Impaired migration and F-actin polymerization in CD34⁺ cells from MDS patients. (A) CD34⁺ cells from healthy donors (normal bone marrow; NBM) and MDS patients were applied to the upper compartment of a microchamber transwell system with 5 μm pores. Migration was induced by 100 ng/mL SDF-1 in HPGM present in the lower compartment of the chamber. Cells were allowed to migrate for 4 hours at 37°C after which the transmigrated CD34⁺ cells were harvested from the lower chamber and counted by FACS analysis. The assay was done in duplicate, and results are expressed as the ratio between transmigrated CD34⁺ cells and the total amount of CD34⁺ cells added to the upper well. The means of 7 MDS patients and 7 healthy donors (NBM) are shown. (B) Isolated CD34⁺ cells were stained with CXCR4-Fitc, or isotype control IgG-Fitc for 20 minutes at 4°C. Cells were washed with phosphate-buffered saline, and the percentage of CXCR4-Fitc positive cells was determined by flow cytometry. The means of 9 MDS patients and 9 healthy donors are shown. (C) Isolated CD34⁺ cells were stimulated with 100 ng/mL SDF-1 for the indicated time. Cells were fixed and permeabilized, and F-actin was stained with 2.5 U/mL OregonGreen 514-Phalloidin. The fluorescence intensity of SDF-1-stimulated cells was determined by FACS analysis and presented as a percentage of that of unstimulated cells. The means of 8 MDS patients and 8 healthy controls (NBM) are shown.