MM HSPCs have disturbed migratory and adhesive properties. (A) Fluorescence micrographs show staining of (i,v) the nucleus, (ii,vi) the actin structure, (iii,vii) CD44, and (iv,viii) a merge in a representative HSC from a healthy donor and an MM patient, respectively. One representative of 4 experiments is shown. (B) For quantification of normal and defective actin assembly, 100 cells were counted and the mean percentages of cells with a normal and defective actin ring structure were determined. (C) Mean fluorescence intensity (MFI) of CD44 was determined by staining with an FITC-conjugated anti-CD44 Ab and flow cytometric analysis. Representative histogram plots of 6 experiments are shown for each subset indicated. Gray line indicates isotype control; black line, healthy donors; and red line, MM patients. (D) Bar charts represent the mean MFI of CD44 in healthy donors (□) and MM patients (■) for each HSPC subset indicated. Error bars represent SEM. (E) Chemotaxis toward stromal-derived factor 1 (SDF-1) in the lower chamber was allowed to continue for 3 hours at 37°C, 5% CO₂ in a humidified atmosphere. The relative transmigration of distinct HSPC subsets of MM patients (■) compared with their healthy counterparts (□) is shown. Highly purified HSPC subsets (5 × 10³ cells each) of 4 healthy donors (□) and 4 MM patients (■) were allowed to adhere on hyaluronic acid (HA)– and fibronectin (FN)–coated glass slides for 3 hours at 37°C, 5% CO₂ in a humidified atmosphere. Mean percentages of adherent cells on (F) HA- and (G) FN-coated glass slides are shown. (H) Equal numbers of BM HSPCs of 6 MM patients and 6 healthy donors were grown in liquid medium supplemented with SCF, TPO, and FLT-3L. On days 0, 2, 3, 6, and 7, viable cell concentrations were determined. Cell-cycle distribution of total BM HSPCs and HSPC subsets was determined ex vivo by staining with FITC-conjugated Ki-67 Ab (intracellular) and Hoechst 33 342 and flow cytometric analysis. The bar charts show the mean percentage of (I) HSPCs and (J) MEP in G₀ and G₁/G₂/S/M phases obtained from the BM of healthy donors (□) and MM patients (■). Error bars represent SEM. The Student t test was used to detect statistically significant differences. *P < .05; **P < .01; ***P < .001.