Extracellular Vesicles: Proteome, miRnome and influence on stem cells

Background.

Recently, extracellular vesicles (EV) have been established as novel vehicles for a pleiotropy of molecules like proteins, coding and non-coding RNAs (Yànez-Mo et al., 2015). Therefore, EVs and their cargo have been proposed to contain biomarkers for various diseases similar to a "message in a bottle" (Lener et al., 2015; Yànez-Mo et al., 2015). In addition, EVs derived from mesenchymal stem cells as well as from dendritic cells have been recognized to have therapeutic potential to avoid tissue damage after stroke or myocardial infarction, as well as in immune modulation (EL Andaloussi et al., 2013; Lee et al., 2013).

EVs do not only populate the immediate environment of their donor cell, but can travel long distances from organ to organ and even from organism to organism (Yànez-Mo et al., 2015). During their transit through the extracellular space and body fluids, EVs prevent the degradation of their cargo (Mulcahy et al., 2014; Yànez-Mo et al., 2015). EVs are instrumental for protein targeting to recipient cells (Mulcahy et al., 2014) but can also be used as delivery system for cargo that is inserted artificially into EVs (EL Andaloussi et al., 2013).

The skin is our largest organ, and a first frontier towards external forces. It is a remarkably well regenerating organ, dependent on pools of stem cells that migrate to the surface and differentiate into epidermal layers (Tobin, 2006). However, it loses functionalities with increasing age leading to poor wound healing and epidermal dysfunctions that make the elderly more vulnerable towards infections, water loss and ability to cope with other stressors including UV irradiation, thereby increasing the probability to develop age-associated diseases (Zouboulis and Makrantonaki, 2011).

Aims and methods.

Our previous research identified selectively secreted and retained extravesicular miRNAs from various cell types including fibroblasts after their entry into cellular senescence. However, changes in the proteome composition during stress or cellular senescence and/or differential packaging of proteins into EVs have not yet been elucidated.

Therefore, our aim is to identify the proteome and confirm selected miRNAs of stressed and senescent fibroblast derived EVs. Furthermore, we will investigate the role of extracellular vesicles (EVs) and their protein and miRNA cargo especially on skin regeneration and homeostasis. Therefore, we will test the targeting of EV cargo from stressed as well as senescent fibroblasts to hair follicle stem cells and test its ability to influence differentiation and proliferation in 2D and 3D human organoid cultures. Then, we will identify stem cell influencing proteins or miRNAs. Hence, components, which change the "aged" stem cell niches, orchestrate the changes in ECM composition and barrier function encountered during wounding or ageing, will be identified and may represent novel therapeutic targets.

Our hypothesis is that (i) the proteome and miRNome cargo of EVs changes with cellular stress and aging, and that (ii) specific proteins or miRNAs impact on stem cell function using hair follicle stem cells as an example.

Therefore, we aim to investigate the composition of EVs secreted by stressed skin cells (fibroblasts and keratinocytes) using miRnomics and proteomics. We will perform mass spectrometry for proteome analysis (co-operation with Friedrich Altmann) as well as Next Generation Sequencing or qPCR panels to identify highly differentially secreted miRNA signature of EVs. Since standardized isolation is still not solved in the field of extracellular vesicle research, we will try to replace differential centrifugation from conditioned media by size exclusion chromatography (cooperation Alois Jungbauer). In contrast to serum contained EVs, not much is yet known on EVs from tissues like the skin, therefore, we also aim to analyze EV composition in vivo by isolating EVs from skin sections and determine their cargo. By applying data-mining and bioinformatic analysis of the cargo, we will identify miRNAs and proteins that influence skin cell physiology including barrier function, ECM composition, regeneration and wound healing under stress conditions, especially human skin stem cells. These proteins and miRNAs will be ranked based on known and predicted target analysis (bioinformatics in collaboration with Heinz Himmelbauer), literature search, preliminary results of the Grillari Lab in order to build testable hypothesis to overexpress or knock-down these candidates and test their influence on human hair follicle stem cell function, on skin homeostasis, as well as to prove the concept that they are actively transferred from one cell type to another. At the end of this project we will have identified EV derived cargo that changes in composition under stress and aging and thus will contribute to our understanding of EV biology and help to answer the question, to what extent EVs might be targets and tools in therapeutic strategies

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