Capture and enrichment of CD34-positive haematopoietic stem and progenitor cells from blood circulation using P-selectin in an implantable device

Abstract

Clinical 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.

Fig 1

Direct capture of blood-borne nucleated cells from circulation using P-selectin and non-coated control surfaces in implanted devices. Following incorporation into the femoral artery of anesthetized rats and 1-h blood perfusion, P-selectin coated tubes (A) showed a significantly greater average concentration of captured nucleated cells than non-coated control tubes (B) [184·6 ± 19·9 cells/mm² for P-selectin tubes (40 μg/ml) vs. 4·7 ± 1·4 cells/mm² for control surfaces (P < 0·01), bar = 50 μm]. (C) Total cell yields from 50 cm implanted tubes with cell adhesion molecule surfaces were significantly greater than the yield from non-specific binding in control tubes (**P < 0·01).

Fig 2

Paired brightfield and epi-fluorescence images of captured cells immunostained for CD34. Comparison of representative paired brightfield and fluorescent images of adherent cells immunolabelled with an antibody against a hematopoietic stem cell surface marker conjugated to quantum dots revealed blood-borne CD34-bright cells (white arrows) distinctly visible among CD34-dark cells on the capture surface (bar = 50 μm). Quantitative analysis confirmed a sixfold enrichment in the purity of CD34-positive cells on P-selectin surfaces over the proportion found in rat whole blood.

Fig 3

Flow-cytometric analysis of the adherent blood-borne cells from implanted tubes indicated a distinct population of CD34-positive cells on the capture surface. (A) Following extraction from the cell-capture tube, adherent mononuclear cells (MNCs) were analysed for surface expression of CD34 (the left-hand region of smaller platelets was excluded from analysis). (B) Immunofluorescent flow cytometric analysis of captured cells revealed a distinct population of MNCs positive for CD34 (arrow, subpopulation represents 38% of total MNCs) when compared to cells stained with isotype control antibody (bold line).

Fig 4

Purity of CD34-positive cells on implanted cell-capture devices with various adhesion-molecule surfaces and in whole blood of granulocyte colony-stimulating factor (GCSF)-mobilized and non-mobilized rats. As expected, the presence of CD34-positive cells was significantly higher in whole blood of GCSF-mobilized rats versus their non-mobilized counterparts (#P < 0·05). Following incorporation into the femoral artery of anesthetized rats and 60-min blood perfusion, purity of CD34-positive cells among mononuclear cells captured using P-selectin was elevated sevenfold over that of whole blood and non-coated control surfaces. CD34⁺ cell-purity in tubes incubated with 40 μg/ml P-selectin was significantly greater than that from capture surfaces coated with less P-selectin, and was similar to surfaces coated with CD34 antibody (*P < 0·05).

Fig 5

Mononuclear cell (MNC) capture and CD34-positive cell purity from implanted cell-capture devices inserted into the rat femoral artery and perfused with a single-pass of blood for 1 h, or incorporated completely into the circulatory system of the rat via arterial-venous shunt for continuous 2-h recirculation. (A) The average capture of MNCs in 50 cm P-selectin tubes (40 μg/ml) was 84 036 cells (178·3 ± 33·8 cells/mm², n = 6) for 1-h single blood-pass and 159 264 cells (338·0 ± 115·0 cells/mm², n = 7) for 2-h recirculation. (B) Purity of captured MNC populations for CD34⁺ cells was 16·2 ± 5·5% and 21·0 ± 6·6% for 1- and 2-h experiments, respectively. The results confirm that targeted cell capture also occurs in a fully implanted arterio-venous recirculation device, and suggest that the amount of capture and degree of purity might be increased with longer times of perfusion.

Fig 6

Bright field microscopic images of erythroid burst-forming units and granulocyte/monocyte colony forming units, expanded from hematopoietic stem cells (HSCs) harvested from implanted cell capture tubes and whole blood. Viable blood-borne HSCs from implanted P-selectin coated tubes (A, C, and E) and rat whole blood (B, D, and F) were expanded in culture for up to 28 d. Robust colonies were observed from HSCs in each group.