Cord blood-derived hematopoietic progenitor cells: in vitro response to hematopoietic growth factors and their recruitment into the S-phase of the cell cycle

In the recent years many studies on the expansion of cord blood (CB)-derived progenitor cells have been performed, whereas less information is available on their cycling status. The objective of this study was to evaluate the cycling status of CB-derived colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC), and their recruitment into the S-phase of the cell cycle in response to a combination of cytokines.CB-derived CFC and LTC-IC were first quantified by standard clonogenic assay and long-term culture, respectively. In a second set of experiments, CB-derived progenitor cells were incubated with interleukin(IL)-3, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) and their cell cycle status assessed both by the cytosine arabinoside (Ara-C) suicide approach and by flow cytometric DNA analysis.We found that only small proportions of both CFC and LTC-IC were in the S-phase of the cell cycle. These estimates were confirmed by flow cytometric DNA analysis, which showed that 96% +/- 2% of CB-derived CD34+ cells were in G0/G1 and only 1.6% +/- 0.4% in the S-phase. Staining of CD34+ cells with an anti-statin monoclonal antibody, a marker of the G0 phase, indicated that among CD34+ cells with a flow cytometric DNA content typical of the G0/G1 phase, 68% +/- 7% of cells were in the G0 phase of the cell cycle. Twenty-four hour incubation with IL-3, SCF and G-CSF significantly increased the proportion of cells in the S-phase for both CFC and LTC-IC without inducing any loss in their number. Flow cytometric DNA analysis also showed an increase of CD34+ cells in the S-phase upon continuous exposure to these cytokines.Our findings indicate that: i) a small number of CB-derived CFC and LTC-IC are in the S-phase of the cell cycle; ii) a substantial number of CD34+ cells with a flow cytometric DNA content typical of the G0/G1 fraction are cycling, as they are found in the G1 phase of the cell cycle; iii) 24-hour incubation with IL-3, SCF and G-CSF can drive a proportion of progenitor cells into the S-phase without reducing their number.