Hematopoietic stem cells (HSC) are characterized by their potential to reconstitute in vivo haematopoiesis in NOD/SCID mice and to give rise to differentiated cells of all haematopoietic lineages. They are usually defined by a CD34+CD38CD90+ antigenic profile; however taking into account the versatility of antigen membrane expression (Lataillade et al., J. Leukoc. Biol. 2005), more reliable methods based on their selective functional/metabolic activities such as ABCG2 activity and Aldehyde Deshydrogenase expression have been developed. Actually, HSC can be purified on basis of Hoechst 33342 die exclusion property due to the membrane ABCG2/BRCP1 transporter expression (SP cells). SP cells could be isolated from a wide variety of human and mammalian tissues and in many cases have been shown to contain multipotent stem cells; SP cells are heterogeneous since the lowest Hoechst fluorescent profile exhibited the highest primitive hierarchical level. Hematopoietic progenitors also expressed ALDH enzymatic activity and the use of BODIPY-AminoAcetAldehyde (BAAA), a fluorescent substrate of the ALDH enzyme, allows analyzing its expression by using Fluorescence Activated Cell Sorting (FACS). It has been reported that there is a close association between high ALDH expression (ALDHbr) and high HSC activity. Based on the hypothesis that the more primitive HSC should express both ALDH high expression level and SP profile, we developed a new multiparamatric flow cytometric assay combining an ALDH/SP co-labelling to identify different haematopoietic progenitor subpopulations. Our strategy allows identifying six lineage negative (Lin) haematopoietic sub-populations from human bone marrow cells according to the co-expression of ALDH levels and SP phenotype: SP+ALDHBr, SPALDHBr, SP+ALDHMid, SPALDHMid, SP+ALDHLo and SP-ALDHLo. We showed that these different sub-populations exhibited specific antigenic patterns and demonstrated variable differentiation properties in long term cultures and clonogenic assays. Whatever the ALDH expression level, SP+ and SPcells expressed the CD34 antigen, however, the SP phenotype identify a higher proportion of CD34+CD38 cells. Furthermore, in contrast to the other subpopulations, Lin-SP+ALDHBr cells were highly enriched with CD34+CD38CD90+ progenitor cells and demonstrated a higher amplification and high clonogenic efficiency in liquid LTC-IC cultures than SP-ALDHBr cells. This subpopulation allowed obtaining all myeloid haematopoietic lineage differentiated cells after 30 days culture in liquid culture, including osteoclastic cells. Interestingly, in contrast to ALDHBr cells, ALDHLo sub-populations were highly enriched with megakaryocytic CD34+CD41+ progenitors that gave rise to CD41+CD42a+ cells. SP+ALDHMid and SP-ALDHMid have intermediate amplification, clonogenic and differentiation abilities as compared with the other subpopulations. In conclusion, our results show that the combined SP/ALDH technology allows discriminating a hierarchy within the human hematopoietic stem/progenitor compartment, the most primitive one being SP+ALDHBr. The stemness of these subpopulations is currently explored by testing their capability to reconstitute long term haematopoiesis in NOD/SCID mice.

Author notes

Disclosure: No relevant conflicts of interest to declare.