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

Abstract

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.

An immobilized nanoparticle-based platform for efficient gene knockdown of targeted cells in the circulation

“…Therefore, PEGylation of the particles is important for stability under flow and target specificity. Consistent with our previous reports, 19,25 large numbers of cells were captured from flow by the PS-DSPE-PEG-NP surface. Although P-selectin-based cell capture is also associated with inflammation of the liver or lung in vivo, our previous work in rats has shown that the risk of systemic inflammatory conditions will be minimal due to the lack of inflammatory chemokines and b-2 integrin ligands on the device surface.…”

“…On the basis of our previous results, 25 we expected that 26 After cellular perfusion, a near monolayer of HL60 cells was tethered (Figure 5d) and accompanied by a thin layer of PS-DSPE-PEG NPs absorbed onto the inner surface of the microtube (Figure 5c). Furthermore, the coated PS-DSPE-PEG NPs were released with the tethered cells by higher shear stress and air embolism from the microtube surface (Figures 5e and f).…”

“…29 To silence genes in circulating cells, an implantable device for siRNA delivery was developed in this study. On the basis of our previous results, 18,19,25,30 P-selectin, an adhesion molecule generated in endothelial and platelet cells and involved in the capture of leukocytes from the bloodstream, 22,31 was selected to construct liposome-based targeted NPs for siRNA delivery. This surface modification endowed the NPs not only with target specificity but also with the ability to adhere onto the inner surface of microtubes.…”

Nanomechanical Control of Cell Rolling in Two Dimensions through Surface Patterning of Receptors

“…Researchers have also investigated the possibility of separating cells based on rolling, including a technique for capturing cells in microfluidic devices 24,25 and separation of cells based on differential rolling velocities in a flow chamber 23 . Separation based on cell rolling is applicable to a wide range of cell types such as leukocytes, hematopoietic stem/progenitor cells, and metastatic cancer cells [19][20][21]25 . Recent work has also shown that co-immobilization of selectins and antibodies that are specific to molecules expressed on the cell surface can affect the rolling behavior of cells, further enhancing the utility of rolling-based separation 26 .…”

Stem cell manipulation, gene therapy and the risk of cancer stem cell emergence

“…1961: Demonstration of the existence of stem cells (2) 1981: First culture of pluripotential cells from mouse embryos (4) 1998: human Embryonic Stem Cell line (6) 2006: Induced Pluripotent Stem cells (iPSC) from mice (7) 2008: -Obtention of iPSC with c-Myc infection (11) -Use of iPSC in an animal model of Parkinson's disease (12) 2010: Obtention of iPSC only with Oct4 and chemical compounds (13) 2016: Complete reconstitution of pupps starting from skin cells (17) 1958: First use of a nuclear transfer approach in tadpole (1) 1974: First transgenic mice (3) 1996: Cloning of Dolly (5) 2007: -Yamanaka's factors to obtain iPSCs (8) -Obtention of humain iPSCs from human somatic cells (9) -Use of iPSC technology to treat sickle cell disease in mice (10) HSC engraftment, including selectin modifications or fucosylation of HSCs (38). Lastly, molecular mechanisms controlling HSC fate determination must be fully deciphered as well as understanding the complexity of HSCs due to their heterogeneity.…”