Abstract
In vitro cell expansion is constrained by default pathways of commitment and differentiation resulting in limited expansion of hematopoietic stem-progenitor cells (HSPCs). Still, several ex vivo manipulations have been reported to achieve expansion of HSPCs by altering cell cycle kinetics and enhancing progression through the G1-S barrier.
We have previously shown that addition of tetraethylenepentamine (TEPA), a polyamine copper chelator, to cytokine-supplemented CD34+ cell cultures modulates cytokine-driven hematopoietic cell fate in vitro, resulting in remarkable expansion of a cell population that displays phenotypic and functional characteristics of HSPCs (Exp Hematol.2004;32 (6):547–55).
The objective of the present study was to evaluate the mechanism leading to expansion of early progenitor cells following short-term exposure to TEPA. To this end, cell cycle profile, tracking of proliferation history, as well as determination of actual numbers of progenitor subsets were studied.
In order to follow the extent of proliferation by tracking the number of cellular divisions, freshly isolated CD34+ cells were labeled with PKH2, a membrane dye that is sequentially diluted during every cell division. Fluorescence intensities of CD34+ and that of a more immature CD34+CD38− cell subset were determined immediately after staining. The cells were then cultured in serum-containing medium and a cocktail of cytokines (SCF, TPO, IL-6, Flt3-ligand, at 50 ng/ml each and IL-3 at 20 ng/ml), with and without TEPA. Total nucleated cells (TNC), purified CD34+ cells and CD34+CD38− cells were analyzed for PKH2 fluorescence intensity during the first two weeks of culture. Cell cycle profile was detected with the DNA intercalating agent propidium iodide, which determines cellular DNA content.
FACS analysis of the cultured cells as well as progenitor cell quantification by immuno-affinity purification revealed comparable expansion levels of TNC and CD34+ cells in both TEPA-treated and control cultures during the first two weeks, as previously published. Although similar CD34+ cell numbers were observed, the mean frequency of CD34+CD38− and CD34+CD38-Lin- cells within the CD34+ cell population was significantly higher in TEPA-treated cultures over the control (0.2 vs. 0.04 and 0.07 vs. 0.01, respectively; n=6, p<0.05). Median PKH2 fluorescence intensity of CD34+CD38− subset was two fold higher in TEPA than in control cultures, demonstrating that early progenitor cells derived from TEPA-treated cultures consistently accomplished less proliferation cycles as compared to early progenitor cells derived from control cultures. This effect was not mirrored by a significant alteration of the cell cycle profile (Control (%): G1=26±14, S=2.6±0.1, G2=0.7±0.4; TEPA(%): G1=29±12, S=1.7±0.9, G2=0.4±0.2).
Taken together, the data suggest that during cycling, the CD34+CD38− phenotype is preserved more successfully in TEPA-treated than in control cultures, suggesting retention of self-renewing potential of early progenitor cells under these culture conditions. This mechanism also supports a role for TEPA in inhibition of early progenitor cell differentiation. Ongoing work is aimed at further defining whether phenotype reversion or self-renewal (or both) lie at the foundation of TEPA-mediated progenitor cell expansion.
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