Proteomics profile of mesenchymal stromal cells and extracellular vesicles in normoxic and hypoxic conditions |
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| Affiliation: | 1. Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil;2. Toxicology Laboratory, Oswaldo Cruz Institute, Rio de Janeiro, Brazil;3. Center for Technological Development in Health, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil;4. National Institute of Science and Technology for Regenerative Medicine, Rio de Janeiro, Brazil;5. Rio de Janeiro Innovation Network in Nanosystems for Health–NanoSaúde, Research Support Foundation of the State of Rio de Janeiro, Rio de Janeiro, Brazil;1. Department of Medicine, University of Calgary, Calgary, Canada, T2N 4N1;2. Department of Laboratory Medicine and Pathology, University of Calgary, Calgary, Canada, T2N 4N1;3. Alberta Precision Laboratories, Calgary, Canada, T2N 4N1;4. Alberta Health Services, Calgary, Canada, T2N 4N1;1. Immunobiology Laboratory, Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi, India;2. Cellular Immunology Laboratory, Department of Zoology, The University of Burdwan, PurbaBardhhaman, India;3. Chittaranjan National Cancer Institute, Kolkata, India;1. School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, China;2. Daxing Research Institute, University of Science and Technology Beijing, Beijing, China;3. Cell Therapy Laboratory, First Hospital of Hebei Medical University, Shijiazhuang, China;4. Department of Immunology, Basic Medical College, Hebei Medical University, Shijiazhuang, China;1. Beijing Key Laboratory of Traditional Chinese Medicine Basic Research on Prevention and Treatment for Major Diseases, Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing, China;2. School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, China;3. Daxing Research Institute, University of Science and Technology Beijing, Beijing, China;1. Sydney Cord Blood Bank, Sydney Children''s Hospital, Randwick, NSW, Australia;2. School of Women''s and Children''s Health, Faculty of Medicine, University of New South Wales, Randwick, NSW, Australia;3. Bone Marrow Transplant Laboratory, Randwick Hospitals, NSW Health Pathology, Randwick, NSW, Australia;4. Queensland Cord Blood Bank at the Mater, Mater Hospital, Raymond Terrace, Queensland, Australia;5. BMDI Cord Blood Bank, Murdoch Children''s Research Institute, Royal Children''s Hospital, Parkville, Victoria, Australia;6. Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia;1. Department of Medicine, Haraldsplass Deaconess Hospital, Bergen, Norway;2. Department of Medicine, Haukeland University Hospital, Bergen, Norway;3. Department of Clinical Science, University of Bergen, Bergen, Norway;4. VID Specialized University, Faculty of Health, Bergen, Norway |
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| Abstract: | Background aimsAlthough bone marrow-derived mesenchymal stromal cells (MSCs) have demonstrated success in pre-clinical studies, they have shown only mild therapeutic effects in clinical trials. Hypoxia pre-conditioning may optimize the performance of bone marrow-derived MSCs because it better reflects the physiological conditions of their origin. It is not known whether changes in the protein profile caused by hypoxia in MSCs can be extended to the extracellular vesicles (EVs) released from them. The aim of this study was to evaluate the proteomics profile of MSCs and their EVs under normoxic and hypoxic conditions.MethodsBone marrow-derived MSCs were isolated from six healthy male Wistar rats. After achieving 80% confluence, MSCs were subjected to normoxia (MSC-Norm) (21% oxygen, 5% carbon dioxide, 74% nitrogen) or hypoxia (MSC-Hyp) (1% oxygen, 5% carbon dioxide, 94% nitrogen) for 48 h. Cell viability and oxygen consumption rate were assessed. EVs were extracted from MSCs for each condition (EV-Norm and EV-Hyp) by ultracentrifugation. Total proteins were isolated from MSCs and EVs and prepared for mass spectrometry. EVs were characterized by nanoparticle tracking analysis. Proteomics data were analyzed by PatternLab 4.0, Search Tool for the Retrieval of Interacting Genes/Proteins, Gene Ontology, MetaboAnalyst and Reactome software.ResultsCell viability was higher in MSC-Hyp than MSC-Norm (P = 0.007). Basal respiration (P = 0.001), proton leak (P = 0.004) and maximal respiration (P = 0.014) were lower in MSC-Hyp than MSC-Norm, and no changes in adenosine triphosphate-linked and residual respiration were observed. The authors detected 2177 proteins in MSC-Hyp and MSC-Norm, of which 147 were identified in only MSC-Hyp and 512 were identified in only MSC-Norm. Furthermore, 718 proteins were identified in EV-Hyp and EV-Norm, of which 293 were detected in only EV-Hyp and 30 were detected in only EV-Norm. Both MSC-Hyp and EV-Hyp showed enrichment of pathways and biological processes related to glycolysis, the immune system and extracellular matrix organization.ConclusionsMSCs subjected to hypoxia showed changes in their survival and metabolic activity. In addition, MSCs under hypoxia released more EVs, and their content was related to expression of regulatory proteins of the immune system and extracellular matrix organization. Because of the upregulation of proteins involved in glycolysis, gluconeogenesis and glucose uptake during hypoxia, production of reactive oxygen species and expression of immunosuppressive properties may be affected. |
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