Cell cycle analysis of the CD133 + and CD133 − cells isolated from umbilical cord blood

Abstract

Background: Umbilical cord blood cells (stem/progenitor cells) exhibit high proliferative capacities leading to a large expansion of cells in appropriate cell culture conditions. The aim of this study was to evaluate by flow cytometry the cycling status of CD133 + and CD133 − cells depending on various culture conditions, such as sera, stem cell factor (SCF), interleukin 3 (IL-3) and interleukin 6 (IL-6). Methods: An immunomagnetic system was used for cell separation. CD133 + and CD133 − cells were seeded in Iscove's Modified Dulbecco's Medium (IMDM) with different serum concentrations and were stimulated with SCF (100 ng/ml), IL-3 (50 ng/ml) and IL-6 (50 ng/ml). Results: Our experiments demonstrated that immediately after separation, 96.75±0.58% of CD133 + cells and 97.04±1.76% of CD133 − cells were in G 0/G 1-phase, while 2.02±0.38% and 0.88±0.52% were in the S-phase, respectively. Our data documented that CD133 + cells are more active than CD133 − cells after the first week of cultivation ( p<0.01). Statistically significant difference was found for CD133 + cells vs. CD133 − cells after second week of cultivation in G 0/G 1- and S-phases under all tested conditions. A combination of 12.5% FCS+12.5% HS yielded the highest cell expansion for CD133 + cells; this was concomitant with highest percentage of S-phase and G 2M-phase. Our data show that the medium with 25% HS was the best for cell expansion and cycling of the CD133 − cells for the first week, followed by the 12.5% FCS+12.5% HS. After 2 weeks of cultivation, obviously 12.5% HS and 12.5% FCS+12.5% HS exhibited similar S-phase amounts in CD133 − cells. A decrease of HS concentrations seemed to stimulate CD133 − cells' S-phase after the second week. Conclusions: Our data indicate that the source and the concentration of the serum used for cultivation have an impact on both cell populations: CD133 + cells are most comfortable with a combination of FCS and HS; CD133 − cells prefer media-containing HS. Cell cycle status may be an important factor for defining cultivation strategies for stem cell expansion.