Folia Biologica
Journal of Cellular and Molecular Biology, Charles University 

Recovery from radiation-induced bone marrow aplasia depends on appropriate cytokine support. The aim of our work was to find a cytokine combination allowing in vitro gamma-irradiated (2.5 Gy) CD34⁺/AC133⁺ haematopoietic stem cells to evade radiation-induced apoptosis and to enhance damage reparation, which should enable proliferation and ex vivo expansion of cells. Cells were isolated using separation in a Cobe separator followed by immunomagnetic selection by antibody against the AC133 antigen. Thus isolated cells were 80% AC133⁺/CD34⁺ and 10% of them expressed the CD33⁺ antigen. Ten thousand of AC133⁺ cells formed 1146 CFU-GM and 304 BFU-E. We proved a high expansion efficiency of cytokine combination SCF + IL-3 + FLT3L in comparison with the combination SCF + IL-3 + IL-11 in both, non-irradiated cells and cells irradiated with a dose of 2.5 Gy. The D₀ value for AC133⁺ cells was determined by the clonogeneity test. The D₀ value for CFU-GM was estimated to be 1.08 Gy and for BFU-E 0.95 Gy. The results of DNA analysis showed that the majority of isolated AC133⁺ cells were in G₀/G₁ of the cell cycle. We proved that the dose of 2.5 Gy induced massive apoptosis (80%) of these cells without progression through the cell cycle, which indicates interphase cell death. Under the influence of cytokine combination (SCF + IL-3 + FLT3L), the surviving 20% of cells entered the cell cycle and, similarly to non-irradiated control cells, on 7th day 35% of cells were in S phase.

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