Cryopreservation and Thawing of Hematopoietic Stem Cells CD34 Induced Apoptosis through Caspases Pathway Activation

BACKGROUD: Cryopreservation, before storage, is an essential step for autologous hematopoietic stem cells transplantation. Currently, the quality of the graft is estimated: by the quantification of CD34 cells after thawing, the percentage of viability of CD34+ cells evaluated by an Annexin V/7AAD staining and a functional CFU-GM assay. The cryopreservation protocols can affect the viability of HSC CD34+ after thawing which could affect the quality of the graft after injection. The cell death could be induced by cells necrosis or apoptosis. Apoptosis, or programmed cell death, involves complex pathways in part Fas ligand (FasL), mitochondrial components, caspases, cathepsins, and calpain. The involvement of apoptosis dependent on caspases activation pathway in the CD34+ quality has not been evaluated. We have conducted a study to assess the extent of apoptosis before and after cryopreservation of PBSC CD34+ cells using a Fluorescent Labelled Inhibitor of Caspases FLICA. We determined the percentage of FLICA positive cells in viable cells (7AAD negative cells) before and after thawing and confronted results to colony forming units potential and to the number of days of clinical aplasia.

METHODS: we tested the induction of caspases-dependent apoptosis before and after cryoconservation in HSC CD34 cells from patients with different hematological malignances: multiple myeloma (n=11), Hodgkin’s (n=2) and non- Hodgkin’s lymphoma NHL (n=5), and amylose (n=1). We evaluated the caspases activation by flow cytometry using the Fluorescent Labelled Inhibitor of Caspases FLICA staining test. Caspases activation status was evaluated in lymphocytes CD3+, monocytes CD14+ and natural killer NK CD56+ cells. Cells were stained in parallel with 7AAD to visualize late dead cells. The correlation with colony forming units CFU-GM and the number of days of aplasia were evaluated.

RESULTS: Caspases-dependent pathway is activated in 22% in CD34 cryoconseved after thawing while this percent was less than 5% in fresh CD34 (p<0.0001). Caspases activation was observed at significantly amounts in other thawing blood cells. Percent of FLICA thawing CD3+, CD56+ and CD14+ cells was 12, 25 and 5% compared to 2, 8 and less than 1% in fresh cells.Induction of caspase-dependent apoptosis in CD34+ after thawing affects clonogenicity. FLICA +CD34 reduced the clonogenicity. Only Flica negative cells are able to differentiate in CFU-GM. Length of aplasia was not affected by the percentage of CD34+ cells engage in a caspases-dependent apoptosis after thawing.

CONCLUSION: Our results suggest that cryopreservation induced apoptosis by a caspases-dependent pathway in all the subset of apheresis product and mainly CD34 cells. These results modify the quality control of the product but do not affect the clinical outcome. The percent of CD3 and CD56 FLICA positive cells could modify the effect of DLI. More data will be presented