Probing the Mitotic History and Developmental Stage of Hematopoietic Cells Using Single Telomere Length Analysis (STELA)

In most somatic cells, telomeres shorten with each round of cell division. As a result telomere length can be used to assess the mitotic history of cells with some important caveats: the telomere length at birth is highly variable (presumably reflecting different alleles of genes regulating telomere length in the germline), telomere losses can be compensated by telomerase and the overall decline in telomere length includes sporadic, variable losses of telomere repeats resulting from damage to telomeric DNA and/or replication errors. The first caveat can be circumvented by testing cells from the same individual and sporadic telomere losses can be analyzed by single telomere length analysis (STELA). It is more difficult to exclude the effect of telomerase on telomere length but we have previously shown that, despite readily detectable expression of telomerase, hematopoietic stem and progenitor cells show a progressive decline in telomere length with cell division and with age. The clinical relevance of telomere shortening is illustrated in several recent studies linking very short telomeres to bone marrow failure and pulmonary fibrosis. We now show that purified human hematopoietic populations from mobilized peripheral blood (MPB) and cord blood (CB) enriched for stem cells (Lin⁻CD34⁺CD38⁻Rho⁻) and successively more mature cells display progressively shorter telomeres, pointing to the utility of this method for studies of the mitotic relationship between various stem and progenitor cells. Ultra-short telomeres were readily observed (and found to be significantly more frequent) in terminally differentiated cell populations of MPB, suggesting that sporadic telomere losses occur more frequently during differentiation. When 1000 Lin⁻CD34⁺CD38⁻Rho⁻ cord blood cells were transplanted into two immuno-deficient mice, the most primitive human hematopoietic cells with a CD34⁺CD38⁻ phenotype lost 3970 and 2790 bp respectively following regeneration in vivo, indicative of ~ 30–80 cell divisions assuming a telomere loss of 50–100 bp/division. Further losses in more differentiated cells were similar to those observed in cells before transplantation. These results illustrate the power of STELA for analysis of telomeres in rare cells and point to a novel strategy to study the turnover and replicative history of cells. Furthermore, these data demonstrate that self-renewal divisions in stem cells rather than additional cell divisions in downstream progenitors are the primary cause of telomere loss following transplantation.