Reactive Oxygen Species Level and GPX3 Expression in Human CD34⁺CD38⁻ Progenitors Harvested From Cord Blood, Adult Bone Marrow and Peripheral Blood

Abstract 4743

Redox signaling has emerged as an important regulator of hematopoietic stem cell (HSC) self-renewal and lifespan. It is established that murine HSCs have a low level of reactive oxygen species (ROS) which is correlated with stem cells properties (Ito K et al, Nat. Medicine, 2006; Jang YY and Sharkis SJ, Blood, 2007). Moreover, it was recently reported that Gpx3, a gene encoding for the ROS scavenger glutathione peroxydase 3, is a determinant of the self-renewal of HSCs (Herault O et al, J. Exp. Med., 2012). All these studies were performed in murine hematopoiesis and the objective of the present study was to quantify the ROS level and the GPX3 expression in human CD34+CD38- progenitor cells (vs CD34+CD38+ cells) harvested from bone marrow, cord blood and peripheral blood.

Human bone marrow (BM) samples were obtained from patients (n=6) undergoing orthopedic surgery (Department of Orthopedic Surgery, University Hospital, Tours, France), ombilical cord bloods were obtained from women (n=5) after vaginal deliveries (Department of Gynecology and Obstetrics, University Hospital, Tours, France), and G-CSF mobilized peripheral blood stem cells were obtained by leukapheresis from patients (n=5) of the Department of Clinical Hematology (University Hospital, Tours, France). All samples were collected from patients informed and consenting following a procedure approved by the ethical committee. The intracellular H₂O₂ level was quantified by flow cytometry. The cells were incubated with 10 μM DCF-DA, 5 μL of APC-Cy7-conjugated anti-CD45 mAb (A20), 2.5 μL of PE-Cy7-conjugated anti-CD34 mAb (8G12) and 2.5 μL of APC-conjugated anti-CD38 mAb (HB7) at 37°C for 10 min and then analyzed. Neutrophils, lymphocytes, and monocytes were identified according to CD45/SSC gating. The subpopulation SSClowCD45intCD34+ has been split into two fractions according to the expression of CD38. The expression of GPX3 was measured by quantitative RT-PCR (vs. GAPDH) in CD34+CD38- and CD34+CD38+ FACS (MoFloTM, Beckman Coulter)-sorted cells using the same gating strategy as previously mentioned for the ROS measurement.

We observed that among the different cell subpopulations, the CD34+CD38- fraction was the one which expressed the lowest level of ROS, which was higher in the CD34+CD38+ fraction in all analyses. This difference in marrow, cord blood and peripheral blood samples was on average (+/−ecm) 3.7+/−0.6, 4.0+/−2.3 and 1.3+/−0.1, respectively. Regarding the GPX3 expression in CD34+ cells, we found a high level in the marrow samples, a moderate level in the cord blood samples and a low level in the peripheral blood samples. The GPX3 expression in CD34+CD38- fraction from bone marrow, cord blood and peripheral blood was on average (+/−ecm) 4.6+/−1.2, 3.2+/−0.4 and 1.3+/−0.1 higher than in CD34+CD38+ cells, respectively.

The ROS level and GPX3 expression observed in human CD34+CD38- progenitors from bone marrow and cord blood are in line with those found in mouse hematopoiesis. It's interresting to note that the mobilization process probably modify these parameters in peripheral blood progenitors. In summary, all these data suggest a key role of GPX3 in the human hematopoiesis and that ROS level could provide a good approach to functionally isolate primitive human HSCs from bone marrow and cord blood, but not from peripheral blood after G-CSF mobilization.