Telomeres, or chromosome ends, in eukaryotic organisms are characterized by tandem repeats of G-rich DNA . In human and other vertebrates, the telomeric sequence contains TTAGGG repeats . During cell replication, there is incomplete DNA synthesis at the 3′ end of chromosomes, and this end-replication problem predicts shortening of the telomeres with cell division [3,, 4]. Indeed, telomere shortening has been observed during in vivo and in vitro aging of normal human somatic cells, such as fibroblasts, keratinocytes, epithelial cells, peripheral blood leukocytes and endothelial cells [5–, 10]. These same cells undergo replicative senescence in vitro, i.e., they become senescent and withdraw from the cell cycle upon reaching the “Hayflick limit,” usually within 30-100 population doublings [11–, 13]. However, cells with essentially unlimited replicative potential such as the reproductive cells, immortal cell lines and cancer tissues have stable telomeres [7, 14–, 16]. The maintenance of telomere lengths in these cells is highly correlated with the presence of telomerase, a ribonucleoprotein enzyme which synthesizes telomeric repeats de novo [17–, 21]. These observations led to the proposal of the telomere hypothesis of cellular aging and immortalization . This hypothesis stipulates that telomere shortening serves as a mitotic clock for replicative senescence, ultimately signaling cell cycle exit, while telomerase activation is necessary for stabilizing telomere lengths, thereby maintaining replicative capacity of self-renewing cells such as cells in the reproductive tissues, tumor cells, and possibly stem cells of various tissue types [5, 14, 19].
The hematopoietic stem cells are usually defined as “self-renewing” multipotential cells which give rise to the myeloid, lymphoid and erythroid cell lineages. The stem cell compartment is small, but the “self-renewing” ability is thought to allow for the maintenance of stem cells despite the staggering daily demand for the production of >1011 mature blood cells [22, 23]. Using a combination of specific cell surface markers, a highly enriched population of the most primitive human hematopoietic progenitors can be isolated from adult bone marrow . These cells, expressing the CD34+CD71loCD45RAlo phenotype, are initially quiescent but can be stimulated to enter cell cycle and proliferate in response to cytokines . To date, there are no culture conditions known that allow these primitive hematopoietic progenitors to self-renew in vitro. The proliferation of such cells seems invariably associated with differentiation, mostly along the myeloid and erythroid pathways. In parallel, there are changes in the cell surface phenotype which are characterized by the activation of CD71 expression and the corresponding downregulation of CD34 expression .
We have previously demonstrated that telomeres shorten in primitive human hematopoietic cells as a function of developmental stage and cytokine-induced proliferation . These data suggest that the proliferative potential of at least some of these cells may be limited and may decrease with age. Recent studies have shown that telomerase activity was detected at low levels in hematopoietic cells and peripheral blood lymphocytes [26–, 28]. Here, we have investigated the expression of telomerase in different subpopulations of normal human bone marrow hematopoietic cells before and after culturing in the presence of cytokines. Using a sensitive polymerase chain reaction (PCR)-based telomerase assay (telomeric repeat amplification protocol, or TRAP) , we found a low basal level of telomerase activity in the most primitive, quiescent hematopoietic cells with the CD34+CD71loCD45RAlo phenotype and the more mature CD34– cells. The cycling CD34+CD71+ progenitors expressed the enzyme activity at a higher level. Interestingly, when the quiescent primitive cells were stimulated with cytokines, telomerase activity was transiently induced in the culture. The presence of telomerase activity, however, was apparently unable to prevent the telomere loss observed previously in similar cultures .