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

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 antiapoptotic 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.LM and BGG was supported by grants from the Spanish Ministry of Science and Innovation (SAF 2010-15239) to BGG and. LMP are supported by FPI fellowships from the Spanish Ministry, and BGG acknowledges support from the ``Ramon y Cajal´´ tenure track programme from the Spanish Ministry of Science and Innovation (RYC2009-04669). AS and AA are fellows of Bolashak International Scholarship, AA, AN, AS are sponsored by KazNMU sponsored program.S

Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells.

Growing evidence suggests that many diseases of aging, including diseases associated with robust changes and adipose deports, may be caused by resident adult stem cell exhaustion due to the process called cellular senescence. Understanding how microRNA pathways can regulate cellular senescence is crucial for the development of novel diagnostic and therapeutic strategies to combat these pathologies. Herein, using integrated transcriptomic and semi-quantitative proteomic analysis, we provide a system level view of the regulation of human adipose-derived stem cell senescence by a subset of mature microRNAs (termed senescence-associated-microRNAs) produced by biogenesis of oncogenic MIR17HG and tumor-suppressive MIR100HG clusters. We demonstrate functional significance of these mature senescence-associated-microRNAs in the process of replicative senescence of human adipose-derived stem cells ex-vivo and define a set of senescence-associated-microRNA gene targets that are able to elicit, modulate and, most importantly, balance intimate connections between oncogenic and senescent events

Unravelling the Basic Calcium Phosphate crystal-dependent chondrocyte protein secretome; a role for TGF-β signaling.

ObjectiveBasic Calcium Phosphate crystals play an active role in the progression of osteoarthritis. However, the cellular consequences remain largely unknown. Therefore, we characterized for the first time the changes in the protein secretome of human OA articular chondrocytes as a result of BCP stimulation using two unbiased proteomic analysis methods.MethodIsolated human OA articular chondrocytes were stimulated with BCP crystals and examined by RT-qPCR and ELISA after twenty-four and forty-eight hours. Forty-eight hours conditioned media were analysed by label-free LC-MS/MS and an antibody array. The activity of BCP dependent TGF-β signalling was analysed by RT-qPCR and luciferase reporter assays. The molecular consequences regarding BCP-dependent TGF-β signalling on BCP-dependent IL-6 were investigated using specific pathway inhibitors.ResultsSynthesized BCP crystals induced IL-6 expression and secretion upon stimulation of human articular chondrocytes. Concomitant induction of catabolic gene expression was observed. Analysis of conditioned media revealed a complex and diverse response with a large number of proteins involved in TGF-β signalling, both in activation of latent TGF-β and TGF-β superfamily members, which were increased compared to non-stimulated OA chondrocytes. Activity of this BCP driven TGF-β signalling was confirmed by increased activity of expression of TGF-β target genes and luciferase reporters. Inhibition of BCP driven TGF-β signalling resulted in decreased IL-6 expression and secretion with a moderate effect on catabolic gene expression.ConclusionBCP crystal stimulation resulted in a complex and diverse chondrocyte protein secretome response. An important role for BCP-dependent TGF-β signaling was identified in development of a pro-inflammatory environment

Analysis of RNA polyadenylation in healthy and osteoarthritic human articular cartilage

ABSTRACTAn important transcript structural element is its 3’ polyadenylated (polyA) tail, which defines the 3’ boundary of the transcript’s genetic information and is necessary for transcript stability. The position of the polyA tail can vary, with multiple alternatively polyadenylated (APA) transcripts existing for a single gene. This can lead to different length transcripts which can vary in their 3’ regulatory domains and even by inclusion or exclusion of protein-coding introns. The distribution of polyA tail location on articular chondrocyte transcripts has not been examined before and this study aimed to be the first to define polyadenylation events in human chondrocytes using age-matched healthy and osteoarthritic knee articular cartilage samples. Total RNA was isolated from frozen tissue samples and analysed using the QuantSeqReverse 3’ RNA Sequencing approach, where each read runs 3’ to 5’ from within the polyA tail into the transcript and will contains a distinct polyA site. Initial analysis of differential expression of overall transcript abundance identified by the reads showed significant disruption to transcript levels when healthy samples were compared to osteoarthritic ones. As we expected, differentially regulated genes were enriched with functionalities that were strongly associated with joint pathology. As part of this analysis, we also identified a substantial number of differentially expressed long non-coding RNAs that had not been linked to osteoarthritis before. Subsequent examination of polyA site data allowed us to deifne the extent of site usage across all the samples. This included identification of chondrocyte genes that exhibited the greatest amount polyA site variation. When comparing healthy and osteoarthritic samples, we found that differential use of polyadenylation sites was modest. However, of the small number of genes affected, there was clear potential for the change in polyadenylation site usage elicited by pathology to have functional relevance. We examined two genes, OSMR and KMT2A, in more detail, defining how APA affects transcript turnover and then, in the case of OSMR, identifying that APA is sensitive to inflammatory cytokine stimulation. Overall, we have characterised the polyadenylation landscape of human knee articular chondrocytes but can conclude that osteoarthritis does not elicit a widespread change in their polyadenylation site usage. This finding differentiates knee osteoarthritis from pathologies such as cancer where APA is more commonly observed.</jats:p

Analysis of RNA Polyadenylation in Healthy and Osteoarthritic Human Articular Cartilage.

Polyadenylation (polyA) defines the 3' boundary of a transcript's genetic information. Its position can vary and alternative polyadenylation (APA) transcripts can exist for a gene. This causes variance in 3' regulatory domains and can affect coding sequence if intronic events occur. The distribution of polyA sites on articular chondrocyte transcripts has not been studied so we aimed to define their transcriptome-wide location in age-matched healthy and osteoarthritic knee articular cartilage. Total RNA was isolated from frozen tissue samples and analysed using the QuantSeq-Reverse 3' RNA sequencing approach, where each read runs 3' to 5' from within the polyA tail into the transcript and contains a distinct polyA site. Differential expression of transcripts was significant altered between healthy and osteoarthritic samples with enrichment for functionalities that were strongly associated with joint pathology. Subsequent examination of polyA site data allowed us to define the extent of site usage across all the samples. When comparing healthy and osteoarthritic samples, we found that differential use of polyadenylation sites was modest. However, in the genes affected, there was potential for the APA to have functional relevance. We have characterised the polyadenylation landscape of human knee articular chondrocytes and conclude that osteoarthritis does not elicit a widespread change in their polyadenylation site usage. This finding differentiates knee osteoarthritis from pathologies such as cancer where APA is more commonly observed

Analysis of RNA Polyadenylation in Healthy and Osteoarthritic Human Articular Cartilage

Polyadenylation (polyA) defines the 3′ boundary of a transcript’s genetic information. Its position can vary and alternative polyadenylation (APA) transcripts can exist for a gene. This causes variance in 3′ regulatory domains and can affect coding sequence if intronic events occur. The distribution of polyA sites on articular chondrocyte transcripts has not been studied so we aimed to define their transcriptome-wide location in age-matched healthy and osteoarthritic knee articular cartilage. Total RNA was isolated from frozen tissue samples and analysed using the QuantSeq-Reverse 3′ RNA sequencing approach, where each read runs 3′ to 5′ from within the polyA tail into the transcript and contains a distinct polyA site. Differential expression of transcripts was significant altered between healthy and osteoarthritic samples with enrichment for functionalities that were strongly associated with joint pathology. Subsequent examination of polyA site data allowed us to define the extent of site usage across all the samples. When comparing healthy and osteoarthritic samples, we found that differential use of polyadenylation sites was modest. However, in the genes affected, there was potential for the APA to have functional relevance. We have characterised the polyadenylation landscape of human knee articular chondrocytes and conclude that osteoarthritis does not elicit a widespread change in their polyadenylation site usage. This finding differentiates knee osteoarthritis from pathologies such as cancer where APA is more commonly observed

MicroRNA Signatures in Cartilage Ageing and Osteoarthritis

Osteoarthritis is the most common degenerative joint disorder. MicroRNAs are gene expression regulators that act post-transcriptionally to control tissue homeostasis. Microarray analysis was undertaken in osteoarthritic intact, lesioned and young intact cartilage. Principal component analysis showed that young intact cartilage samples were clustered together; osteoarthritic samples had a wider distribution; and osteoarthritic intact samples were separated into two subgroups, osteoarthritic-Intact-1 and osteoarthritic-Intact-2. We identified 318 differentially expressed microRNAs between young intact and osteoarthritic lesioned cartilage, 477 between young intact and osteoarthritic-Intact-1 cartilage and 332 between young intact and osteoarthritic-Intact-2 cartilage samples. For a selected list of differentially expressed microRNAs, results were verified in additional cartilage samples using qPCR. Of the validated DE microRNAs, four—miR-107, miR-143-3p, miR-361-5p and miR-379-5p—were selected for further experiments in human primary chondrocytes treated with IL-1β. Expression of these microRNAs decreased in human primary chondrocytes treated with IL-1β. For miR-107 and miR-143-3p, gain- and loss-of-function approaches were undertaken and associated target genes and molecular pathways were investigated using qPCR and mass spectrometry proteomics. Analyses showed that WNT4 and IHH, predicted targets of miR-107, had increased expression in osteoarthritic cartilage compared to young intact cartilage and in primary chondrocytes treated with miR-107 inhibitor, and decreased expression in primary chondrocytes treated with miR-107 mimic, suggesting a role of miR-107 in chondrocyte survival and proliferation. In addition, we identified an association between miR-143-3p and EIF2 signalling and cell survival. Our work supports the role of miR-107 and miR-143-3p in important chondrocyte mechanisms regulating proliferation, hypertrophy and protein translation