Effects of a 2-step Culture with Cytokine Combinations on Megakaryocytopoiesis and Thrombopoiesis from Carbon-ion Beam-irradiated Human Hematopoietic Stem/progenitor Cells

To evaluate whether the continuous treatment of two cytokine combinations is effective in megakaryocytopoiesis and thrombopoiesis in hematopoietic stem/progenitor cells exposed to heavy ion beams, the effects of a 2-step culture by a combination of recombinant human interleukin-3 (IL-3) + stem cell factor (SCF) + thrombopoietin (TPO), which just slightly protected against carbon-ion beam-induced damages, and a combination of IL-3 + TPO, which selectively stimulated the differentiation of the hematopoietic stem/progenitor cells to megakaryocytes and platelets, were examined. CD34(+)-hematopoietic stem/progenitor cells isolated from the human placental and umbilical cord blood were exposed to carbon-ion beams (LET = 50 keV/microm) at 2 Gy. These cells were cultured under three cytokine conditions. The number of megakaryocytes, platelets and hematopoietic progenitors were assessed using a flow cytometer and a clonogenic assay at 14 and 21 days after irradiation, respectively. However, the efficacy of each 2-step culture was equal or lower than that of using the IL-3 + SCF + TPO combination alone and the 2-step culture could not induce megakaryocytes and platelets from hematopoietic stem/progenitor cells exposed to high LET-radiation such as carbon-ion beams. Therefore, additional cytokines and/or hematopoietic promoting compounds might be required to overcome damage to hematopoietic cells by high LET radiation.