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 × 104 to 7.1 × 104) than antibody devices (approximately 1.74 × 104 to 2.61 × 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.
SummaryClinical infusion of haematopoietic stem and progenitor cells (HSPCs) is vital for restoration of haematopoietic function in many cancer patients. Previously, we have demonstrated an ability to mimic physiological cell trafficking in order to capture CD34-positive (CD34 + ) HSPCs using monolayers of the cell adhesion protein P-selectin in flow chambers. The current study aimed to determine if HSPCs could be captured directly from circulating blood in vivo. Vascular shunt prototypes, coated internally with P-selectin, were inserted into the femoral artery of rats. Blood flow through the cell capture device resulted in a wall shear stress of 4-6 dynes/cm 2 . After 1-h blood perfusion, immunofluorescence microscopy and flow cytometric analysis revealed successful capture of mononuclear cells positive for the HSPC surface marker CD34. Purity of captured CD34 + cells showed sevenfold enrichment over levels found in whole blood, with an average purity of 28%. Robust cell capture and HSPC enrichment were also demonstrated in devices that were implanted in a closed-loop arterio-venous shunt conformation for 2 h. Adherent cells were viable in culture and able to differentiate into burst-forming units. This study demonstrated an ability to mimic the physiological arrest of HSPCs from blood in an implantable device and may represent a practical alternative for adult stem cell capture and enrichment.
Hematopoietic stem cell therapy is used to treat both malignant and non-malignant diseases, and enrichment of the hematopoietic stem and progenitor cells (HSPCs) has the potential to reduce the likelihood of graft vs host disease or relapse, potentially fatal complications associated with the therapy. Current commercial HSPC isolation technologies rely solely on the CD34 surface marker, and while they have proven to be invaluable, they can be time-consuming with variable recoveries reported. We propose that selectin-mediated enrichment could prove to be a quick and effective method for recovering HSPCs from adult bone marrow (ABM) on the basis of differences in rolling velocities and independently of CD34 expression. Purified CD34+ ABM cells and the unselected CD34- ABM cells were perfused over immobilized P-, E-, and L-selectin-IgG at physiologic wall shear stresses, and rolling velocities and cell retention data were collected. CD34+ ABM cells generally exhibited lower rolling velocities and higher retention than the unselected CD34- ABM cells on all three selectins. For initial CD34+ ABM cell concentrations ranging from 1% to 5%, we predict an increase in purity ranging from 5.2% to 36.1%, depending on the selectin used. Additionally, selectin-mediated cell enrichment is not limited to subsets of cells with inherent differences in rolling velocities. CD34+ KG1a cells and CD34- HL60 cells exhibited nearly identical rolling velocities on immobilized P-selectin-IgG over the entire range of shear stresses studied. However, when anti-CD34 antibody was co-immobilized with the P-selectin-IgG, the rolling velocity of the CD34+ KG1a cells was significantly reduced, making selectin-mediated cell enrichment a feasible option. Optimal cell enrichment in immobilized selectin surfaces can be achieved within 10 min, much faster than most current commercially available systems.
Current hematopoietic stem cell (HSC) separation methods utilize immunomagnetic techniques where antibodies are conjugated to paramagnetic or iron-dextran particles so that the cells can be precipitated using a magnetic field. Antibodies are usually targeted against CD34 antigen, a known HSC marker that is lost as the cell matures into a terminally differentiated blood cell. While this method is able to produce stem cell purities greater than 80% in most cases, cell yield is usually low (<50%) and there is evidence to suggest that not all HSCs express CD34 and hence, cannot be selected using these methods. Our long-term aim is to move away from immunologic isolation methods and instead focus on general HSC behavior in the presence of specific proteins. Selectins are a group of adhesion molecules that have been shown to selectively retard HSC rolling in comparison to more mature blood cells so that, under the right conditions, HSC isolation from whole blood should be possible. Since this functional method is independent of specific HSC surface marker, it would enable isolation of all HSC sub-populations as well as improve the overall yield. We have used two geometries for HSC isolation using selectins. The first assembly was a commercially available parallel plate flow chamber with rectangular cross-section. This proved to be inefficient for cell separation due to the emergence of dead zones within the chamber and excessive cell accumulation along the tubing and fittings that comprised the inlets and outlets. We have evaluated this chamber and two redesigns using finite elements software. Ultimately we decided on a simpler approach — to replace the flow chamber with a capillary tube, which reduced unwanted cell accumulation due to gravity or dead zones while increasing the effective area for separation, and hence allowing for better HSC isolation.
Clinical isolation and infusion of hematopoietic stem cells (HSCs) is a vital step for restoration of hematopoietic function in many cancer patients. Previously, we have demonstrated an ability to capture HSCs using monolayers of P‐selectin in parallel plate flow chambers. The aim of the current study was to determine if HSCs could be captured directly from circulating blood in vivo. Biocompatible micro‐tubes, coated internally with P‐selectin, were inserted into the femoral artery of rats mobilized with granulocyte colony stimulating factor. Blood flow through the device was verified and wall shear stress was measured to be 4–6 dynes/cm2. In devices removed after 1‐hr blood perfusion, immunofluorescence microscopy and flow cytometric analysis revealed successful capture of mononuclear cells positive for the HSC‐surface marker CD34. Purity of captured CD34+ cells represented a 7‐fold enrichment over levels found in whole blood, with an average purity of 28%. Robust cell capture and HSC enrichment were also demonstrated in P‐selectin coated devices implanted in a closed‐loop arterial‐venous shunt conformation and perfused for 2 hrs. Furthermore, adherent cells were viable in culture and differentiated into burst forming units. The study demonstrates an ability to mimic the physiologic arrest of HSCs from blood in an implantable device and may represent a practical alternative for HSC capture and enrichment.
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