Telomere dynamics in hematopoietic stem cells

Hematopoietic stem cells (HSCs) are known to possess an exceptionally high replicative potential (Szilvassy SJ 2003). However, although they express detectable levels of telomerase, previous murine studies have suggested that HSCs still experience telomere shortening and are not immortal (Allsopp RC 2003). To further clarify the role of aging and clonal exhaustion in HSCs we used murine models to investigate the relationship between the number of cells transplanted, telomerase status, the length of their telomeres and their long term exhaustion. We initiated our studies by investigating the replicative potential of titrated numbers of highly purified HSCs. Results showed that bone marrow stemming from a single HSC could only be serially transplanted twice whereas bone marrow stemming from 10 HSCs could be serially transplanted at least three times. In order to better understand HSC replicative potential, we modified Flow-FISH for murine telomere length measurements. Our Flow-FISH protocol enables us to measure telomere lengths at different timepoints within individual mice, providing us with a unique insight into the telomere length status. With the primary goal of clarifying the role of aging and clonal exhaustion in HSC we aimed to investigate the relationship between telomerase status and HSC exhaustion. We set out to characterize the HSCs of TERT-KO mice that lack the mTERT gene encoding the telomerase enzyme (Erdmann N 2004). A comparison of the frequency of Kit⁺Sca⁺Lineage ̄ cells between the TERT-KO and its wild type counterpart revealed no difference. To our knowledge no studies have explored the rate of telomere shortening of HSC within individual mice over time. Using our optimised Flow-FISH based method we studied the rate of telomere shortening within primary BM transplant recipients, in relation to telomerase status. We find that telomere length is maintained over a nine month period post transplantation, regardless of telomerase status. Furthermore, serially transplanting titrated numbers of wild type and telomerase deficient WBM does not result in telomere shortening. However, despite the apparent maintenance of telomere length, reconstitution levels decrease with each transplantation regardless of transplant dose and telomerase status. These findings suggest the presence of telomere independent barriers, HSC dilution (Iscove NN 1999), alternative lengthening of telomeres or low HSC turnover following WBM transplantation.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat