Murine Side Population Cells Contain Cobblestone Area-forming Cell Activity in Mobilized Blood

Primitive hematopoietic stem cells (HSCs) can be purified from murine bone marrow by sorting Hoechst 33342-effluxing side population (SP) cells. The aim of this study was to establish whether SP cells from peripheral blood contain primitive HSCs and whether this is altered in mice following mobilization. SP cells were analyzed and isolated from bone marrow and blood of mice after mobilization; the HSC content of isolated SP cells was determined through surrogate cobblestone area-forming cell (CAFC) assays. SP cells in normal blood were not found in the high Hoechst dye effluxing portion of the SP tail, did not express the stem cell markers c-Kit and CD34, and did not have measurable CAFC activity. In contrast, SP cells in mobilized blood expressed both stem cell markers, contained cells in the high dye efflux portion of the SP tail, and displayed significant day- 28 to day-35 CAFC activity with 165- to 334-fold enrichment. In comparison to mobilized blood SP cells, normal marrow SP cells contained a higher proportion of cells expressing c-Kit and CD34 and had a greater percentage of cells in the high Hoechst dye-effluxing portion of the SP tail. Analysis of SP cells in the bone marrow after mobilization revealed a decrease in the frequency of SP cells, in expression of c-Kit and Sca+ CD34⁺/CD34^-, and in day-7 to day-35 CAFC activity, consistent with mobilization into blood. We conclude that murine SP cells mobilized into blood contain primitive hematopoietic stem cell activity (day-28 to day-35 CAFC activity). This model offers a means to study the mechanisms of mobilization of primitive stem cells directly in a murine model.