EPCR-expressing marrow correlates with HSC phenotype and shows significant CFU activity. (A) Correlation of expression level of EPCR to HSC phenotype was analyzed by performing multiparameter flow cytometry and gating the indicated intensity of EPCR expression (denoted by colored horizontal bars with percentage of total cells indicated) and replotting gated cells to display the indicated parameters. Frame color of each graph denotes the histogram gate in which plotted cells are found (black frame indicates total bone marrow). Each graph uses the identical set of gates for the specified combination of parameters set using isotype control antibodies. Hoechst profiles are shown with identical SP and MP gates, with top and bottom text indicating percentage of plotted cells falling into MP or SP gates, respectively. Replotting of EPCR-positive cells (gated in the top histogram) to display levels of other markers analyzed (bottom plots) shows that EPCR expression correlates well with enrichment for Sca-1 and c-Kit expression and depletion of CD34 and lineage marker staining. In the experiment shown, nonspecific antibody binding as measured by isotype control using the identical EPCRhi gate accounted for approximately 10% of EPCR-positive cells analyzed (n = 3). Numbers in panels Aii-Aiv represent the percentage of total cells found in each quadrant. (B) Populations gated in panel A were sorted and subjected to methylcellulose-based colony formation assays (CFU) to determine the relative proportion of each progenitor type found within various EPCR-expressing subfractions. Although accounting for only 1.4% of total bone marrow, EPCR intermediate–expressing cells contained a large proportion of colony-forming activity in the marrow. EPCR high–expressing cells, which were most highly enriched for cells exhibiting HSC immunophenotype, showed very little progenitor activity (n = 9). Error bars represent standard deviation.
