Replicative Senescence of Mesenchymal Stem Cells: A Continuous and Organized Process

Abstract

Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. These cells are usually culture expanded prior to their application. However, a precise molecular definition of MSC and the sequel of long-term in vitro culture are yet unknown. In this study, we have addressed the impact of replicative senescence on human MSC preparations. Within 43 to 77 days of cultivation (7 to 12 passages), MSC demonstrated morphological abnormalities, enlargement, attenuated expression of specific surface markers, and ultimately proliferation arrest. Adipogenic differentiation potential decreased whereas the propensity for osteogenic differentiation increased. mRNA expression profiling revealed a consistent pattern of alterations in the global gene expression signature of MSC at different passages. These changes are not restricted to later passages, but are continuously acquired with increasing passages. Genes involved in cell cycle, DNA replication and DNA repair are significantly down-regulated in late passages. Genes from chromosome 4q21 were over-represented among differentially regulated transcripts. Differential expression of 10 genes has been verified in independent donor samples as well as in MSC that were isolated under different culture conditions. Furthermore, miRNA expression profiling revealed an up-regulation of hsa-mir-371, hsa-mir-369-5P, hsa-mir-29c, hsa-mir-499 and hsa-let-7f upon in vitro propagation. Our studies indicate that replicative senescence of MSC preparations is a continuous process starting from the first passage onwards. This process includes far reaching alterations in phenotype, differentiation potential, global gene expression patterns, and miRNA profiles that need to be considered for therapeutic application of MSC preparations.

MiR-146a as marker of senescence-associated pro-inflammatory status in cells involved in vascular remodelling

“…Several independent lines of evidence have indicated a critical role for miRs, as biomarkers of cellular senescence, in both replicative and stress-induced modulation of in vitro cellular senescence (Lafferty-Whyte et al 2009;Poliseno et al 2008;Wagner et al 2008;Li et al 2009Li et al , 2010Olivieri et al 2009). Human umbilical vein endothelial cells (HUVECs) have been extensively used as an in vitro endothelial model representing common functional and morphological features of the in vivo endothelial cells (Unterluggauer et al 2007).…”

Material‐Driven Fibronectin Assembly Promotes Maintenance of Mesenchymal Stem Cell Phenotypes

“…When using MSCs for regenerative medicine, it is important to obtain a suffi cient number of cells that maintain pluripotency without compromising MSC senescence. [ 3,4 ] Material systems that mimic the natural niche environment of MSCs may offer an alternative to the use of complex cocktails of soluble factors used in the culture media. Previous studies show that the cell/material interface plays an essential role on MSC function and differentiation, encompassing promising approaches to manipulate differentiation of stem cells ranging from chemistry, [5][6][7] surface modifi cations, [ 8,9 ] topography, [10][11][12] stiffness, [ 13,14 ] and even dynamic material properties such as stress relaxation.…”

The role of the microenvironment on the fate of adult stem cells

“…The frequency of bipotent clones (with osteo-and chondrogenic potential) decreased with age, and a higher relative frequency of bipotent clones was found in younger donors compared to older donors. Conversely, older donors had a relatively higher frequency of tri-potent clones [51]. Several studies have also assessed tissue regeneration in young mice compared to aged mice.…”

“…However, after a certain number of cell divisions, SCs enter senescence and stop proliferating, showing an enlarged and flattened cell shape [95]. Previous studies have shown that human MSCs exhibit reduced differentiation potentials in vitro and that the replicative senescence of MSC preparations begins with the first passage [51]. Thus, the difficulty in the long-term expansion of MSCs using standard culture systems without the loss of their SC properties suggests that a critical feature of their microenvironment that is necessary for the retention of SC properties is absent in these culture systems.…”