CD133 immunomagnetic separation: effectiveness of the method for CD133(+) isolation from umbilical cord blood.

BACKGROUND AIMS: Umbilical cord blood (UCB) is a rich source of stem cells, the characterization and isolation of which requires specific stem cell markers and reliable and reproducible protocols.
METHODS: We assessed CD133 isolation in 39 UCB samples, using a commercial immunomagnetic cell-sorting protocol, and, because of its non-reproducibility, we applied optimized protocols in an effort to improve it. These included extra-labeling of the selected CD133(+) subpopulation and indirect labeling using anti-phycoerythrin (PE) microbeads, goat anti-mouse IgG microbeads or a combination of both. The CD34 isolation was used as a control.
RESULTS: The mononuclear cell fraction expressed 0.53±0.06% CD133. The corresponding value for CD34 was 1.64±0.15%. Following the manufacturer's instructions, the CD34 isolation resulted in a population expressing 93±1.25% CD34 while, after the corresponding process, CD133(+) expression ranged from 10% to 85% (median 60%). The optimized isolation protocols did not result in improved CD133(+) yield. The variation in the purity of the CD133 population cannot be attributed to the different clones of CD133 used, because they do not cross-block, while other factors such as glycosylation, which could possibly interfere, do not apply in normal hematopoietic stem cells (HSC).
CONCLUSIONS: CD34 isolation by the immunomagnetic method results in highly pure CD34(+) population, while the efficient and reproducible yield of a pure CD133(+) population is not feasible. Therefore quantification of the positive cells should follow each isolation procedure in order to confirm the number of CD133(+) cells.