3.1. Effects onmigration
Chemotaxis of CD34+ cells towards a gradient of SDF-1α and CXCR4 agonists or antagonists was assessed in a transwell migration assay. The number of migratory cells increased continuously with the SDF-1α concentration of up to 500 ng/mL SDF-1α . In contrast, neither the agonist CTCE-0214 nor the antagonists CTCE-9908 or AMD3100 induced any significant migration under these conditions (Figure 2(a)). However, a further increase in the concentration of CTCE-0214 (100 μg/mL) resulted in a moderate increase in migration of CD34+ cells.
3.2. Effects on podia formation
Polarization and podia formation are prerequisites for directed locomotion of HPC. We have analyzed the podia formation of CD34+ cells upon 4-hour treatment with SDF- 1α, CXCR4 agonists or CXCR4 antagonists. Consistent with our previous report 15% (±2%) of the cells demonstrated an elongated morphology with a prominent uropod in the control experiments . The percentage of polarized cells with a prominent uropod increased under treatment with SDF-1α in a concentration-dependent manner (up to 40%± 10% in SDF-1α 500 ng/mL). Surprisingly, a significant increase in podia-forming cells was also achieved by the agonist CTCE-0214 or the antagonist CTCE-9908. Thus, these compounds might induce podia formation, probably via cytoskeletal rearrangements, but did not induce directed migration in concentrations between 10 ng/mL to 500 ng/mL. AMD3100 did not affect uropod formation (Figure 2(b)).
3.3. Effects on survival and proliferation
CD34+ cells were cultured in a culture medium supplemented with a cytokine cocktail (Epo, IL-3, IL-6, GM-CSF, SCF, bFGF, and IGF-1) as described before [28, 29]. A high cell viability was observed and less than 1% of the cell population was PI positive upon treatment with any of the CXCR4 agonists or antagonists. Most of the cells divided up to six times after five days and cell division kinetics were not affected by the compounds (Figures 3(a) and 3(b)). Thus, we reasoned that a cytokine deprived culture condition might be more suitable for analysis of survival and proliferation. Culturing of enriched CD34+ cells in RPMI-1640 medium with 20% FCS for seven days without any supplements resulted in a reduced cell viability. According to forward scatter (FSC) and side scatter (SSC), the cell population could be clearly distinguished and represented about 30% of the counted events. About 3.1% ± 0.9% of these cells were positive for propidium iodide (Figure 3(a)). The addition of SDF-1α, CTCE-0214, CTCE-9908, or AMD3100 (500 ng/mL each) did not have any significant impact on the cell number according to counted events, nor on the percentage of PI positive cells (3.9% ± 0.7%; 3.0% ± 0.4%; 2.0% ± 1.3%; 3.4%±1.2%; 3.3%±0.5%, resp.). Of the remaining cells approximately 50% have divided as determined by dilution of CFSE dye. SDF-1α and its analogs did not have any significant effect on proliferation of CD34+ cells although there was a tendency towards more cell divisions with CTCE-0214 (Figure 3(c)).
3.4. Effects on adhesion
The SDF-1α/CXCR4 pathway plays a significant role in homing and mobilization of hematopoietic stem cells. Human mesenchymal stromal cells from bone marrow (BM-MSC) represent a surrogate model for studying molecular mechanisms of adhesion of human HPC towards the cellular determinants of the niche. This heterotypic cell-cell adhesion was analyzed using a novel adhesion assay . By means of gravitational force, HPCs were separated into those adherent to the feeder layer and those that dropped to the original covering glass upon inversion of the culture preparations. Furthermore, we have analyzed adhesion of HPC to a protein coated glass surface without feeder layer cells. About 20% ± 1% of the cells demonstrated unspecific binding to BSA and 28% ± 6% remained attached to fibronectin coated surfaces . In contrast, 74% ± 10% of the CD34+ adhered to BM-MSC under control conditions without addition of SDF-1α analogs. As demonstrated in Figure 4, there was a significant reduction in cell adhesion upon treatment with SDF-1α (55% ± 13%; P = .014), CTCE-0214 (46% ± 16%; P = .7) and AMD3100 (32% ± 11%; P = 7 × 10−5). The peptide antagonist CTCE-9908 did not have an effect on cell adhesion (74% ± 8%).
3.5. Effects on surface expression of CXCR4
Various reports demonstrated that SDF-1α induced internalization of the CXCR4 receptor [38–40]. In this study, we have analyzed surface expression of CXCR4 and CD34 either under treatment with CXCR4 agonists and antagonist (n = 4) or after two additional washing steps upon treatment with CXCR4 agonists and antagonists (n = 4). In four individual experiments without additional washing steps, 41.6%±7.9% of the cells within the CD34+ fraction expressed CXCR4 on the cell surface and this proportion was even higher in the residual CD34− cells (61.4% ± 14.9%, P = .07). Pretreatment with SDF-1α reduced the CXCR4 expression within the CD34+ cells (30.0% ± 13.9%, P = .13). Detection of CXCR4 was significantly reduced upon pretreatment with AMD3100 (6.5%±0.8%, P = .002). The peptide agonist CTCE-0214 did not show any effect on CXCR4 expression (43.9% ± 12.9%), whereas the peptide antagonist CTCE-9908 enhanced the proportion of CXCR4+ cells (47.0% ± 6.1%, P = .02). A representative experiment of four is shown in Figure 5. Additional washing steps with ice-cold PBS to remove SDF-1α agonists and antagonists prior to antibody staining revealed the same tendency, although CXCR4 detection was higher upon removal of AMD3100 (control 32.8% ± 3.3%; SDF-1α 20.7% ± 5.2%, P = .06; CTCE-0214 31.2% ± 5.2%; CTCE- 9908 33.9% ± 4.7%; AMD3100 22.5% ± 6.1%, P = .02). In addition, cells were permeabilized upon treatment with the SDF-1α agonists and antagonists. In the control, nearly all cells (98.45%) were positive for CXCR4 and no decrease in CXCR4 detection was observed after cytokine stimulation.