Thrombopoietin responsiveness reflects the number of doublings undergone by megakaryocyte progenitors

Abstract

AbstractTo assess the variation of thrombopoietin (TPO) responsiveness associated with megakaryocyte (MK) progenitor amplification, TPO dose-response curves were obtained for normal human, single-cell plated CD34+CD41+ cells. The number of MKs per well was determined in situ and expressed as number of doublings (NbD). Dose-response curves of the mean frequency of clones of each size versus log TPO concentration showed highly significant differences in the TPO concentration needed for half-maximum generation of clones of different sizes (TPO50): 1.89 ± 0.51 pg/mL for 1 MK clones; 7.75 ± 0.81 pg/mL for 2 to 3 MK clones; 38.5 ± 5.04 pg/mL for 4 to 7 MK clones, and 91.8 ± 16.0 pg/mL for 8 to 15 MK clones. These results were consistent with a prediction of the generation-age model, because the number of previous doublings in vivo was inversely correlated with the number of residual doublings in vitro. TPO responsiveness decreased in vitro by a factor of 3.5 per doubling, reflecting the recruitment of progressively more ancestral progenitors. In support of this hypothesis, the more mature CD34+CD41+CD42+ cell fraction had a lower TPO50 (P < .001), underwent fewer NbD (P < .001), and expressed a 2.8-fold greater median Mpl receptor density (P < .001) than the CD34+CD41+CD42– fraction. Progenitors that have completed their proliferative program have maximum factor responsiveness and are preferentially induced to terminal differentiation.