G-CSF-mobilized CD34+ peripheral blood stem cells are significantly less apoptotic than unstimulated peripheral blood CD34+ cells: role of G-CSF as survival factor

The mechanism of release of CD34⁺ cells into the peripheral blood (PB) after mobilization treatment with chemotherapy and/or growth factors is not clearly understood. Growth factors may induce increased proliferation and self renewal within the stem cell compartment. It is possible that they alter adhesion molecule profiles or other progenitor:stroma interactions, to allow release of these cells into the periphery. However, CD34⁺ cells are present in the PB under steady-state conditions, albeit in low number. Growth factors such as granulocyte colony-stimulating factor (G-CSF) may promote the survival of CD34⁺ cells in the PB by suppressing apoptosis. In order to test this hypothesis, we have quantitated apoptotic cells in the CD34⁺ fraction of peripheral blood stem cell (PBSC) collections, using two-colour flow cytometry, after staining with anti-CD34 antibody and the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD). 7AAD differentially stains live, apoptotic and dead cells, due to the altered accessibility of DNA in each subpopulation.
We have shown a significant reduction in the proportion of apoptotic cells in the CD34⁺ population mobilized by G-CSF compared to CD34⁺ cells in unstimulated PB, consistent with the theory that G-CSF is acting, at least in part, by suppressing apoptosis. In addition, we found that G-CSF mobilized CD34⁺ cells are less apoptotic than CD34⁺ cells of unstimulated normal bone marrow, indicating that, at the doses used, G-CSF is significantly altering the survival capacity of the mobilized cells.