Macrophages are passengers in CGS expansion cultures. (A) Cytopathology evidence for macrophages. Cytospins were prepared from day 14 cord blood CD34⁺ Y* CGS expansion cultures and stained by Wright-Giemsa. The representative slide shows the presence of morphologically distinct macrophage (Mac) and their tight physical association with developing erythroblasts reminiscent of erythroid islands. The presence of an enucleated RBC is also indicated. (B) Immunophenotyping evidence for macrophages. Cells from day 14 cord blood CD34⁺ Y* CGS expansion cultures were stained for the activated macrophage cell surface marker CD206 and analyzed by flow cytometry. The representative histogram shows the presence of 5% CD206⁺ cells, and that only 20% of these also express viral vector GFP. Gates were set based on staining controls. The percentage of cells in each quadrant is indicated. (C) Cytopathology of CD206⁺ cells. Cells expressing CD206 were sorted and analyzed by cytospin and Wright-Giemsa staining. The representative slide shows a preponderance of large macrophages with large foamy cytoplasm and blue nuclei. (D) Contribution of macrophages to expansion cultures. The relative frequency of CD235a⁺/CD41a⁻ erythroid cells and activated CD206⁺ macrophages were tracked over time in cord blood CD34⁺ cultures expanded with the Y* CGS by immunofluorescent staining and flow cytometry. The relative frequency of cells expressing vector GFP is shown for the 2 populations early and late in culture. Data represent the mean ± SE from 3 independent experiments. P values are based on the t test across all time points.