Abstract
Sulfated glycans such as fucoidan have been shown to induce rapid hematopoietic progenitor stem cell (HPCs) mobilization in mice and primates. Our previous clinical study has demonstrated up-regulation of CXCR4 on peripheral CD34+ cells in volunteers who had received oral doses of 75% pure fucoidan. The aim of this study was to investigate the in vitro effects of fucoidan on a cytokine mediated CD34+ cell expansion system and to characterize the fucoidan effects on CD34+ proliferation and differentiation using a factorial design experiment and flow cytometry. Isolex selected CD34+ cells harvested from multiple myeloma patients were used in the study. There is institutional approval for the in vitro growth of CD34+ cells from mobilized peripheral blood.
Fractional (25−1 resolution V, 16 runs, 2 blocks, E=ABCD) and full (24, 16 runs, 2 blocks) factorial design experiments were used to investigate the effects of depyrogenated fucoidan (DP-GFS) and cytokines on the growth of CD34+ cells. The cells were grown in 24 well dishes (1 ml/well) at an inoculum density of 20,000 cells/ml in serum free media and cultured for 10 days in a cytokine cocktail including G-CSF, FL, SCF, TPO and SDF1 each at two levels 100 ng/ml or 1000 ng/ml. DP-GFS was used in the cultures at four different levels including 0, 10, 100 and 500 μg/ml. Cells were then harvested and analyzed using three color flow cytometry and a panel of antibodies that includes CD15, CD14, CD41a, CD38, CD34, and CXCR4.
The in vitro effect of DP-GFS on CXCR4 expression was opposite to that found in vivo using 75% fucoidan. At 100 ng/ml and at saturating levels of FL, SCF, TPO and G-CSF (1000 ng/ml), DP-GFS at 10 μg/ml was observed to reduce the number of CD34+ cells produced per input CD34+ cell (1.09±0.07 versus 0.84±0.05, n=16, p=0.002) and have a negative effect on the percentage of CD34+ cells that express CXCR4 (87.2±1.8 versus 77.4±2.3, n=16, p=0.001). There was no significant effect of DP-GFS on the total cellular output nor other cell subsets examined (CD41a+CD14−, CD15+, CD14+). Our in vitro results are more consistent with a recent clinical study that demonstrates down-regulation of CXCR4 receptors on CD34+ cells in G-CSF or GM-CSF mobilized blood (Dlubek et al, 2006). We hypothesize that fucoidan acts in vitro and in vivo via different mechanisms.
