Influence of aging on proliferation, pluripotency, immunogenic profiles from bone marrow mesenchymal stem cells

Abstract

Mesenchymal stem cells (MSCs) are highly relevant for regeneration of mesoderm tissues such as bone and cartilage. The promising role of MSCs in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age. In this research we have studied and treated to understand how aging influences in proliferation and pluripotency capacities from these cells and also into their immunogenic potential. Six age groups from bone marrow mesenchymal stem cells of Wistar rats were studied (newborn, infant, young, pre-pubertal, pubertal and adult). Quantitative proteomic assay was performance by iTRAQ-8-plex and the proteins statistically significant modulated were grouped in pluripotency, proliferative and metabolism processes. Proliferation makers, CD117 and Ki67 were measure by flow cytometry assay. Real time polymerase chain reaction analysis of pluripotency markers Rex1, Oct4, Sox2 and Nanog were done. Biological differentiation was realized using specific mediums for 14 days to induce osteogenesis, adipogenesis and chondrogenesis and differentiated cells were analysed using histochemical techniques. Enzymatic analysis of several enzymes as L-lactate dehydrogenase and glucose-6-phosphate isomerase were done to validate iTRAQ data. To deeply study these differences we have analyzed by Next Generation Sequencing six age groups from bone marrow mesenchymal stem cells. A total of 9628 genes presented differences of expression among age groups and those genes were grouped into metabolic pathways. We focused our research in young, pre-pubertal and adult groups which presented the highest amount of genes differentially expressed related with inflammation mediated by chemokine and cytokine signalling pathway when compared with newborn group which was used as a control. Afterwards, extracellular vesicles from those groups were isolated and characterized by nanoparticle tracking analysis and flow cytometry and several micro-RNAs were checked by qRT-PCR because of their relationship with the pathway of interest. Since miR-21-5p was statistically significant highest in extracellular vesicles from mesenchymal stem cells of pre-pubertal group, we realized a functional experiment inhibiting it expression and investigating the modulation of Toll-Like Receptor 4 and their link to damage-associated molecular patterns. Aging affects proliferation, pluripotency and immunogenic profiles of bone marrow mesenchymal stem cells. Also its affects production, content of pro-inflammatory miRs and affectivity of bone marrow mesenchymal stem cell-derived extracellular vesicles. These findings are important to the understanding about influence of the aging on mesenchymal stem cells and to advance in the development EV-based therapies.