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

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 G0/G1-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 G0/G1- 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 G2M-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.