Two widely used functional HSCs markers: (1) so-called Side Population (SP) based on Hoechst efflux ability and (2) ALDH – aldehyde dehydrogenase activity. Dual staining protocol for both (SP+ALDH) markers and their correlation with the surface phenotype was published in 2007 by Dominique Bonnet group. They concluded that:
Hoechst exclusion ability does not seem to be the method of choice for the isolation of human hematopoietic stem cells.
The later study, published a year ago, investigating the relationships between functional and surface HSCs markers in details.
Here, we demonstrate the advantage of combining SP and ALDH selection to identify and purify human SP/ALDHBr cells that exhibit a primitive phenotype, characterized by the canonical CD34+CD38Low/-CD90+ phenotype, quiescent/G0 status, a high proliferative potential in liquid culture, and capability of engrafting and generating human CD45+ SP cells in NOD-SCID mice. Thus, our data indicate that dual SP and ALDH technologies refine the Lin-CD34+CD38Low/- progenitor compartment and define a new HSC/HPC distribution within this compartment.
In comparison with Bonnet study:
By using combined SP and ALDH technology, we demonstrated that a fraction of Lin- human BM cells coexpresses both SP and ALDH functionalities, suggesting an overlap between these two markers. Indeed, all ALDH fractions contained SP cells, but with at least a fourfold higher percentage of SP cells in the ALDHBr subset (21.55% ± 8.57%) than in Lin- cells (2%-5%). This proportion was three- to fivefold higher than that described by Pearce and Bonnet, who showed that 0.04% of ALDH+ BM MNCs were SP cells. However, considering the percentage of Lin- cells (1%-2%) in MNCs, our results are in agreement with their data. Most importantly, we demonstrated the advantage of performing combined SP and ALDH selection on Lin- cells rather than on total MNCs, significantly improving the purification efficiency of cells that coexpress SP and ALDH stem cell markers.
A small overlap between the SP and ALDH subsets was reported by Pearce and Bonnet, suggesting that the identification of human stem cells was better achieved via ALDH activity than via Hoechst exclusion. Our data demonstrate that the coupled SP/ALDH method allows for the identification of an overlap between SP and ALDH cells that mainly concerns the primitive CD34+CD38Low/-CD90+ cells, suggesting that this combined method could be used to isolate primitive human hematopoietic cells.
The also assessed single and dual (SP/ALDH) stained subsets in xenotransplant assay, studied quiescence and unveil hierarchy of HSCs based on surface and functional markers.
Honestly, I didn’t understand the huge advantage of dual SP/ALDH stained HSCs compared with conventional surface markers (Lin-/CD34+/CD38-/CD90+) because there is an overlap. But this study is very interesting methodologically.
You can find the article in open access.