Abstract
Mesenchymal stem cells promising role in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age. 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 using an 8-plex iTRAQ labeling and the proteins differentially expressed 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 or chondrogenesis and immunostain analysis of differentiated cell resulting were done. Enzimoimmunoassay analysis of several enzymes as L-lactate dehydrogenase and glucose-6-phosphate isomerase were also done to validate iTRAQ data. Taking together these results indicate for the first time that mesenchymal stem cells have significant differences in their proliferative, pluripotency and metabolism profiles and those differences are age depending.
Highlights
MSCs populations from different ageing groups were characterized by flow cytometry to check percentage of positive cells for mesenchymal markers, CD29 and CD90, and to check that were negative for hematopoietic markers (CD34, CD45) (Fig. 1A)
The mesenchymal markers were not as abundant as published by Harting et al.[22] these cells were able to adhere to the plastic plate and this is an intrinsic characteristic of mesenchymal stem cells and all of the groups were able to differentiate towards several mesoderm lineages (Fig. 2A,B). iTRAQ analysis is an adequate technique to study complex samples like we have used in this work[23]
The cut-off for significant fold-change was determined based on the 2 biological replicates of two iTRAQ experiments which were chosen based on the following criteria: it contained more than 3 unique peptides (> 95%) and p value < 0 .05 for the 114/119 reporter ions
Summary
MSCs populations from different ageing groups were characterized by flow cytometry to check percentage of positive cells for mesenchymal markers, CD29 and CD90, and to check that were negative for hematopoietic markers (CD34, CD45) (Fig. 1A). We did not observe statistical significant differences between the mesenchymal markers into the different MSCs aging group studied (Fig. 2C). These were coincident with results published by Jin et al.[6] indicating that MSCs have similar levels of surface antigen expression included MSCs from different tissues. The cut-off for significant fold-change was determined based on the 2 biological replicates of two iTRAQ experiments which were chosen based on the following criteria: it contained more than 3 unique peptides (> 95%) and p value < 0 .05 for the 114/119 reporter ions. The fold-change thresholds of > 1.20 or < 0.80 was set to identify true differences between expression of reporter io
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