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Analysis of the telomere length of subpopulations of primary human hematopoietic cells

University of British Columbia

Telomeres are structures at the ends of eukaryotic chromosomes; they protect the ends from degradation and end-to-end fusions. Mammalian telomeres consist of a tandem array of G-rich repeats, with a length of two to 12 kb in human somatic cells. Somatic cell telomere length shortens with each cell division, leading to senescence or apoptosis. Critically short telomeres can lead to genomic instability, thus in cells that divide continually, such as germ cells, telomere length is maintained by telomerase. In a rare autosomal dominant form of dyskeratosis congenita, patients carry a mutation in the RNA template of telomerase, resulting in half maximal telomerase activity. Patients with this genotype die of aplastic anemia, indicating that maintenance of telomere length is likely critical, and may be particularly important in hematopoietic stem cells. Telomere length can be measured by a quantitative fluorescence in situ hybridisation and flow cytometry based method (Flow- FISH). In the present study, this technique was used to measure telomere length in lymphocytes and in "candidate" stem cell populations isolated from eight cadaveric marrow samples from normal adults (aged 14 to 48 years). Telomere length analysis of B and T cells - two populations that undergo activation-induced telomerase up-regulation - revealed that CD20⁺ B cells in the bone marrow had longer telomeres than CD3⁺ T cells (p<0.002). The CD34⁺CD38⁻ populations from each of the eight donors also had longer telomeres than the T cell subsets. The telomerase activity in T lymphocyte populations thus appears insufficient to prevent telomere shortening during differentiation from the pluripotent stem cell. To compare telomere length in different subsets of primitive hematopoietic ceils, we analysed FACS-purified CD34⁺CD38⁻ and CD34⁺CD38⁺ populations. The CD34⁺CD38⁻ cells had significantly longer telomeres than the CD34⁺CD38⁺ cells (p<0.02, n=8). The Side Population (SP) cells identified by FACS as Hoechst 33342'° cells did not have significantly longer telomeres than the CD34⁺CD38⁻ population. In two donor samples, additional populations were sorted, and the data is consistent with the proposed hierarchical pattern of these cell populations, with CD34⁺CD38⁻ thought to have the greatest proliferative potential and the longest telomeres. This data supports the hypothesis that hematopoietic cells with the greatest proliferative potential will have the longest telomere length. It is still unclear if a hematopoietic cell population exists within the SP with "germ-line" or "fetal" length telomeres. While we cannot exclude that a small subset of more primitive cells capable of maintaining long telomere length exists within the sorted SP cells that were analysed, we conclude that telomere length declines in the majority of the primitive hematopoietic cells examined here.

Thesis/Dissertation

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2009-10-05

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http://hdl.handle.net/2429/13637

Medical Genetics

University of British Columbia

UBCV

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