Curiously, we have identified unanticipated changes in expression of cell cycle genes in the CML quiescent cells, which merit further investigation. We have also identified a number of unexpected genes as being more than 5 fold changed in the quiescent cells compared to dividing cells for both normal and CML samples. Specifically, there is a large cohort of genes preferentially expressed in quiescent normal or CML cells, which encode members of the chemokine family of proteins. Work is ongoing to establish the relevance, if any, of these genes to stem cell quiescence.