Abstract
Hematopoietic stem/progenitor cells (HSPCs), which are present in small numbers in hematopoietic tissues, can differentiate into all hematopoietic lineages and self-renew to maintain their undifferentiated phenotype. HSPCs are extremely sensitive to oxidative stressors such as anti-cancer agents, radiation, and the extensive accumulation of reactive oxygen species (ROS). The quiescence and stemness of HSPCs are maintained by the regulation of mitochondrial biogenesis, ROS, and energy homeostasis in a special microenvironment called the stem cell niche. The present study evaluated the relationship between the production of intracellular ROS and mitochondrial function during the proliferation and differentiation of X-irradiated CD34+ cells prepared from human placental/umbilical cord blood HSPCs. Highly purified CD34+ HSPCs exposed to X-rays were cultured in liquid and semi-solid medium supplemented with hematopoietic cytokines. X-irradiated CD34+ HSPCs treated with hematopoietic cytokines, which promote their proliferation and differentiation, exhibited dramatically suppressed cell growth and clonogenic potential. The amount of intracellular ROS in X-irradiated CD34+ HSPCs was significantly higher than that in non-irradiated cells during the culture period. However, neither the intracellular mitochondrial content nor the mitochondrial superoxide production was elevated in X-irradiated CD34+ HSPCs compared with non-irradiated cells. Radiation-induced gamma-H2AX expression was observed immediately following exposure to 4 Gy of X-rays and gradually decreased during the culture period. This study reveals that X-irradiation can increase persistent intracellular ROS in human CD34+ HSPCs, which may not result from mitochondrial ROS due to mitochondrial dysfunction, and indicates that substantial DNA double-strand breakage can critically reduce the stem cell function.
Highlights
Mitochondria, the organelles that produce the energy molecule adenosine triphosphate by oxidative phosphorylation, are a source of reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, which are generated during respiratory metabolism in vivo
To evaluate the radiosensitivity of myeloid hematopoietic progenitors, CD34+ Hematopoietic stem/progenitor cells (HSPCs) exposed to 0.5–7 Gy were assayed for burst-forming unit-erythroid (BFU-E); colony-forming unitgranulocyte macrophage (CFU-GM); colony-forming unit-granulocyte erythroid, macrophage, and megakaryocyte (CFU-Mix); and colony-forming cells (CFCs) using methylcellulose semisolid culture supplemented with a hematopoietic cytokine combination comprising recombinant human erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and stem cell factor (SCF)
On day 3, an approximately 7-fold increase compared to the initial input was observed in the culture exposed to 4 Gy. These findings show that the overproduction of intracellular ROS was induced following X-irradiation of CD34+ HSPCs, but that this was not derived from the mitochondria
Summary
Mitochondria, the organelles that produce the energy molecule adenosine triphosphate by oxidative phosphorylation, are a source of reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, which are generated during respiratory metabolism in vivo. The abnormal accumulation of endogenous or exogenous ROS by the mitochondrial electron transport chain or in response to low linear energy transfer ionizing radiation, such as X-rays, may cause lipid peroxidation, protein denaturation, or DNA mutations [4,5,6]. Hematopoietic stem cells are usually present in a special microenvironment called the stem cell niche, which maintains the stemness by controlling the ROS generation [7,8,9,10] In this niche, physiological levels of intracellular ROS affect the endogenous growth signals, cell survival, proliferation, and differentiation of HSPCs with the production of many cytokines [2,11,12]. Information about the relationship between the radiosensitivity of HSPCs and mitochondrial function is limited
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