Talk list. Definition: open questions. Functions: manyfold. The road ahead: GMP, open questions. From discovery. To basic science
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1 Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles* * and from the NIH exrna Communication Consortium Ciro Tetta MD
2 Talk list Definition: open questions From discovery Functions: manyfold To basic science The road ahead: GMP, open questions To clinical application Yanez-Mo, M et al, Biological activities of EVs and their physiological functions, JEV 2015; 4: Quesenberry PJ et al. Potential fucntional applications of EVs: a report by the NIHCommon Fund Extracellular RNA Communication Consortium. JEV 2015; 4:27575
3 Documents Documents Documents EV research has been of interest for over a decade, but with major growth beginning in Extracellular vesicle total 4,058 documents Microvesicle total 501 documents 600 Exosome total 3,249 documents Scopus search and data analysis (Search terms in Abstract, Title, Keywords, from , all document types, all subject areas ) 3
4 DEFINITION 4
5 Extracellular vesicles Exosomes Cell-derived Vesicles Microvesicles Apoptotic bodies Camussi G. et al. Kidney Int 2010; Tetta C. et al. Endocrine,
6 Size Distribution of Extracellular Vesicles György B, et al. Cell Mol Life Sci 2011;68:
7 Nothing goes lost! From Byproduct to Product Open questions: what is the golden standard technique/s? What do we mean by that?
8 FUNCTION 8
9 The biogenesis and uptake of exrna. Exocytosis is commonly mediated via clathrin-coated pits after which endocytic vesicles progress from early to late endosomes also referred to as multivesicular bodies. ExRNA can be detected in the exosomes or in the microvesicles associated to proteins or as part of lipoprotein particles, particularly HDL. Open questions: how exrnas are released for functional activity upon entry into recipient cells Patton JG, JEV
10 EVs from stem /progenitor cells as a paracrine / endocrine mechanism EVs may mediate a horizontal transfer of genetic information Murine Embryonic Stem Cells - Ratajczak J et al. Leukemia Yuan A et al. PLoS One 2009 Adult Stem Cells - EVs - human EPC-derived EVs: proangiogenesis on endothelial cells - Deregibus et al. Blood human EPC-derived EV microrna: - Cantaluppi et al. Cell Transplant 2012; Kidney Int human MSC-derived EV mrna - Bruno et al. JASN human MSC-derived EV microrna - Collino et al. PloS One human liver SC-derived EV mrna - Herrera et al. J Cell Mol Med. 2010
11 Pro-angiogenetic and repair effect
12 Characterization of MSC-derived MVs MVs shuttle selected patterns of mrna and microrna (GEO d.b. accession GSE12243) A total of 365 transcripts were found*, 132 were associated to Entrez Gene identifiers by IPA 6.0 analysis, including: -mrna characteristic of mesenchymal cell lineages, such as neural, osteogenic, epithelial and hematopoietic; -mrna involved in the control of transcription, cell proliferation and immune regulation. * 42 were co-expressed in MV and cells Collino et al. PLoS One 2010
13 Collino et al. PLoS One 2010 Expression of RNA-binding proteins in MVs RNPs in MSC (stress granules) RNPs in MVs Western blot analysis TEM of MVs Immunogold analysis MV as compartimentalization site for RNA
14 Characterization of mirnas Precursor mirnas Mature mirnas mrna degradation by mirnas Collino et al. PLoS One 2010
15 EVIDENCE FOR MV-MEDIATED TRANSFER OF GENETIC INFORMATION A proof of transfer in target cells of mrna delivered from MVs derives from: -experiments showing the transfer of reporter genes. MVs derived from human stem cells may deliver both in vitro and in vivo human mrna -Bruno et al. JASN 2009 Herrera et al. J Cell Mol. Med experiments showing translation into protein of reporter mrna such as the green fluorescence protein mrna (GFP). Cells targeted with MVs carrying GFP mrna transiently produced the GFP proteins Deregibus et al. Blood 2007; Aliotta et al. 2010
16 EVIDENCE FOR MV-MEDIATED TRANSFER OF GENETIC INFORMATION MV-mediated transfer of specific mirnas to target cells (mtec) Selective expression of mirnas (in a-amanitin-treated mtec) Internalization of PKH26-labeled MVs in mtec (by fluorescence-labeled reporter mirnas) Internalization of PKH26-labeled MVs in mtec Reduction of expression of target proteins of specific mirnas PTEN mir-21 cyclin D1 mir-100, mir-99,mir-223 Bcl-2 mir-34, mir-181b, mir-16 Collino et al PLoS One 2010
17 EVs Protect Against Acute Tubular Injury
18 EVs Protect from Lethal Cisplatin-induced Renal Injury Effect of EVs on in vitro expression of gene involved in apoptosis in tubular cells treated with cisplatin: CIS +MV vs CIS Up-regulated Down-regulated BCL2 BCL2L1(BCL-XL) BIRC8 LTA CASP8 CASP1 BCL2L11 TP73 HRK CARD6
19 Bidirectional exchange of information between injured cells and stem cells EV-mediated reprogramming of renal resident cells by transfer of specific mrnas, mirnas and proteins -Deregibus et al. Blood Bruno et al. J Am Soc Nephrol Gatti et al. NDT 2011 & Proteins -Dooner et al. Stem Cells Dev Aliotta et al. Exp Hematology Quesenberry et al. Stem Cells Dev Open questions: how exrnas are released for functional activity upon entry into recipient cells? Are the epigenetic effects really all due to exrna transfer or only partly?
20 PEOPLE MARIE CURIE ACTIONS Marie Curie Industry-Academia Partnerships and Pathways (IAPP) Call: FP7-PEOPLE-2013-IAPP Role of Extracellular Vesicles from Stem Cells in organ injury (EVStemInjury) IAPP2013 EVStemInjury Project Catenion meeting Mar 2014
21 The partners PUBLIC SECTOR PRIVATE SECTOR
22 Potential clinical utility of EVs Considerations for regulatory requirements GMP Protocol of isolation to be defined (optimal cell source culturing, isolation, characterization markers, cost-effective production / automation, optimal dosing), Size range for EVs Biochemical markers (for EVs broadly and for specific preparations) Purity of EV preparation and absence of undesired components Sterility of cell growth and EV isolation environments Optimal EV isolation methods (including for scalability, purity, and particular EV subsets) Development of EV stabilization methods Optimal storage conditions for EV stability Analytical methods and standards for all of the above Serum-free media use (likely to be compulsory)
23 Potential clinical utility of EVs Considerations for regulatory requirements Consistency The preparative method results in a heterogeneous population. Further separation to a homogenous population would not be technically possible because there are no definite size, density or protein markers that can reliably define a specific population. EV product should not have an absolute requirement for characterization of cell-surface markers, content, or for homogeneity but rather should be based on the definition and description of a size-defined, functionally effective population isolated using a robust, reproducible and validated process.
24 Potential clinical utility of EVs Considerations for regulatory requirements Safety - The safety profile needs to be demonstrated in preclinical studies. Efficacy - Functional potency test relevant to the proposed application. Dose and delivery - This would depend on efficacy and safety profiles, bioavailability and specific formulation of the therapeutic product e.g. the EV product may be cryopreserved, lyophilized, used in suspension, or combined into a matrix. 24
25 GMP Production: what do we need - Isolation Protocols for large recovery: - UC / SEC / FFF / Polymeric P - FAQ: HOW DO THEY AFFECT THE BIOLOGICAL ACTIVITIES OF EV? - Criteria for EV characterization: reproducible functional tests (forget about homogeneity!) - FAQ: WHICH ARE THE CRITERIA? - Validated potency assays designed for different app - The assays qualified for potenecy should be neither too sophisticated, time-consuming nor too expensive. A point that remains to be solved is quantitation of EVs 25
26 Translational Centre of Regenerative Medicine University of Torino M C. Tetta V. Fonsato F. Collino M.. Herrera V.M. Navarro F. Figliolini M. Tapparo C. Cavallari M.C. Deregibus F. Antico C. Contursi G. Camussi S. Bruno C. Grange V. Cantaluppi C. Pasquino M. Delena A. Iavello A.Carpanetto A.Pitino S.Previdi Cell-Factory: M.Gunetti Administrative Office G. Ferri
27 27
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