Clonal heterogeneity in growth kinetics of CD34+CD38- human cord blood cells in vitro is correlated with gene expression pattern and telomere length.

Human hematopoietic stem cells (HSCs) are characterized by an extensive proliferative capacity that decreases from fetal liver to cord blood (CB) to adult bone marrow. In previous studies, it was demonstrated that the proliferative capacity of individual CD34+CD38- HSC clones is correlated with their growth kinetics in vitro and that HSC turnover in vivo can be estimated by telomere-length measurements. The present study was aimed at the characterization of the clonal composition of CD34+CD38- human umbilical CB cells in terms of growth kinetics, telomere length, and gene expression profile. For this purpose, individual CD34+CD38- CB cells were sorted into 96-well plates containing serum-free medium supplemented with six growth factors. During expansion, cell numbers in each individual well were scored in 3-day intervals. Once sufficient cell numbers were achieved, telomere length was measured by flow fluorescence in situ hybridization (flow FISH). In a second set of experiments, gene expression and colony-forming capacity were analyzed in slowly growing clones as compared with fast-growing clones, using linear amplification and oligonucleotide microarrays (HG-U133A; Affymetrix). Individual CD34+CD38- cells from CB displayed an extensive functional heterogeneity in growth kinetics. Among highly proliferative clones, the most slowly growing clones were characterized by the longest telomeres. Furthermore, significant differences in gene expression were detected between slow- and fast-growing clones, whereas no significant difference in colony-forming capacity was observed. These data provide further evidence for a functional hierarchy in the human HSC compartment and suggest a link between telomere length and proliferation capacity of individual HSC clones.

Normalization of previously shortened telomere length under treatment with imatinib argues against a preexisting telomere length deficit in normal hematopoietic stem cells from patients with chronic myeloid leukemia.

Telomeres are composed of TTAGGG repeats and associated proteins. In somatic cells, telomere repeats are lost with each cell division, eventually leading to genetic instability and cellular senescence. In previous studies, we described substantial and disease stage-specific telomere shortening in peripheral blood (PB) leukocytes from patients with chronic myeloid leukemia (CML). Here, we sought to determine whether age-adjusted telomere length in PB granulocytes (deltaTEL(gran)) is associated with response to treatment with the selective tyrosine kinase inhibitor imatinib. A total of 517 samples from 206 patients in chronic phase (CP), accelerated phase (AP), and blast crisis (BC) before and up to 706 days after initiation of imatinib therapy (median: 144 days) were analyzed by quantitative fluorescence in situ hybridization of interphase cells in suspension (Flow-FISH); telomere fluorescence was expressed in molecular equivalents of soluble fluorochrome units (MESF). Telomere length in samples from start of treatment up to day 144 was significantly shorter (mean +/- SE; -1.5 +/- 0.3 kMESF) compared to samples from patients treated for more than 144 days (-0.8 +/- 0.3 kMESF, p = 0.035). In patients with repeated measurements, a significant increase in telomere length under treatment was observed. Median telomere length in major remission was found to be significantly longer compared to patients without response to treatment measured either by cytogenetics (n = 246, p < 0.05), interphase FISH (n = 204, p = 0.002), or quantitative RT-PCR (n = 371, p < 0.05). In conclusion, the increase in telomere length under treatment with imatinib reflects a shift from Ph+ to Ph- cells in the PB of patients with CML.