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Stromal cell-derived factor-1alpha (SDF-1alpha) has pleiotropic effects on hematopoietic progenitor …
Biology Articles » Biotechnology » Red Biotechnology » The Many Facets of SDF-1alpha CXCR4 Agonists and Antagonists on Hematopoietic Progenitor Cells » Figures
Figure 1: SDF-1a/CXCR4 axis and interaction with small molecules. The chemokine stromal cell-derived factor-1 alpha (SDF-1a or CXCL12) is secreted by stromal cells with various effects on hematopoietic progenitor cells (HPCs). It binds to the CXCR4 receptor and this interaction can be influenced by the peptide agonist CTCE-0214 as well as by the peptide antagonists CTCE-9908 or the nonpeptide antagonist AMD3100.
Figure 2: Effects on cell migration and podia formation.The effect of the SDF-1a, CXCR4 agonist, and CXCR4 antagonists on migration of human CD34+ cells was assessed in transwell migration experiments. The fold increase in migration upon treatment with these compounds was determined in relation to the corresponding control in four to eight independent experiments. SDF-1a induced chemoattraction in a dose-dependent manner. The same concentration of the peptide agonist CTCE-0214 and the peptide antagonist CTCE-9908 did not have any significant effect on migration of CD34+ cells although there was a significant effect using much higher concentrations of CTCE-0214. (a) *P
Figure 3: Effects on Proliferation. CD34+ cells were stained with CFSE and subsequently grown for 5 days in culture medium with a cytokine cocktail (Epo, IL-3, IL-6, GM-CSF, SCF, bFGF, and IGF-1) or for 7 days in culture medium without cytokine supplements. Cultivation with the cytokine cocktail resulted in a very high viability of > 95%, whereas without cytokines the cell population could be distinguished according to forward scatter (FSC) and side scatter (SSC) from cell fragments (black spots: PI positive; grey spots: PI negative). (a) The number of cell divisions was estimated within the PI negative cell population by the dilution of CFSE dye.Whereas the CD34+ cells demonstrated a high proliferation with the cytokine cocktail, only about 50% of the cells proliferated without these cytokines. (b) SDF-1a, the peptide agonist CTCE-0214, the peptide antagonist CTCE-9908, and the nonpeptide antagonist AMD3100 did not have a significant effect on the fraction of proliferating cells (c) nor on the percentage of PI negative cells (three independent experiments).
Figure 4: Effects on adhesion. Adhesion of CD34+ cells was either analyzed on protein-coated surfaces (white bars) or on mesenchymal stromal cells (MSCs) from human bone marrow (black bars) in culture media supplemented with SDF-1a, CTCE-0214, CTCE- 9908, or AMD3100. Adhesion was significantly reduced by SDF-1a, peptide agonist CTCE-0214, as well as by the mobilization agent AMD3100 (three independent experiments; ‡P = .014; #P = .007; †P = .7 × 10-4; ¶P = .002).
Figure 5: Effects on surface levels of CXCR4 detected by flow cytometry. Surface expression of CXCR4 (phycoerythrin; PE) and CD34 (allophycocyanin, APC) was analyzed on the surface of CD34+ enriched cells upon treatment with CXCR4 agonist and antagonist without additional washing steps. Demarcation of autofluorescence is indicated by black lines. The proportion of CXCR4+ cells was higher in residual CD34- cells compared to the CD34+ fraction. CXCR4 detection was reduced upon treatment with SDF-1a and AMD3100. The peptide agonist CTCE-0214 did not affect the proportion of CXCR4+ cells, whereas the peptide antagonist CTCE-9908 increased this proportion. Concentration of cytokines was 500 ng/mL. Representative plots of four independent experiments are shown.
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