Proliferative properties of human umbilical cord blood megakaryocyte progenitor cells to human thrombopoietin

We evaluated the effects of human thrombopoietin (TPO) alone, or in combination with other several hematopoietic factors, on megakaryocyte colony growth from human umbilical cord blood CD34⁺ cells in serum-depleted agar cultures. The addition of TPO alone had a concentration-dependent effect on the number of pure megakaryocyte colonies and of megakaryocytes per colony (colony size). The maximally stimulating concentration of TPO generated a greater number of megakaryocyte colonies and larger megakaryocyte colony size compared with the stimulation observed with an optimal concentration of human interleukin-3 (IL-3) or stem cell factor (SCF). At the high concentration of TPO that yielded the maximum colony numbers, a substantial proportion of megakaryocyte colonies contained 100 or more cells per colony. The combination of TPO plus SCF resulted in a synergistic enhancement of both the number and size of megakaryocyte colonies. Among the combinations of TPO plus other cytokines tested, IL-3 plus TPO had a modest effect on megakaryocyte colony numbers. The generation of megakaryocyte colonies from subpopulations of CD34⁺ cells was further examined. The addition of TPO alone induced a greater number of megakaryocyte colonies from CD34⁺CD41⁺ cells compared with CD34⁺CD41^- cells, and TPO plus IL-3 exerted a synergistic effect on the number of megakaryocyte colonies only from CD34⁺CD41^- cells. In contrast to the effects on colony numbers, TPO induced larger megakaryocyte colony sizes from CD34⁺CD41^- cells, compared with CD34⁺CD41⁺ cells. In the case of HLA-DR expression, TPO and IL-3, administered singly or in combination, induced similar megakaryocyte colony numbers and sizes from CD34⁺DR⁺ and CD34⁺DR^- subpopulations. Ploidy analysis revealed that the majority of megakaryocytes generated from cord blood CD34⁺ cells in serum-free liquid cultures containing TPO displayed 2N and 4N values, suggesting that they were immature. These results indicate that, compared with IL-3 and SCF, TPO has more potent proliferative effect on human cord blood megakaryocyte progenitors, leading to greater numbers of megakaryocyte progenitors triggered for both growth and cell division, and synergizes with SCF to enhance megakaryocyte colony growth.