{"title":"Functional single-cell analyses of mesenchymal stromal cell proliferation and differentiation using ALDH-activity and mitochondrial ROS content","authors":"","doi":"10.1016/j.jcyt.2024.04.003","DOIUrl":null,"url":null,"abstract":"<div><h3>Baskground</h3><p>Previous research has unveiled a stem cell-like transcriptome enrichment in the aldehyde dehydrogenase-expressing (ALDH<sup>high</sup>) 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.</p></div><div><h3>Methods</h3><p>To address this, the proliferative and differentiation potential of individual ALDH<sup>high</sup> and ALDH<sup>low</sup> MStroCs incubated at low oxygen concentrations, estimated to mimic stem cell niches (0.1% O<sub>2</sub>), were assayed using single-cell clonal analysis, compared to standard conditions (20% O<sub>2</sub>).</p></div><div><h3>Results</h3><p>We confirm that a high proliferative capacity and multi-potent MSCs are enriched in the ALDH<sup>high</sup> MStroC population, especially when cells are cultured at 0.1% O<sub>2</sub>. 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<sup>low</sup> fraction. Furthermore, single-cell assays performed after combined cell sorting based on ALDH and MSX showed that the MSX<sup>low</sup> MStroC population is enriched in stem/progenitor cells in all conditions, irrespective of ALDH expression or culture oxygen concentration. Importantly, the ALDH<sup>high</sup>MSX<sup>low</sup> MStroC fraction exposed to 0.1% O<sub>2</sub> 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<sup>high</sup> fraction, which exclusively resides in the ALDH<sup>low</sup> MStroC population.</p></div><div><h3>Conclusion</h3><p>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.</p></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cytotherapy","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1465324924006017","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Baskground
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.
Conclusion
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.
期刊介绍:
The journal brings readers the latest developments in the fast moving field of cellular therapy in man. This includes cell therapy for cancer, immune disorders, inherited diseases, tissue repair and regenerative medicine. The journal covers the science, translational development and treatment with variety of cell types including hematopoietic stem cells, immune cells (dendritic cells, NK, cells, T cells, antigen presenting cells) mesenchymal stromal cells, adipose cells, nerve, muscle, vascular and endothelial cells, and induced pluripotential stem cells. We also welcome manuscripts on subcellular derivatives such as exosomes. A specific focus is on translational research that brings cell therapy to the clinic. Cytotherapy publishes original papers, reviews, position papers editorials, commentaries and letters to the editor. We welcome "Protocols in Cytotherapy" bringing standard operating procedure for production specific cell types for clinical use within the reach of the readership.