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P-Selectin–Coated Microtube for Enrichment of CD34⁺ Hematopoietic Stem and Progenitor Cells from Human Bone Marrow

¹ Department of Biomedical Engineering, ² Department of Medicine, Wilmot Cancer Center, University of Rochester, ³ CellTraffix Inc, Rochester, NY.

^aAddress correspondence to this author at: Department of Biomedical Engineering, University of Rochester, Room 218, Goergen Building, Rochester, NY 14627. Fax 585-276-1999; e-mail mike_king{at}urmc.rochester.edu.

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 10⁴ to 7.1 x 10⁴) than antibody devices (approximately 1.74 x 10⁴ to 2.61 x 10⁴).

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.

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