Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome and secretome analysis of mechanically stretched osteocytic cells reveals activation of signalling pathways implicated in bone regeneration and DNA-repair systems

ABSTRACT: Osteocytes are secretory bone cells that can play an important role in regenerative rehabilitation through their secretome. Making use of RNA-seq and bioinformatics we examined the transcriptome of mouse and human osteocytic bone cells cultured under cyclic tension delivered by a computer-controlled bioreactor. We discovered that a single bout of tensile stretch is enough to activate signalling pathways or processes implicated in bone tissue regeneration, and DNA-repair systems which are relevant to cancer. This results are valuable to the scientific community by allowing to inform future exercise-based regenerative rehabilitation protocols targeting musculoskeletal disorders or cancer.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Mus musculus

SUBMITTER: Livia Santos

PROVIDER: E-MTAB-9487 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:BACKGROUND: Ischemia/reperfusion injury (IRI) is the leading cause of acute kidney injury (AKI). The current standard of care focuses on supporting kidney function, stating the need for more efficient and targeted therapies to enhance repair. Mesenchymal Stromal Cells (MSCs) and their secretome, either as conditioned medium (CM) or extracellular vesicles (EVs), have emerged as promising options for regenerative therapy, however, their full potential in treating AKI remains unknown.METHODS: In this study, we employed an in vitro model of chemically-induced ischemia using antimycin A combined with 2-deoxy-D-glucose to induce ischemic injury in proximal tubule epithelial cells. Afterwards, we evaluated the effects of MSC secretome, CM or EVs obtained from adipose tissue, bone marrow and umbilical cord, on ameliorating the detrimental effects of ischemia. To assess the damage and treatment outcomes, we analyzed cell morphology, mitochondrial health parameters (mitochondrial activity, ATP production, mass and membrane potential) and overall cell metabolism by metabolomics.RESULTS: Our findings show that ischemic injury caused cytoskeletal changes confirmed by disruption of the F-actin network, energetic imbalance as revealed by a 50% decrease in the oxygen consumption rate, increased oxidative stress, mitochondrial dysfunction and reduced cell metabolism. Upon treatment with MSC secretome, the morphological derangements were partly restored and ATP production increased by 40-50%, with umbilical cord-derived EVs being most effective. Furthermore, MSC treatment led to phenotype restoration as indicated by an increase in cell bioenergetics, including increased levels of glycolysis intermediates, as well as an accumulation of antioxidant metabolites.CONCLUSION: Our in vitro model effectively replicated the in vivo-like morphological and molecular changes observed during ischemic injury. Additionally, treatment with MSC secretome ameliorated proximal tubule damage, highlighting its potential as a viable therapeutic option for targeting AKI.

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Project description:Canine adipose-derived mesenchymal stem cells (cAD-MSCs) show therapeutic promise for their regenerative potential, particularly in their secretome. However, concerns arise regarding the impact of in vitro cultivation need for storing therapeutic doses, prompting this study to comprehensively explore the impact of in vitro aging on gene expression and secretome composition. The study involved collecting abdominal adipose tissue samples from nine healthy female dogs, from which cAD-MSCs were extracted and cultured. Stem cells were validated through trilineage differentiation assays and flow cytometry immunophenotyping. Gene expression profiling, using RT-qPCR array and cAD-MSCs secretome LC-MS/MS analysis, were conducted at passages 3 and 6 to reveal gene expression and protein composition alterations during in vitro culture. Gene expression profiling of in vitro aged cAD-MSCs revealed expression alterations, while significant downregulation was observed in two MSC-associated genes. Proteomic analysis revealed 10% distinctively expressed proteins, and several up- and downregulations. Grouping these proteins with biologically significant ones, Gene Ontology Panther Pathway analysis revealed that P3 proteins were significantly associated with cytoskeletal regulation, nicotinic acetylcholine receptor signaling, inflammation mediated by chemokine and cytokine signaling, Wnt signaling, and CCKR signaling map. In contrast, P6 proteins were linked to the xanthine and guanine salvage pathway, adenine and hypoxanthine salvage pathway, and blood coagulation pathway. To the best of our knowledge, this study presents the first original perspective on the changes in secretome composition that occur when cAD-MSCs age in vitro. Our findings highlight significant changes that, in conclusion, indicate the regenerative potential of cAD-MSCs and that their secretome may be compromised due to in vitro aging. Consequently, our study suggests a preference for earlier passages when considering these cells for therapeutic applications.

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Transcriptome and secretome analysis of mechanically stretched osteocytic cells reveals activation of signalling pathways implicated in bone regeneration and DNA-repair systems

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