Transcriptional profiling of interleukin-2-primed human adipose derived mesenchymal stem cells revealed dramatic changes in stem cells response imposed by replicative senescence

Abstract

Inflammation is a double-edged sword with both detrimental and beneficial consequences. Understanding of the mechanisms of crosstalk between the inflammatory milieu and human adult mesenchymal stem cells is an important basis for clinical efforts. Here, we investigate changes in the transcriptional response of human adipose-derived stem cells to physiologically relevant levels of IL-2 (IL-2 priming) upon replicative senescence. Our data suggest that replicative senescence might dramatically impede human mesenchymal stem cell (MSC) function via global transcriptional deregulation in response to IL-2. We uncovered a novel senescence-associated transcriptional signature in human adipose-derived MSCs hADSCs after exposure to pro-inflammatory environment: significant enhancement of the expression of the genes encoding potent growth factors and cytokines with anti-inflammatory and migration-promoting properties, as well as genes encoding angiogenic and anti-apoptotic promoting factors, all of which could participate in the establishment of a unique microenvironment. We observed transcriptional up-regulation of critical components of the nitric oxide synthase pathway (iNOS) in hADSCs upon replicative senescence suggesting, that senescent stem cells can acquire metastasis-promoting properties via stem cell-mediated immunosuppression. Our study highlights the importance of age as a factor when designing cell-based or pharmacological therapies for older patients and predicts measurable biomarkers characteristic of an environment that is conducive to cancer cells invasiveness and metastasis.

Acute Genotoxic Stress-Induced Senescence in Human Mesenchymal Cells Drives a Unique Composition of Senescence Messaging Secretome (SMS)

“…SEN hADSCs were obtained by treating the hADSCs at PD9 with 50 µg/ml final concentration of bleomycin diluted from 10mg/ml bleomycin stock in complete xeno-free media for 2 hrs at 37°C as previously described (in [27,31] and shown schematically in Figure 2A). Following the treatment, the medium was aspirated, the cells were washed twice with PBS and fresh complete xeno-free media was replaced.…”

“…The pH-dependent senescence-associated β-galactosidase activity (SA-β-Gal) assay was performed as described in the manufacturer's kit (BioVision) and previously published [10,27,29]. Briefly, the cultured hADSCs were fixed in fixative solution for 15 min at room temperature, washed twice with PBS and stained with X-Gal containing supplement overnight at 37°C.…”

“…Such resident senescent MSC might exert "bystander" effects inside the tumor microenvironment through their capacity to lock immunocompetent cells in a quiescent, non-proliferative state [25,26] thus helping tumors to evade immune surveillance. In addition, the "bystander" senescent MSC can promote an environment that supports tumor neovascularization and metastasis through the release of angiogenic, migratory and anti-apoptotic factors [27].…”

“…[27]. 100 ng of total RNA of each sample was used to construct libraries for sequencing on Ion Proton (Life Technologies, USA) following the manufacturer's instructions.…”

“…Sequence reads were mapped to the reference human genome assembly hg19 (GRCh37) using the Torrent Mapping Alignment Program (TMAP, Life Technologies). The quality of the four condition-specific combined RNA-seq runs was evaluated by comparing the expected counts of ERCC spike-in RNA sequences, obtained from the manufacturer's website, against the observed counts of RNA-seq tags that map to the same sequences as described in [27]. Initial gene expression levels were taken as the sum of exon-mapped reads for individual NCBI RefSeq gene models (c), and lowly expressed genes (read counts per million<1) were removed from subsequent analyses.…”