Magnetic-Assisted Fractionation of Bone Marrow Cells into Subsets Differing in CD45 Expression Levels, Surface Phenotypes and Functional Properties

Abstract

Cells of higher organisms express numerous cell surface proteins, and their spectrum and level of expression are directly related to cells’ functions. The technology of mass cell selection based on the surface protein expression levels may be highly important both for basic research and cell therapy applications. We have previously developed a method of magnetic selection of cells differing in surface marker expression levels, which we term here MACS-MEL (Magnetic-Assisted Cell Selection by Marker Expression Levels). The method demonstrated its effectiveness in the artificial model system, namely retrovirally transduced NIH 3T3 cells. However, whether it was also applicable to complex natural cell populations remained unclear. In the current study, we validated the MACS-MEL approach by separating mouse bone marrow (BM) cells into fractions according to the expression of pan-hematopoietic marker CD45. In the basic protocol, two-stage fractionation of CD45+ cells from BM was performed using selection of cells consecutively with 2 μL and 8 μL of anti-CD45 magnetic beads, resulting in isolation of CD45high and CD45int cell populations. To explore in full the potential of the method, the extended protocol was also tested, where a third selection stage with 30 μL of anti-CD45 beads was added. The isolated cell fractions were analyzed by flow cytometry for CD45 expression, as well for CD11b, Gr-1, CD117, CD115 and CD19 markers, while their in vitro progenitor function was assessed by quantitating colony-forming units (CFUs) in methyl cellulose. The results of analysis demonstrate that the isolated cell fractions significantly differed both in their surface phenotypes and CFU potential. In particular, cell fractions with progressively reduced CD45 expression were characterized by decreasing expression of myeloid differentiation markers CD11b and Gr-1, as well as B-lymphoid marker CD19. The expression of stem/progenitor cell marker CD117, on the contrary, significantly increased. The CFU frequency also strongly correlated with decrease in CD45 expression, while the differentiation potential of CFUs differed substantially in various cell fractions. In general, our results demonstrate that less differentiated hematopoietic cells in mouse BM studied using in vitro tests are characterized by lower CD45 expression levels, in full accordance with data obtained in human system. Successful validation of the MACS-MEL in a BM system, characterized by existence of multiple cell types and high phenotypic and functional heterogeneity, demonstrated the effectiveness, simplicity and affordability of this method. The MACS-MEL approach can be applied for mass selection of cells based on differential marker expression and may yield cell subsets suitable for advanced cell therapy applications.