Project description:We report small RNA sequencing technology for high-throughput profiling of microRNA content within small extracellular vesicles isolated from nonsenescent and senescent human dermal fibroblasts.
Project description:We report small RNA sequencing technology for high-throughput profiling of microRNA content within small extracellular vesicles isolated from nonsenescent and senescent human umbilical cord mesenchymal stem cells (hUCMSCs)
Project description:Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. However, the altered secretome of senescent cells can promote the growth of the surrounding cancer cells. Although extracellular vesicles (EVs) have emerged as new players in intercellular communication, their role in the function of senescent cell secretome has been largely unexplored. Here, we show that exosome-like small EVs (sEVs) are important mediators of the pro-tumorigenic function of senescent cells. sEV-associated EphA2 secreted from senescent cells binds to ephrin-A1 that is highly expressed in several types of cancer cells and promotes cell proliferation through EphA2/ephrin-A1 reverse signalling. sEV sorting of EphA2 is increased in senescent cells due to its enhanced phosphorylation resulting from oxidative inactivation of PTP1B phosphatase. Our results demonstrate a novel mechanism of reactive oxygen species (ROS)-regulated cargo sorting into sEVs, which is critical for the potentially deleterious growth-promoting effect of the senescent cell secretome.
Project description:Small RNA sequencing of small extracellular vesicles secreted by nonsenescent vs. senescent human umbilical cord mesenchymal stem cells
Project description:Ultraviolet B (UVB) radiation is a major contributor to skin photo-ageing. Although mainly absorbed by the epidermis, UVB photons managing to penetrate the upper dermis affect human dermal fibroblasts (HDFs), leading, among others, to the accumulation of senescent cells. In vitro studies have shown that repeated exposures to subcytotoxic UVB radiation doses provoke HDFs’ premature senescence shortly after the end of the treatment period. Here, we found that repetitive exposures to non-cytotoxic UVB radiation doses after several days lead to mixed cultures, containing both senescent cells and fibroblasts resisting senescence. “Resistant” fibroblasts were more resilient to a novel intense UVB radiation stimulus. RNA-seq analysis revealed that ERCC6, encoding Cockayne syndrome group B (CSB) protein, is upregulated in resistant HDFs compared to young and senescent cells, also confirmed at the protein level. CSB was found to be a key molecule conferring protection towards UVB cytotoxicity, as siRNA-mediated CSB loss-of-expression rendered HDFs significantly more susceptible to a high UVB radiation dose. In accordance, cells from a CSB-deficient patient were found to be dramatically more photosensitive. UVB-resistant HDFs remained normal (able to undergo replicative senescence) and non-tumorigenic. Even though they formed a distinct population in-between young and senescent cells, resistant HDFs retained numerous tissue-impairing characteristics of the senescence-associated secretory phenotype, including increased matrix metalloprotease activity and promotion of epidermoid tumor xenografts in immunodeficient mice. Collectively, here we describe a novel subpopulation of HDFs showing increased resistance to UVB-mediated premature senescence, as well as undesirable traits that may negatively affect skin homeostasis.
Project description:Acetylated proteome data of young human dermal fibroblasts (HDFs) [Young], old HDFs [Old], FTO-knockdown HDFs [shFTO], KAT8-overexpression HDFs [KAT8] and KAT8-overexpressed HDFs after FTO deprivation [shFTO_KAT8] were analysed by LC‒MS/MS.
Project description:To identify cellular senescence markers using senescent fibroblast-derived extracellular vesicles (EVs), we collected small EVs by three different methods: size exclusion chromatography (SEC), affinity column for phosphatidylserine (PS), and immunoprecipitation (IP) using antibodies against tetraspanin proteins (CD9, CD63 and CD81), from young and senescent fibroblasts to proteomic analysis. In addition, small EVs were collected from culture supernatants of cells derived from patients with Werner's syndrome, a known Progeria, and healthy individuals, and subjected to proteomic analysis.
Project description:A growing body of evidence in mammalian cells indicates that secreted vesicles can be used to mediate intercellular communication processes by transferring various bioactive molecules, including mRNAs and microRNAs. Based on these findings, we decided to analyze whether T. cruzi-derived extracellular vesicles contain RNA molecules and performed a deep sequencing and genome-wide analysis of a size-fractioned cDNA library (16M-bM-^@M-^S40 nt) from extracellular vesicles secreted by noninfective epimastigote and infective metacyclic trypomastigote forms. Our data show that the small RNAs contained in these extracellular vesicles originate from multiple sources, including tRNAs. In addition, our results reveal that the variety and expression of small RNAs are different between parasite stages, suggesting diverse functions. Taken together, these observations call attention to the potential regulatory functions that these RNAs might play once transferred between parasites and/or to mammalian host cells. Small RNAs profiles (16-40 nt) of epimastigote-derived extracellular vesicles, metacyclic trypomastigote-derived extracellular vesicles and metacyclic trypomastigote parental cells.
Project description:To define the characteristics of human oral mucosa fibroblasts (hOFs), we analyzed the gene expression of hOFs compared with that of human dermal fibroblasts (hDFs), and that of hOF-derived induced pluripotent stem cells (hOF-iPSCs). In this dataset, we found that 5,738 probes and 5672 probes were differently expressed in hOFs and hDFs respectively, compared with those in hOF-iPSCs at more than 2-fold levels. In contrast, only 434 probes are differently expressed between hOFs and hDFs.
Project description:Senescent cell extracellular vesicles (senEVs)are novel and underappreciated components of the senescence secretome. To understand the role that senEVs play in inflammatory responses to senescence, we developed and validated a novel engraftment-based senescence model that allowed us to genetically block the release of senEVs in vivo. We found that senEVs orchestrate prompt removal of senescent cells and inhibit tumor recurrence. Recruitment of MHC-II+ antigen presenting cells into the senescence microenvironment was significantly decreased in the absence of senEVs. Inhibition of senEV release changed the primary target of senescent cell signaling from antigen presenting cells to neutrophils. Antigen presenting cells recruited and activated CCR2+ TH17 cells, which in turn inhibited B cell activation. Through multimodal transcriptional and proteomic analysis, we identified 6 ligands that were specific to senEVs, suggesting a role in promoting cell adhesion. Overall, these results indicate that senEVs complement the activity of secreted inflammatory mediators in recruiting and activating distinct immune cell subsets for efficient removal of senescent cells.