Abstract
Hematopoietic stem cells (HSCs) that have a potential to differentiate into all lineages of hematopoietic cells are present in the hematopoietic niche, which maintains HSC stemness. Cytokine stimulation of HSCs promotes the cell cycle, and HSCs proliferate and differentiate thereafter and mature into functional blood cells. However, a lack of cytokine stimulation prevents the survival of HSCs, finally leading to apoptosis and cell death. Therefore, HSCs exposed to radiation under cytokine-free conditions causes an increase in cell death-related factors, but little information has been obtained to date regarding the precise mechanisms involved in this process. In the present study, we investigated the effect of ionizing radiation on the survival and clonogenic potential of human hematopoietic stem/progenitor cells under cytokine-free conditions. After informed consent was obtained from the legal guardian of the patient, hematopoietic mononuclear cells were separated from placental/umbilical cord blood, and CD34+ cells were enriched using magnetic cell sorting. The purified CD34+ cells were irradiated by X-rays with a dose of 0.5 or 2 Gy at a dose rate of 85–90 cGy/min, and were thereafter cultured in serum-free medium with no cytokine stimulation. After 12, 24 or 48 hours in culture, the cells were harvested from the culture and the surviving cells were quantified. Changes in cell-surface antigens during in vitro culture and the cell cycle status were also analyzed by flow cytometry. The clonogenic potential of harvested cells was analyzed using the methylcellulose culture technique. As a result, the surviving fraction cells decreased with the increasing period in cell culture, and the expression of early hematopoiesis-related antigens such as CD110 and Tie-2 were altered. In addition, the number of colony-forming cells was also decreased with increasing culture period and radiation dose. In the cell cycle analyses, treated cells shifted into the subG1 phase from the G0/G1 phase. Because CD34+ cells are a heterogenous population, their radiosensitivity may vary among subtypes. Currently, the subsequent analyses are undergoing investigation in our laboratory.
