Enrichment and purification of hematopoietic stem and progenitor cells (HSPCs) is important in transplantation therapies for hematological disorders and for basic stem cell research. Primitive CD34+ HSPCs have demonstrated stronger rolling adhesion than mature CD34- mononuclear cells on selectins. We have exploited this differential rolling behavior to capture and purify HSPCs from human bone marrow and rat peripheral blood by perfusing samples through selectin-coated microtubes.

Bone marrow- mononuclear cells were perfused through the cell capture microtubes coated with adhesion molecules. The device lumen was washed and captured cells were visualized and estimated by video microscopy. Adherent cells were eluted by high shear, calcium free buffer and air embolism. Immunofluorescence staining followed by flow cytometry was used to analyze CD34+ HSPCs. CD34+ HSPC purity of cells captured in adhesion molecule-coated microtubes was significantly higher than the fraction of CD34+ cells found in bone marrow- mononuclear cells (2.5 °" 0.8%). P-selectin coated surfaces yielded 16-20% CD34+ cell purity, while anti-CD34 antibody coated surfaces yielded 12-18%. Although the CD34+ cell purities were comparable between selectin and antibody surfaces, the total number of CD34+ HSPCs captured was significantly higher in P-selectin devices (~5.7-7.1 x 104) when compared to the antibody device (~1.74-2.61 x 104). Furthermore, analysis for cells positive for CD133, a surface marker for more primitive HSPCs, depicted approximately 10-14 fold enrichment in P-selectin samples over control bone marrow mononuclear cells. The captured cells were viable and exhibited in vitro colony forming capabilities.

In a parallel study we aimed at capturing HSPCs directly from circulating blood in vivo. Vascular shunt prototypes, coated internally with P-selectin, were inserted into the femoral artery GCSF mobilized rats. After one-hour blood perfusion, a successful capture of mononuclear cells was observed and analysis of such cells revealed a significant enrichment for CD34+ HSPCs. Blood flow through the cell capture device resulted in a wall shear stress of 4-6 dynes/cm2. Purity of captured CD34+ cells showed 7-fold enrichment (average purity = 28 „b 4%) over levels found in the control mobilized peripheral blood mononuclear cells (average purity = 3 „b 0.5%). Robust cell capture and HSPC enrichment were also observed in microtubes that were implanted in a closed-loop arterio-venous shunt conformation for two hours. Adherent cells were viable and were able to differentiate into burst forming units in culture.

Thus, P-selectin can be used in a compact flow device to capture and enrich HSPCs. This study supports the hypothesis that flow-based adhesion molecule-mediated capture may be a viable physiologic approach to the capture and purification of HSPCs.

References:

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2. Narasipura SD, Wojciechowski JC, Charles N, et al. P-selectin- coated microtube for enrichment of CD34+ hematopoietic stem and progenitor cells from human bone marrow. Clin Chem 2008;54:77-85.

3. Charles N, Liesveld JL, King MR. Investigating the feasibility of stem cell enrichment mediated by immobilized selectins. Biotechnol Prog 2007;23:1463-72.

4. Wojciechowski JC, Narasipura SD, Charles N, et al. Capture and enrichment of CD34-positive haematopoietic stem and progenitor cells from blood circulation using P-selectin in an implantable device. Br J Haematol 2008;140:673-81.