A High ALDH Activity Identifies Both Normal and Leukemic Stem Cells.

Aldehyde Dehydrogenase (ALDH) is a cytosolic enzyme that is responsible for the oxidation of intracellular aldehydes. Elevated levels of ALDH have been demonstrated in murine and human progenitor cells when compared to other haematopoietic cells and this is thought to be important in chemoresistance. A method to assess ALDH activity in viable cells has been developed and has recently been made commercially available in a kit format. Here, we confirmed the use of the ALDH substrate kit to identify cord blood stem/progenitor cells. Cells with a high ALDH activity accounted for only 0.82%±0.39% of mononucleated cells in cord blood (range = 0.35–1.29%, n=17). When cells negative for lineage antigens (Lin^neg - 99.3%±0.35% pure) were analyzed, 71.1%±9.1% possessed a high ALDH activity (n=9). We can now report that Lin^neg/CD34⁺ and Lin^neg/ALDH⁺ are essentially overlapping populations. Indeed, 93.3%±3.4% of Lin^neg/CD34⁺ cells are ALDH⁺, and 94.3%±2.5% of Lin^neg/ALDH⁺ cells are positive for the CD34 antigen. A small proportion of Lin^neg cells were positive for ALDH, but negative for CD34 (3.4%±1.9%) and a larger proportion (28.4%±7.7%) were Lin^neg/CD34^neg/ALDH^neg cells. Lin^neg/CD34^neg/ALDH^neg cells were almost exclusively CD7⁺, indicating that this subset probably represents NK progenitors. In addition, the majority of Lin^neg/CD34^neg/ALDH⁺ cells co-express CD38 indicating that they are also committed cells. The remaining candidate Lin^neg/CD34^neg/ALDH⁺/CD38^neg stem cells account for 0.19% of lineage negative cells and approximately 0.0006% to 0.002% of mononuclear cells.

We then progressed to the analysis of malignant hematopoietic cells with a high ALDH activity. In contrast to the remarkable consistency of cord blood ALDH labeling, the staining of AML samples gave more varied profiles. One pattern (pattern-1, 6/17 of AML samples tested) was very similar to the cord blood profile; ALDH⁺ cells were rare (0.16%±0.14%), had a low/medium side scatter and were almost exclusively CD34⁺ (87.1%±9.2%). In another group of patients (pattern-2, 6/17) ALDH⁺ cells were much more frequent 13.9%±11.8%, had a higher side scatter and were not exclusively CD34⁺ (41.1%±9.2%). In a similar proportion of patients (pattern-3) we could not detect any ALDH activity above the level of inhibitor controls.

When FACSorted and injected into NOD/SCID mice, ALDH⁺ cells from pattern-2 gave rise to unilineage myeloid engraftment in 2 separate experiments (four mice), suggesting a leukemic origin. However, when ALDH⁺ cells from pattern-1 were injected into sublethally irradiated NOD/SCID mice, multilineage engraftment was observed (two patients, two mice).

Hence, it seems that a high ALDH activity may be used to identify non-malignant stem cells within some AML samples. In addition, a high ALDH activity also identifies some patients’ leukemic stem cells. The incidence of normal or leukemic stem cells with an extremely high ALDH activity may have important implications for resistance to chemotherapy. Identification and isolation of leukemic cells on the basis of ALDH activity provides a tool for their isolation and further analysis.