Reactive oxygen species levels differentiate CD34⁺ human progenitors based on CD38 expression

Low reactive oxygen species (ROS) levels are well-established characteristics of mouse hematopoietic stem cells (HSCs). However, little is known about these levels in human HSCs. This study aimed at quantifying ROS levels in human CD34⁺ CD38^low and CD34⁺ CD38^high human progenitors from bone marrow, cord blood and cells mobilized for autologous HSC transplantation. A specifically devised multiparameter flow cytometry method was used to quantify ROS levels (H₂ DCFDA staining) in sub-populations of primary cells. Results were confirmed by assessing gene expression level of the ROS scavenger GPX3, a key determinant of HSC self-renewal, in sorted CD34⁺ CD38^low and CD34⁺ CD38^high cells. CD34⁺ CD38^low cells from bone marrow and cord blood displayed significantly lower levels of ROS than CD34⁺ CD38^high cells and other leukocytes. Moreover, the correlation between ROS and GPX3 expression was verified in sorted CD34⁺ CD38^low and CD34⁺ CD38^high cells. These results confirm, in human, data previously reported in mice. Moreover, the flow cytometry assay we developed could allow for a more precise enumeration of repopulating primitive progenitors in the course of HSC transplantation.