The combined use of Hoechst efflux ability and aldehyde dehydrogenase activity to identify murine and human hematopoietic stem cells

Objective

In murine hematopoietic tissue, direct repopulation experiments have demonstrated that the side population (SP) represents a remarkable enrichment of hematopoietic stem cells. Human SP has been phenotyped as negative for lineage antigens as well as CD34. However, in the 9 years since the original publication, no long-term hematopoietic reconstitution has been reported for the adult human SP/CD34⁻ subset. Elevated levels of aldehyde dehydrogenase (ALDH) have been demonstrated in murine and human progenitor cells when compared to other hematopoietic cells.

Methods

Here, we report the phenotype of human cord blood SP cells. We established the technique of simultaneous phenotyping, Hoechst exclusion, and ALDH labeling on murine tissues. We then performed the simultaneous analysis of phenotype, SP, and ALDH activity on human cord blood and bone marrow cells. Finally, we analyzed the phenotype and functional potential of human cord blood ALDH⁺ cells to determine whether Lin⁻/CD34⁻ cells are identified via this technique.

Results

We demonstrate that human Lin⁻/CD34⁻/ALDH⁺ cells are capable of long-term repopulation. Although the SP technique identifies cells that overlap with the ALDH⁺ cell population, this is restricted to the CD34⁺ cell subset.

Conclusion

Hoechst exclusion ability does not seem to be the method of choice for the isolation of human hematopoietic stem cells.