Dataset Information

Individual response to mTOR inhibition in delaying replicative senescence of mesenchymal stromal cells.

ABSTRACT: BACKGROUND AIMS:Delaying replicative senescence and extending lifespan of human mesenchymal stromal cells (MSCs) may enhance their potential for tissue engineering and cell based therapies. Accumulating evidence suggests that inhibitors of the mTOR signaling pathway, such as rapamycin, constitute promising pharmacological agents to retard senescence and extend stemness properties of various progenitor cell types. Here, we investigated whether the ability of rapamycin to postpone replicative senescence varies among bone marrow MSC samples (BM-MSCs) derived from different healthy donors, and explored the molecular mechanisms that drive rapamycin-mediated lifespan increment. METHODS:BM-MSCs at early passages were serially passaged either in absence or continuous presence of rapamycin and the number of cell population doublings until growth arrest was measured. The inhibition of mTOR signaling was assessed by the phosphorylation status of the downstream target RPS6. The expression levels of several senescence and pluripotency markers at early and late/senescent passages were analyzed by RT-qPCR, flow cytometry and western blot. RESULTS:We found that the lifespan extension in response to the continuous rapamycin treatment was highly variable among samples, but effective in most BM-MSCs. Despite all rapamycin-treated cells secreted significantly reduced levels of IL6, a major SASP cytokine, and expressed significantly higher levels of the pluripotency marker NANOG, the expression patterns of these markers were not correlated with the rapamycin-mediated increase in lifespan. Interestingly, rapamycin-mediated life-span extension was significantly associated only with repression of p16INK4A protein accumulation. CONCLUSIONS:Taken together, our results suggest that some, but not all, BM-MSC samples would benefit from using rapamycin to postpone replicative arrest and reinforce a critical role of p16INK4A protein downregulation in this process.

SUBMITTER: Antonioli E

PROVIDER: S-EPMC6354956 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The need for an autologous cell source for bone tissue engineering and medical applications has led researchers to explore multipotent mesenchymal stromal cells (MSC), which show stem cell plasticity, in various human tissues. However, MSC with different tissue origins vary in their biological properties and their capability for osteogenic differentiation. Furthermore, MSC-based therapies require large-scale ex vivo expansion, accompanied by cell type-specific replicative senescence, which affects osteogenic differentiation. To elucidate cell type-specific differences in the osteogenic differentiation potential and replicative senescence, we analysed the impact of BMP and TGF-β signaling in adipose-derived stromal cells (ASC), fibroblasts (FB), and dental pulp stromal cells (DSC). We used inhibitors of BMP and TGF-β signaling, such as SB431542, dorsomorphin and/or a supplemental addition of BMP-2. The expression of high-affinity binding receptors for BMP-2 and calcium deposition with alizarin red S were evaluated to assess osteogenic differentiation potential. Our study demonstrated that TGF-β signaling inhibits osteogenic differentiation of ASC, DSC and FB in the early cell culture passages. Moreover, DSC had the best osteogenic differentiation potential and an activation of BMP signaling with BMP-2 could further enhance this capacity. This phenomenon is likely due to an increased expression of activin receptor-like kinase-3 and -6. However, in DSC with replicative senescence (in cell culture passage 10), osteogenic differentiation sharply decreased, and the simultaneous use of BMP-2 and SB431542 did not result in further improvement of this process. In comparison, ASC retain a similar osteogenic differentiation potential regardless of whether they were in the early (cell culture passage 3) or later (cell culture passage 10) stages. Our study elucidated that ASC, DSC, and FB vary functionally in their osteogenic differentiation, depending on their tissue origin and replicative senescence. Therefore, our study provides important insights for cell-based therapies to optimize prospective bone tissue engineering strategies.

Project description:Bone marrow mesenchymal stromal cells (BMSCs) are multipotent cells able to self-renew and differentiate, depending on the microenvironment, into adipocytes and osteoblasts. These cells have a limited number of replications and enter replicative senescence during in vitro expansion. The role of DNA methylation (DNAm) assumes importance in cell function and commitment; however, its exact contribution to BMSC differentiation and replicative senescence is still unclear. We performed a genome-wide DNAm analysis on BMSCs cultured in vitro at early passages and induced to differentiate into adipocytes and osteoblasts, and on replicative senescent BMSCs and HUVECs, to identify DNAm patterns of senescence and differentiation. We also compared BMSCs and HUVECs in replicative senescence and found that, in both cellular systems, genome-wide hypomethylation was accompanied by a higher-than-expected overlap of differentially methylated positions (DMPs) and concordance in terms of direction of the change. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on lineage-independent senescence-associated DMPs revealed 16 common pathways, including Insulin resistance, Molecule adhesion, and Wnt/β-catenin signaling. In both adipogenesis and osteogenesis, we observed a general demethylation of CpG sites compared with undifferentiated BMSCs with a higher number of DMPs in osteogenesis. KEGG analysis resulted in 30 pathways enriched in osteoblasts and only 2 in adipocytes when compared to undifferentiated cells. When comparing differentiated BMSCs with senescent ones, osteogenesis exhibited a greater overlap with senescence in terms of number of DMPs and direction of methylation change compared to adipogenesis. In conclusion, this study may be useful for future research on general mechanisms that occur in replicative senescence and furthermore to identify trajectories of BMSC differentiation and common aspects of differentiated and senescent cells.

Dataset Information

Individual response to mTOR inhibition in delaying replicative senescence of mesenchymal stromal cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets