P-Selectin coated microtube for enrichment of CD34+ hematopoietic stem and progenitor cells from human bone marrow.

BACKGROUND: Enrichment and purification of hematopoietic stem and progenitor cells (HSPCs) is important in transplantation therapies for hematologic disorders and in basic stem cell research. Primitive CD34+ HSPCs have demonstrated stronger rolling adhesion on selectins than mature CD34- mononuclear cells (MNCs). We have exploited this differential rolling behavior to capture and purify HSPCs from bone marrow by perfusing MNCs through selectin-coated microtubes.
METHODS: Bone marrow MNCs were perfused through the cell-capture microtubes coated with adhesion molecules. We washed the device lumen and visualized and estimated captured cells by video microscopy. Adherent cells were eluted by high shear, calcium-free buffer, and air embolism. We used immunofluorescence staining followed by flow cytometry to analyze CD34+ HSPCs.
RESULTS: CD34+ HSPC purity of cells captured in adhesion molecule-coated devices was significantly higher than the fraction of CD34+ cells found in bone marrow MNCs [mean (SE) 2.5% (0.8%)]. P-selectin-coated surfaces yielded 16% to 20% CD34+ cell purity, whereas antibody-coated surfaces yielded 12% to 18%. Although CD34+ cell purity was comparable between selectin and antibody surfaces, the total number of CD34+ HSPCs captured was significantly higher in P-selectin devices (approximately 5.7 x 10(4) to 7.1 x 10(4)) than antibody devices (approximately 1.74 x 10(4) to 2.61 x 10(4)).
CONCLUSIONS: P-selectin can be used in a compact flow device to capture HSPCs. Selectin-mediated capture of CD34+ HSPCs resulted in enrichment approximately 8-fold higher than the CD34+ cell population from bone marrow MNCs. This study supports the hypothesis that flow-based, adhesion molecule-mediated capture may be a viable alternative approach to the capture and purification of HSPCs.