Previous research has unveiled a stem cell-like transcriptome enrichment in the aldehyde dehydrogenase-expressing (ALDH) mesenchymal stromal cell (MStroC) fraction. However, considering the heterogeneity of MStroCs, with only a fraction of them presenting bona fide stem cells (MSCs), the actual potency of ALDH as an MSC-specific selection marker remains an issue. To address this, the proliferative and differentiation potential of individual ALDH and ALDH MStroCs incubated at low oxygen concentrations, estimated to mimic stem cell niches (0.1% O), were assayed using single-cell clonal analysis, compared to standard conditions (20% O). We confirm that a high proliferative capacity and multi-potent MSCs are enriched in the ALDH MStroC population, especially when cells are cultured at 0.1% O. Measurements of reduced/oxidized glutathione and mitochondrial superoxide anions with MitoSoX (MSX) indicate that this advantage induced by low oxygen is related to a decrease in the oxidative and reactive oxygen species (ROS) levels in the stem cell metabolic setup. However, ALDH expression is neither specific nor exclusive to MSCs, as high proliferative capacity and multi-potent cells were also found in the ALDH fraction. Furthermore, single-cell assays performed after combined cell sorting based on ALDH and MSX showed that the MSX MStroC population is enriched in stem/progenitor cells in all conditions, irrespective of ALDH expression or culture oxygen concentration. Importantly, the ALDHMSX MStroC fraction exposed to 0.1% O was almost exclusively composed of genuine MSCs. In contrast, neither progenitors nor stem cells (with a complete absence of colony-forming ability) were detected in the MSX fraction, which exclusively resides in the ALDH MStroC population. Our study reveals that ALDH expression is not exclusively associated with MSCs. However, cell sorting using combined ALDH expression and ROS content can be utilized to exclude MStroCs lacking stem/progenitor cell properties.< Réduire