Cryopreservation and thawing of hematopoietic stem cell CD34-induced apoptosis through caspase pathway activation: Key role of granulocytes

Introduction: Cell damage inescapably occurs during both the freezing and the thawing graft processes for autologous hematopoietic stem cell (HSC) transplantation. To estimate HSC injury, a quality control is performed including: (i) CD34(+) quantification; (ii) percentage of CD34(+) viability and (iii) evaluation of HSC functional ability to form colony forming unit - granulocyte macrophage (CFU-GM). Apoptosis involves complex pathways such as caspase enzymes. Here, we assess the extent of apoptosis that is caspase-dependent before and after cryoconservation of CD34(+), using a Fluorescent Labeled Inhibitor of CAspases (FLICA). Methods: Caspase pathway activation status was evaluated in 46 patients (multiple myeloma [n = 24], lymphoma [n = 22]), by flow cytometry, using a 7-aminoactinomycin-D (7AAD)/FLICA staining test, in CD34(+), CD3(+), CD14(+) and CD56(+) cells. Viable 7AAD(-)/FLICA(+) cells were then correlated with various parameters. Results: We showed a significant caspase pathway activation, with 23% CD34(+)/7AAD(-)/FLICA(+) cells after thawing, compared with the 2% described in fresh CD34(+) cells (P < 0.0001). Moreover, caspase pathway was significantly activated in thawing CD3(+), CD56(+) and CD14(+) cells. We also report a significant correlation between the rate of CD34(+)/7AAD(-)/ FLICA(+) cells and post-thawing granulocytes count (P = 0.042) and their potential to be differentiated into CFU-GM (P = 0.004). Discussion: Our results show substantial cell death, induced by the increase of caspase pathway activation, secondary to the thawing process, and across all study cell types. This observation may affect the immune response quality during recipient aplasia, without detecting a clinical impact. Moreover, caspase pathway activation through CD3(+) and CD56(+) subpopulations could modify the therapeutic result of donor lymphocytes infusion (DLI).