Hematopoietic stem cells (HSCs) constitute a reservoir of undifferentiated cells that can be committed, upon appropriate stimuli, in the haematic lineages. Although residing in a bone-marrow hypoxic microenvironment (niche) and mainly relying on anaerobic glycolysis, HSCs are endowed with mitochondria. Recently, specific interest has been focused on HSCs mitochondria and on their role as reactive oxygen species (ROS) generators during the early phases of commitment. Indeed, consolidated evidences highlight the importance of redox signalling in the homeostasis of fundamental processes in cell adaptive biology and particularly in controlling the balance between self-renewal and differentiation of stem cells. HSCs constitutively generate low levels of ROS produced by both mitochondrial respiratory chain and NADPH oxidases (NOXs). ROS would act as secondary messengers, modulating the expression of master transcription factors leading or (pre)conditioning stem cells towards differentiation. Myelodisplastic syndrome (MDS) is characterized by disturbance of the HSC differentiation and most MDS patients are treated with iron chelators to compensate for the iron overload consequent to the blood cell transfusion-based standard therapy. Intriguingly, a robust percentage of patients, treated with the iron chelator deferasirox (DFX), recover correct HSCs differentiation whereas other chelators, like deferoxamine (DFO) did not. In the presented study we investigated the effect of DFX and DFO on the redox homeostasis of hematopoietic stem/progenitor cells (HSPCs) in order to get insights on the differential effect of iron chelators in rescuing altered hematopoiesis.
Human HSPCs were isolated from peripheral blood (PB) or bone marrow (BM) of G-CSF-treated or untreated healthy donors, respectively, by immuno-selection against the specific markers CD133 and CD34 and resulted >99% immunophenotypically homogeneous. Mitochondrial respiratory activity was measured in intact HSPCs by high resolution oxymetry. Morpho-functional features of HSPC-mitochondria, expression of the HSPC-surface commitment markers and ROS level were analyzed by laser scanning confocal microscopy (LSCM) and flow cytometry using specific probes or antibodies. HSPCs were treated with 100 mM DFX or DFO for 24 hrs.
Measurement of oxygen consumption rate as well as molecular analyses in PB-HSPCs confirmed a poor mitochondrial oxidative phosphorylation phenotype. LSCM imaging of mitochondria in either PB- and BM-HSCs displayed a punctuate rather than interconnected network. However, co-staining of mitochondria and CD34/CD133 stemness-markers revealed a striking inverse correlation. Finally HSPCs produced DCF-detectable and DPI-inhibitable ROS attributable to constitutive NOX activity and related to stabilization of the hypoxia-inducibile factor (HIF1a) under normoxic condition. DFX treatment of HSPCs resulted in a significant up-regulation of ROS level whereas no significant change was observed following DFO treatment. Importantly, the DFX-mediated ROS production was insensitive to treatment with low NOX-inhibiting concentration of DPI but was abrogated by high concentration of DPI thus pointing to mitochondria as ROS source.
Our results show that HSCs in the early phase of commitment undergo a progressive increase of mitochondrial mass indicating the need of a bioenergetic up-regulation to cope with the oncoming energy-demanding proliferative/differentiative phenotype. Redox signaling, mediated by ROS production and likely triggered by changes in the environmental oxygen tension, appears to be essential in regulating HSC self-renewal and preservation of pluripotency. DFX treatment, by modulating ROS production, might lead to activation of redox-sensitive key factors able to restore the hematopoietic function in MDS patients. This effect seemingly is independent on the iron-chelating property of DFX but pertains to additional pharmacological properties that warren further investigation.
No relevant conflicts of interest to declare.
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