Figure 2

EML CD34 cell subsets exhibit distinct multilineage differentiation patterns, cell cycle profiles and proliferation kinetics.

(a) Colony-forming cell (CFC) assay analysis of the differentiation capacity of total EML cells and purified CD34^neg, CD34^low, CD34^int and CD34^high EML cell subsets into erythroid, megakaryocytic and granulocyte-macrophage lineages (n = 15). The histograms and the table show the numbers of granulocyte-macrophage (CFU-GM), megakaryocytic (CFU-Meg) and erythroid (BFU-E) colonies generated by total EML cells and each EML CD34 cell subset (10³ cells/well) in the presence of cytokines IL-3, Tpo and Epo, respectively. Data are mean ± SD. * P value < 0.05, ** P value < 0.001. (b) Analysis of cell cycle profiles of total EML cells and CD34^neg, CD34^low, CD34^int and CD34^high EML cell subsets using Propidium Iodide, Hoechst 33342 and Ki-67 vs 7-AAD staining (n = 3–5). Data are shown as mean ± SD. * P value < 0.05, ** P value < 0.001. (c) Flow cytometry analysis of proliferation kinetics of total EML cells by CFSE labeling. The grey-shaded histogram on the left side of the panel shows unlabeled cell auto-fluorescence and the limits of detectable cell divisions. The histogram outlined in red on the right side of the panel shows CFSE signal in EML cells immediately after the labeling. The overlay of CFSE signal at 24 hour time points following labeling shows EML cell division history. During each cell division CFSE is partitioned equally among daughter cells, resulting in a corresponding uniform decrease in CFSE fluorescence among all dividing cells. Synchronous cell divisions result in a corresponding uniform decrease in CFSE signal among all dividing cells and narrow fluorescence histograms. (n = 2). (d) Analysis of proliferation kinetics of CD34^neg, CD34^low, CD34^int and CD34^high EML cell subsets by CFSE labeling. The overlay of CFSE signal at 24 hour time points following labeling shows division history and proliferation kinetics of each EML cell subset. The cells within CD34^neg subset exhibited more asynchronous and slower division at each time point (see also Supplementary Fig. S5), as shown by varying levels or CFSE fluorescence resulting in broader and overlapping histograms at different time points after CFSE labeling (n = 4).