CD133 Immunomagnetic Separation: How Effective Is the Method for CD133⁺ Cell Isolation from Umbilical Cord Blood?.

Umbilical cord blood (UCB) is a rich source of stem cells. Characterisation of stem cell subpopulations residing in UCB requires specific stem cell markers and reliable and reproducible protocols. We assessed the feasibility of CD133 positive cells’ isolation using immunomagnetic cell sorting protocols. The separation was based on the manufacturer’s instructions (Myltenyi Biotec). We compared these results with the corresponding ones of CD34 isolation. Two additional protocols were also applied to increase the efficiency of the CD133⁺ yield. The first one included an extra-labeling of the selected CD133 subpopulation from the first column with CD133 microbeads before their application to the second column. The second protocol was an indirect labelling procedure, using CD133-PE monoclonal antibody and either anti-PE microbeads or goat anti-mouse IgG microbeads as well as a combination of them. The CD133 isolation was performed in 18 UCB samples, while the CD34 in 17 samples. The volume of the samples used for CD133⁺ isolation was 38.5 ± 2.1ml and the respective one for the CD34⁺ protocol was 40.4 ± 2.8ml. The mononuclear cell fraction had 1.29 ± 0.16 x10⁸ cells and 0.53 ± 0.06% of them expressed CD133. The corresponding values for samples used for CD34 isolation were 2.8 ± 0.3 x10⁸ mononuclear cells and 1.64 ± 0.15% CD34. Following the manufacturer’s instructions for CD133 isolation, the number of cells obtained was 21.33 ± 3.3x10⁴ (median 18 x10⁴) and CD133 expression ranged from 10–85% (median=60%). Concerning the CD34 isolation, the number of cells obtained was 54.3 ± 2.1x10⁴ and 93 ± 1.25% expressed CD34. Applying the extra-labeling method, the number of cells obtained was 8±4 x10⁴ and these cells were CD133⁺ at a percentage of 32.2±4.1%. Applying the indirect immunomagnetic separation the results were as follow: using anti-PE microbeads only, 3 ± 3 x10⁴ cells were retrieved and the percentage of CD133⁺ was 13.8±9.7. With the use of goat anti-mouse IgG microbeads the cell number obtained was 13±1 x10⁴ of which CD133⁺ were just 2.0± 2%. In the combination method, the cells initially isolated with anti-PE microbeads and subsequently with goat anti-mouse IgG microbeads were 20 x 10⁴, of which only 18.5% were CD133⁺. Finally, labeling the cells initially with anti-PE microbeads and consecutively with goat anti-mouse IgG microbeads followed by column isolation yielded 16x10⁴ cells and 19.1% CD133 positive. In conclusion CD34 isolation by the immunomagnetic method results in highly pure CD34⁺ population confirming the reliability and reproducibility of the method. On the other hand, with the currently available CD133 isolation kit, efficient and reproducible yield of pure CD133 positive population is not feasible.