Effect of age on pro-inflammatory miRNAs contained in mesenchymal stem cell-derived extracellular vesicles

Developers

J. Fafián-Labora, I. Lesende-Rodriguez, J. Mateos, M. C. Arufe et al.

Description of the technology

The promising role of mesenchymal stem cells (MSCs) in cell-based therapies and tissue engineering appears to be limited due to a declination of their regenerative potential with increasing donor age. In other words, stem cells possess significant age-dependent differences in their immune-response profile. In process of this technology development, these differences were analyzed by next-generation sequencing (NGS) of samples from bone marrow mesenchymal stem cells of six age groups − newborn, infant, young, pre-pubertal, pubertal and adult − to evaluate the modifications of gene expression during ageing. The research was focused on young, pre-pubertal and adult groups, which presented the highest amount of differentially expressed genes related to inflammation mediated by chemokine and cytokine signalling pathways compared with the newborn group, which was used as a control. A total of 9,628 genes presenting differential expression between age groups were grouped into metabolic pathways.

Recently, the role of micro-RNAs in ageing and immunosenescence has been reported and their relevance to extracellular vesicles from MSCs affecting their therapeutic potential. Extracellular vesicles (EVs), such as exosomes or micro-vesicles, are released by cells into the environment as sub-micrometre particles enclosed by a phospholipid bilayer. EVs have been found to mediate interactions between cells, mediate non-classical protein secretion, facilitate processes such as antigen presentation, participate in trans-signalling to neighbouring cells and in the transfer of RNAs and proteins. The detection of low copy numbers of mRNA and small RNAs, including micro-RNAs (miRNA), in EVs from mouse and human mast cell lines (MC/9 and HMC-1, respectively) has added much research interest impetus to the field. While mRNA and miRNA in EVs are inactive, they have the potential to be active when EVs are transfected into nearby cells. Studies indicate that the EV miRNA expression profile may be of diagnostic/therapeutic potential

In the studies, supporting this technology, the extracellular vesicles extracted from samples of each group were characterized by nanoparticle tracking and flow cytometry analysis, and several micro-RNAs were verified by quantitative real-time polymerase chain reaction because of their relationship with the pathway of interest. Since miR-21-5p showed the highest statistically significant expression in extracellular vesicles from mesenchymal stem cells of the pre-pubertal group, a functional experiment was conducted, which inhibited its expression and investigating the modulation of Toll-Like Receptor 4 (TLR4) and their link to damage-associated molecular patterns (DAMP). The Toll-like receptors, an important component of innate and adaptive immune responses, are expressed in MSCs and their derived EVs during ageing. TLR4 signalling contributes to response specificity, leading to increased transcription of NF-κ B and AP-1 target genes like IL-8, IL-6, IL-1β, TNF-α, and IFN-β. DAMPs are molecules that have a physiological role inside but acquire additional functions when exposed to the extracellular environment, and they can be secreted by living cells undergoing a life-threatening stress.

Together, these results indicated for the first time that mesenchymal stem cell-derived extracellular vesicles have significant age-dependent differences in their immune profiles.

Practical application

These results provide insight into the mechanism involved in MSC ageing and suggest possible interventions in miRNAs to maintain function of MSCs and their derived extracellular vesicles prior to in vivo transplantation or as pharmacological agents in disease. Particularly, the inhibition of miR-21 produces an over-expression of Wnt5a accompanied by a decrease of the LMNA/C senescence marker, suggesting a role of miR-21 in self-renewal and increasing of pluripotent capacities of the MSCs.

Technology is promising for application in all fields, where stem cells can be used, and has a high potential for maintaining the stem cell functionality

Laboratories

• Grupo de Terapia Celular y Medicina Regenerativa (TCMR-CHUAC). CIBER-BBN/ISCIII. Servicio de Reumatología, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), SERGAS, Departamento de Medicina, Facultade de Oza, Universidade de A Coruña (UDC), A Coruña (Spain)
• Grupo Fisiopatología Endocrina, Nutricional y Médica (FENM-CHUAC), Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), SERGAS, Departamento de Medicina, Facultade de Oza, Universidade de A Coruña (UDC), A Coruña (Spain)
• Cardiology Department, Health in Code, A Coruña (Spain)
• Experimental Rheumatology, Radboudumc University Medical Center, Nijmegen (The Netherlands)

Links

Publications

• Fafián-Labora, J. et al. «Effect of age on pro-inflammatory miRNAs contained in mesenchymal stem cell-derived extracellular vesicles." 7 Scientific Reports (2017): 43923.
• Fafián-Labora, J. et al. «Influence of age on rat bone-marrow mesenchymal stem cells potential." 5 Sci Rep (2015): 16765.