■先前的研究揭示了在醛脱氢酶表达(ALDHhigh)间充质基质细胞(MStroC)部分中的干细胞样转录组富集。然而,考虑到MStroCs的异质性,其中只有一小部分提供真正的干细胞(MSC),ALDH作为MSC特异性选择标记的实际效力仍然是一个问题.
方法:要解决这个问题,在低氧浓度下孵育的单个ALDHhigh和ALDHlowMStroCs的增殖和分化潜能,估计模拟干细胞生态位(0.1%O2),使用单细胞克隆分析进行测定,与标准条件(20%O2)相比。
结果:我们确认在ALDHhighMStroC群体中富集了高增殖能力和多潜能的MSCs,特别是当细胞在0.1%O2下培养时。用MitoSoX(MSX)测量还原/氧化的谷胱甘肽和线粒体超氧化物阴离子表明,低氧诱导的这种优势与干细胞代谢设置中氧化和活性氧(ROS)水平的降低有关。然而,ALDH表达对MSCs既不是特异性的也不是排他性的,在ALDHlow部分中也发现了高增殖能力和多能细胞。此外,基于ALDH和MSX的联合细胞分选后进行的单细胞测定表明,MSXlowMStroC群体在所有条件下都富含干/祖细胞,无论ALDH表达或培养氧浓度。重要的是,暴露于0.1%O2的ALDHhighMSXlowMStroC部分几乎完全由真正的MSCs组成。相比之下,在MSXhigh部分中既没有检测到祖细胞也没有干细胞(完全没有集落形成能力),仅存在于ALDHlowMStroC人群中。
结论:我们的研究表明,ALDH表达并不完全与MSCs相关。然而,使用组合的ALDH表达和ROS含量的细胞分选可用于排除缺乏干/祖细胞性质的MStroCs。
UNASSIGNED: Previous research has unveiled a stem cell-like transcriptome enrichment in the aldehyde dehydrogenase-expressing (ALDHhigh) 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.
METHODS: To address this, the proliferative and differentiation potential of individual ALDHhigh and ALDHlow MStroCs incubated at low oxygen concentrations, estimated to mimic stem cell niches (0.1% O2), were assayed using single-cell clonal analysis, compared to standard conditions (20% O2).
RESULTS: We confirm that a high proliferative capacity and multi-potent MSCs are enriched in the ALDHhigh MStroC population, especially when cells are cultured at 0.1% O2. 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 ALDHlow fraction. Furthermore, single-cell assays performed after combined cell sorting based on ALDH and MSX showed that the MSXlow MStroC population is enriched in stem/progenitor cells in all conditions, irrespective of ALDH expression or culture oxygen concentration. Importantly, the ALDHhighMSXlow MStroC fraction exposed to 0.1% O2 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 MSXhigh fraction, which exclusively resides in the ALDHlow MStroC population.
CONCLUSIONS: 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.