L AS DISTINTAS EXPRESIONES DE LA CIUDADANÍA

The discovery of specialized EVs carrying regulatory molecules between cells has been the hallmark of a recently discovered novel form of intercellular communication. Us- ing comparative proteomics, genomics and bioinformatics analyses we showed that osteo-

§ Chapter 7

blasts secrete EVs containing the signature content of their parental cells while being selec- tively enriched with a unique set of bioactive cargo. With carefully designed studies, we showed that EVs regulate the expression of cell growth-related HSPC and bone- metastasizing prostate cancer genes, and stimulate their growth in vitro. However, the mo- lecular mechanisms behind EV-mediated crosstalk still remain to be explored. Thorough integrated biochemical and genetic analyses are necessary to identify the biologically active EV components and study their physiological relevance in vivo.

The increasing knowledge on EV biology has brought along therapeutic opportu- nities to be exploited clinically. We principally investigated the possibility of using osteo- blast-EVs in stem cell therapies, and consequently demonstrated their potency for ex vivo expansion of UCB-derived HSPCs suitable for transplantations. However, this approach comes with a caveat: EVs stimulate the proliferation and maintenance of HSPCs in short- term cultures. Our current knowledge on EV content coupled with further investigation on the key molecular mechanism that stimulate HSPC proliferation may give us clues to ma- nipulate EVs to stimulate long-term maintenance of the stem cells in vitro. This is especial- ly important to develop improved expansion strategies that will meet the clinical need. Moreover, understanding the key players that regulate HSPC fate holds the promise of us- ing EVs to manipulate other types of stem cells for various regenerative medicine applica- tions in the future.

The comprehensive EV data presented throughout this thesis serves as an exten- sive osteoblast-EV cargo repository that offers a great source for non-invasive diagnostics as well as facilitates the development of cell-free strategies for therapeutic applications. We showed that the osteoblast-EV protein content is greatly dependent on the stage of mineral- ization. Therefore, EVs pose as perfect candidates for biomarker discovery useful for the early detection of bone diseases. Furthermore, thanks to their endogenous and biocompati- ble nature EVs are ideal virus-free vector alternatives for safer gene therapy applications. We anticipate that an elaborate understanding of the mechanism of EV cargo loading will enable us to develop tailored EVs containing therapeutic cargo to treat a broad variety of pathological conditions.

With increasing number of studies deciphering the complexity of EV biology, this newly emerging scientific field rapidly advances beyond basic science. In this thesis, we presented our in vitro efforts to uncover the intricate interactions networks that underlie the function of EVs secreted by osteoblasts. To move forward, we need to identify new re- search strategies and invest in developing optimized technologies that enhances our knowledge required to bring osteoblast-EVs towards clinical applications.

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Appendix A