Hypoxia/hypercapnia prevents iron-dependent cold injuries in cord blood stem and progenitor cells.

BACKGROUND: Cold-induced cell injuries are associated with an increase in the cellular labile iron pool (LIP) followed by lipid peroxidation and alteration of mitochondrial function, which lead to cell death. Recently, we showed that incubation in a hypoxic/hypercapnic (HH) gas mixture improved the survival of a population of cord blood hematopoietic progenitors and CD34+ hematopoietic progenitor and stem cells in severe hypothermia. To explain the underlying mechanism, here we test if this HH-induced cytoprotection in cold conditions is associated with the level of LIP and lysosome stability. METHODS: Cord blood CD34+ cells were incubated in air (20% O2/0.05% CO2) or in the hypoxic (5% O2)/hypercapnic (9% CO2) atmosphere for 7days at 4°C and analyzed. RESULTS: Incubation in HH condition maintained the day 0 (D-0) level of LIP detected using a bleomycin-dependent method. This was associated with preservation of lysosome integrity and a higher cell survival. Conversely, in the air condition LIP was significantly increased. Also, the presence of a moderate concentration of iron chelator deferoximine improves the conservation of total CD34+ cells and committed progenitors in air condition. Pre-treatment of CD34+ cells with the lysomotropic agent imidazole induces significant decrease in the lysosomal stability and in all conditions. This is associated with an important decrease of survival of conserved cells and an increase in the cellular LIP level. DISCUSSION: Our study showed that HH gas mixture cytoprotection during hypothermia maintains lysosome stability, which enables preservation of the cellular chelatable iron in the physiological ranges. These findings suggest a way to optimize cell conservation without freezing.

A new clinical-scale serum-free xeno-free medium efficient in ex vivo amplification of mesenchymal stromal cells does not support mesenchymal stem cells.

BACKGROUND: We evaluated a new serum-free, xeno-free medium (Xuri, GE HealthCare) in ex vivo cultures for amplification of mesenchymal stromal cells (MStroC) in comparison with classical culture supplemented with fetal calf serum and basic fibroblast growth factor. STUDY DESIGN AND METHODS: MStroC and mesenchymal stem cell (MSC) proliferative capacities were studied in bulk cultures and single-cell cultures with assay of secondary replating capacity of individual clones. Flow-cytometric phenotype analysis and proliferative history analysis were also performed. RESULTS: In cultures initiated with previously amplified and cryopreserved MStroC from human marrow, Xuri medium enabled a total cell expansion fold comparable to one obtained in control fetal calf serum (FCS)-supplemented culture. However, both the number and the proliferative capacity of colony-forming unit-fibroblast were greatly reduced in Xuri medium cultures. This is even more evident in single-cell cultures, where, in rare positive wells, only several cells were found in Xuri cultures, compared to abundant cell content in FCS and α-minimal essential medium cultures. Replating these single-cell clones in secondary cultures (FCS in both cases) revealed a total exhaustion of MSC proliferative capacity after Xuri primary culture. CONCLUSION: Since in both conditions after a 7-day bulk culture, similar immunophenotype and proliferative history were found when the standard MSC immunophenotype panel was employed, the loss of proliferative capacity in Xuri medium shows that it cannot maintain functional MSC population. This is a drastic example showing that the real MSC activity can be completely unrelated to the immunophenotype considered as MSC phenotype.