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

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 hypoth esis that flow-based, adhesion molecule-mediated capture maybe a viable alternative approach to the capture and purification of HSPCs. (c) 2007 American Association for Clinical Chemistry.