Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow

Abstract

The hematopoietic stem cell (HSC) is a unique cell type found in bone marrow, which has the capacity for both self-renewal and differentiation into all blood lineages. The identification of genes expressed specifically in HSCs may help identify gene products vital to the control of self-renewal and/or differentiation, as well as antigens capable of forming the basis for improved methods of stem cell isolation. In previous studies, we identified a number of genes that appeared to be differentially expressed in murine bone marrow-derived HSCs, using microarray technology. We report here that one of those genes, encoding the murine endothelial protein C receptor (EPCR), is expressed at high levels within the bone marrow in HSCs. Bone marrow cells isolated on the basis of EPCR expression alone are highly enriched for hematopoietic reconstitution activity, showing levels of engraftment in vivo comparable to that of stem cells purified using the most effective conventional methods. Moreover, evaluation of cell populations first enriched for stem cell activity by conventional methods and subsequently fractionated on the basis of EPCR expression indicates that stem cell activity is always associated with EPCR-expressing cells. Based on our findings, we believe EPCR represents the first known marker that 'explicitly' identifies hematopoietic stem cells within murine bone marrow.

Figure 1.

Specific expression of EPCR by SP cells. (A) RNA from sorted side population (SP) or main population (MP) cells was extracted and subjected to multiplex RT-PCR to detect expression of EPCR message. EPCR message was easily amplified from SP cell RNA but was nearly undetectable within a similar quantity of MP RNA (n = 3). (B) C57BL/6 total bone marrow was stained with Hoechst 33342 dye to identify the SP and further stained with mAb 1560, a monoclonal antibody specific for EPCR. Gray histograms denote level of staining produced by isotype control antibody. Red and blue histograms represent specific staining by mAb 1560. EPCR protein is readily detectable at the cell surface of SP cells with very little expression detectable in MP (n = 5).

Figure 2.

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 EPCR^hi 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.

Figure 3.

Hematopoietic reconstitution potential of EPCR-positive cells and other purified stem cell populations. (A) Competitive repopulation experiments showing the contribution to blood chimerism made by indicated numbers of cells purified by different methods (n = 4 to 12 mice per group). (B) Kinetics of 1000 HSC engraftment of mice shown in panel A over the course of study. Error bars represent standard deviation.

Figure 4.

EPCR expression accurately identifies HSC activity. (A) Flow cytometry of total bone marrow stained with both Sca-1 and EPCR antibodies showing that EPCR identifies a distinct subfraction of Sca-1^hi cells that constitutes approximately 10% of the Sca-1^hi population. Black, green, and red text indicates the percentage of total cells found to be Sca-1^hi, Sca-1^hi EPCR^–, and Sca-1^hi EPCR⁺, respectively (left panel). Right panel indicates level of engraftment 4 months after transplantation of 100 cells sorted using the gates shown in the left panel in competition with 200 000 unfractionated bone marrow competitor cells. EPCR expression clearly fractionates all stem cell activity from the Sca-1^hi population. (B) Gating logic used to isolate the 500 SP cells, either positive or negative for EPCR expression, that were transplanted in competitive repopulation experiments against 200 000 unfractionated bone marrow competitor cells (left panels). Engraftment of cells sorted 4 months after transplantation showing little engraftment activity by SP cells lacking EPCR (right panels). (C) Gating logic used to isolate 500 EPCR-expressing cells exhibiting either SP or MP dye-efflux phenotype (left panel). Repopulation of sorted cells 4 months after transplantation in competition with 200 000 unfractionated bone marrow cells showing significant engraftment by EPCR-expressing MP cells (right panel). Error bars represent standard deviation.