Publications by authors named "Eva Rohde"

41 Publications

Extracellular vesicles from human multipotent stromal cells protect against hearing loss after noise trauma in vivo.

Clin Transl Med 2020 Dec;10(8):e262

GMP Unit, Spinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria.

The lack of approved anti-inflammatory and neuroprotective therapies in otology has been acknowledged in the last decades and recent approaches are heralding a new era in the field. Extracellular vesicles (EVs) derived from human multipotent (mesenchymal) stromal cells (MSC) can be enriched in vesicular secretome fractions, which have been shown to exert effects (eg, neuroprotection and immunomodulation) of their parental cells. Hence, MSC-derived EVs may serve as novel drug candidates for several inner ear diseases. Here, we provide first evidence of a strong neuroprotective potential of human stromal cell-derived EVs on inner ear physiology. In vitro, MSC-EV preparations exerted immunomodulatory activity on T cells and microglial cells. Moreover, local application of MSC-EVs to the inner ear significantly attenuated hearing loss and protected auditory hair cells from noise-induced trauma in vivo. Thus, EVs derived from the vesicular secretome of human MSC may represent a next-generation biological drug that can exert protective therapeutic effects in a complex and nonregenerating organ like the inner ear.
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http://dx.doi.org/10.1002/ctm2.262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7752163PMC
December 2020

Weiss Response to Sengupta et al. (DOI: 10.1089/scd.2020.0095).

Stem Cells Dev 2020 12;29(24):1533-1534

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

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http://dx.doi.org/10.1089/scd.2020.0114DOI Listing
December 2020

Re: "Exosomes Derived from Bone Marrow Mesenchymal Stem Cells as Treatment for Severe COVID-19" by Sengupta et al.

Stem Cells Dev 2020 07 10;29(14):877-878. Epub 2020 Jun 10.

GMP Unit, Spinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), University Hospital, Salzburg, Austria.

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http://dx.doi.org/10.1089/scd.2020.0089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374615PMC
July 2020

International Society for Extracellular Vesicles and International Society for Cell and Gene Therapy statement on extracellular vesicles from mesenchymal stromal cells and other cells: considerations for potential therapeutic agents to suppress coronavirus disease-19.

Cytotherapy 2020 09 16;22(9):482-485. Epub 2020 May 16.

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany. Electronic address:

Statement: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight.
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http://dx.doi.org/10.1016/j.jcyt.2020.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229942PMC
September 2020

Upregulation of mitotic bookmarking factors during enhanced proliferation of human stromal cells in human platelet lysate.

J Transl Med 2019 12 30;17(1):432. Epub 2019 Dec 30.

Department of Transfusion Medicine, University Hospital of Salzburg (SALK), Paracelsus Medical University, Salzburg, Austria.

Background: Innovative human stromal cell therapeutics require xeno-free culture conditions. Various formulations of human platelet lysate (HPL) are efficient alternatives for fetal bovine serum (FBS). However, a consistent lack of standardized manufacturing protocols and quality criteria hampers comparability of HPL-products. Aim of this study was to compare the biochemical composition of three differential HPL-preparations with FBS and to investigate their impact on stromal cell biology.

Methods: Stromal cells were isolated from bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC) and cultured in medium supplemented with pooled HPL (pHPL), fibrinogen-depleted serum-converted pHPL (pHPLS), mechanically fibrinogen-depleted pHPL (mcpHPL) and FBS. Biochemical parameters were analyzed in comparison to standard values in whole blood. Distinct growth factors and cytokines were measured by bead-based multiplex technology. Flow cytometry of stromal cell immunophenotype, in vitro differentiation, and mRNA expression analysis of transcription factors SOX2, KLF4, cMYC, OCT4 and NANOG were performed.

Results: Biochemical parameters were comparable in all pHPL preparations, but to some extent different to FBS. Total protein, glucose, cholesterol and Na were elevated in pHPL preparations, K and Fe levels were higher in FBS. Compared to FBS, pHPL-based media significantly enhanced stromal cell propagation. Characteristic immunophenotype and in vitro differentiation potential were maintained in all four culture conditions. The analysis of growth factors and cytokines revealed distinct levels depending on the pre-existence in pHPL, consumption or secretion by the stromal cells. Interestingly, mRNA expression of the transcription and mitotic bookmarking factors cMYC and KLF4 was significantly enhanced in a source dependent manner in stromal cells cultured in pHPL- compared to FBS-supplemented media. SOX2 mRNA expression of all stromal cell types was increased in all pHPL culture conditions.

Conclusion: All pHPL-supplemented media equally supported proliferation of WAT- and UC-derived stromal cells significantly better than FBS. Mitotic bookmarking factors, known to enable a quick re-entry to the cell cycle, were significantly enhanced in pHPL-expanded cells. Our results support a better characterization and standardization of humanized culture media for stromal cell-based medicinal products.
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http://dx.doi.org/10.1186/s12967-019-02183-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936143PMC
December 2019

Extracellular Vesicles Can Deliver Anti-inflammatory and Anti-scarring Activities of Mesenchymal Stromal Cells After Spinal Cord Injury.

Front Neurol 2019 29;10:1225. Epub 2019 Nov 29.

Institute of Experimental Neuroregeneration, Paracelsus Medical University, Salzburg, Austria.

Spinal cord injury is characterized by initial neural tissue disruption that triggers secondary damage and extensive non-resolving inflammation, which aggravates loss of function and hinders recovery. The early onset of inflammation following traumatic spinal cord injury underscores the importance of acute intervention after the initial trauma. Injections of mesenchymal stromal cells (MSCs) can reduce inflammation following spinal cord injury. We asked if extracellular vesicles (EVs) can substitute the anti-inflammatory and anti-scarring activities of their parental MSCs in a rat model of contusion spinal cord injury. We report that MSC-EVs were as potent as the parental intact cells in reducing the level of neuroinflammation for up to 2 weeks post-injury. Acute application of EVs after spinal cord injury was shown to robustly decrease the expression of pro-inflammatory cytokines in the spinal cord parenchyma in the very early phase of secondary damage. Moreover, the anti-scarring impact of MSC-EVs was even more efficient than the parental cells. We therefore conclude that anti-inflammatory and anti-scarring activities of MSC application can be mediated by their secreted EVs. In light of their substantial safety and druggability advantages, EVs may have a high potential in early therapeutic treatment following traumatic spinal cord injury.
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http://dx.doi.org/10.3389/fneur.2019.01225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896947PMC
November 2019

Single-use IgE-selective immunoadsorber column for the treatment of severe atopic dermatitis.

J Clin Apher 2020 Jan 22;35(1):50-58. Epub 2019 Nov 22.

Department of Transfusion Medicine, Paracelsus Medical University Hospital Salzburg, Salzburg, Austria.

Background: Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease with unmet therapeutic need in a critical cohort of recalcitrant cases. Immunoadsorption (IA) aims at an immunomodulatory depletion of pathogenic serum mediators and has recently revealed promising clinical results for the treatment of AD.

Objective: To determine efficacy, sustainability, safety, and clinical impact of IgE selective IA in AD using a single-use IgE immunoadsorber column.

Methods: This open-label pilot study comprised five patients (mean SCORAD 67.9 ± 11.4, range 52.2-81.9; mean serum IgE level 5904 ± 5945 U/mL, range 1000-15 600 IU/mL) who underwent IgE-selective IA. Three patients continued prior therapy with systemic immunosuppressive drugs during IA as an add-on therapeutic approach. All patients received three courses of IA. The first course consisted of three consecutive daily treatments followed by two sequences with two consecutive applications. All courses were performed on a monthly regimen.

Results: IA proved efficacy in selectively depleting serum IgE levels in all participants (mean reduction by cycle of 81% ± 12%, range 64%-93%). It further led to a clinically relevant and sustained improvement of AD with a maximum decline in SCORAD and EASI scores by up to 35% and 52%, respectively, compared to baseline. Scores persisted below baseline for at least 12 weeks beyond the last IA. The intervention was also well tolerated with no severe adverse events during a total of 35 procedures.

Conclusion: Data of this preliminary trial indicates clinical efficacy, feasibility, safety as well as tolerability of IgE-selective IA in AD.
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http://dx.doi.org/10.1002/jca.21759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003837PMC
January 2020

Heparin Differentially Impacts Gene Expression of Stromal Cells from Various Tissues.

Sci Rep 2019 05 10;9(1):7258. Epub 2019 May 10.

Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University of Salzburg, Salzburg, Austria.

Pooled human platelet lysate (pHPL) is increasingly used as replacement of animal serum for manufacturing of stromal cell therapeutics. Porcine heparin is commonly applied to avoid clotting of pHPL-supplemented medium but the influence of heparin on cell behavior is still unclear. Aim of this study was to investigate cellular uptake of heparin by fluoresceinamine-labeling and its impact on expression of genes, proteins and function of human stromal cells derived from bone marrow (BM), umbilical cord (UC) and white adipose tissue (WAT). Cells were isolated and propagated using various pHPL-supplemented media with or without heparin. Flow cytometry and immunocytochemistry showed differential cellular internalization and lysosomal accumulation of heparin. Transcriptome profiling revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis, depending on stromal cell origin. The influence of heparin on the WNT, PDGF, NOTCH and TGFbeta signaling pathways was further analyzed by a bead-based western blot revealing most alterations in BM-derived stromal cells. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells, indicating compatibility for clinically applied cell products.
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http://dx.doi.org/10.1038/s41598-019-43700-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510770PMC
May 2019

Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications.

J Extracell Vesicles 2019 29;8(1):1609206. Epub 2019 Apr 29.

Institute of Medical Biology, Agency for Science, Technology and Research, Singapore, Singapore.

Small extracellular vesicles (sEVs) from mesenchymal stromal/stem cells (MSCs) are transiting rapidly towards clinical applications. However, discrepancies and controversies about the biology, functions, and potency of MSC-sEVs have arisen due to several factors: the diversity of MSCs and their preparation; various methods of sEV production and separation; a lack of standardized quality assurance assays; and limited reproducibility of and functional assays. To address these issues, members of four societies (SOCRATES, ISEV, ISCT and ISBT) propose specific harmonization criteria for MSC-sEVs to facilitate data sharing and comparison, which should help to advance the field towards clinical applications. Specifically, MSC-sEVs should be defined by quantifiable metrics to identify the cellular origin of the sEVs in a preparation, presence of lipid-membrane vesicles, and the degree of physical and biochemical integrity of the vesicles. For practical purposes, new MSC-sEV preparations might also be measured against a well-characterized MSC-sEV biological reference. The ultimate goal of developing these metrics is to map aspects of MSC-sEV biology and therapeutic potency onto quantifiable features of each preparation.
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http://dx.doi.org/10.1080/20013078.2019.1609206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6493293PMC
April 2019

Manufacturing and characterization of extracellular vesicles from umbilical cord-derived mesenchymal stromal cells for clinical testing.

Cytotherapy 2019 06 9;21(6):581-592. Epub 2019 Apr 9.

GMP Laboratory, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria; Department of Transfusion Medicine, Paracelsus Medical University (PMU), Salzburg, Austria; Research Program Nanovesicular Therapies, Paracelsus Medical University (PMU), Salzburg, Austria; Celericon Therapeutics G.m.b.H., Paracelsus Medical University (PMU), Salzburg, Austria.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) may deliver therapeutic effects that are comparable to their parental cells. MSC-EVs are promising agents for the treatment of a variety of diseases. To reach the intermediate goal of clinically testing safety and efficacy of EVs, strategies should strive for efficient translation of current EV research. On the basis of our in vitro an in vivo findings regarding the biological actions of EVs and our experience in manufacturing biological stem cell therapeutics for routine use and clinical testing, we discuss strategies of manufacturing and quality control of umbilical cord-derived MSC-EVs. We introduce guidelines of good manufacturing practice and their practicability along the path from the laboratory to the patient. We present aspects of manufacturing and final product quality testing and highlight the principle of "The process is the product." The approach presented in this perspective article may facilitate translational research during the development of complex biological EV-based therapeutics in a very early stage of manufacturing as well as during early clinical safety and proof-of-concept testing.
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http://dx.doi.org/10.1016/j.jcyt.2018.12.006DOI Listing
June 2019

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

Authors:
Clotilde Théry Kenneth W Witwer Elena Aikawa Maria Jose Alcaraz Johnathon D Anderson Ramaroson Andriantsitohaina Anna Antoniou Tanina Arab Fabienne Archer Georgia K Atkin-Smith D Craig Ayre Jean-Marie Bach Daniel Bachurski Hossein Baharvand Leonora Balaj Shawn Baldacchino Natalie N Bauer Amy A Baxter Mary Bebawy Carla Beckham Apolonija Bedina Zavec Abderrahim Benmoussa Anna C Berardi Paolo Bergese Ewa Bielska Cherie Blenkiron Sylwia Bobis-Wozowicz Eric Boilard Wilfrid Boireau Antonella Bongiovanni Francesc E Borràs Steffi Bosch Chantal M Boulanger Xandra Breakefield Andrew M Breglio Meadhbh Á Brennan David R Brigstock Alain Brisson Marike Ld Broekman Jacqueline F Bromberg Paulina Bryl-Górecka Shilpa Buch Amy H Buck Dylan Burger Sara Busatto Dominik Buschmann Benedetta Bussolati Edit I Buzás James Bryan Byrd Giovanni Camussi David Rf Carter Sarah Caruso Lawrence W Chamley Yu-Ting Chang Chihchen Chen Shuai Chen Lesley Cheng Andrew R Chin Aled Clayton Stefano P Clerici Alex Cocks Emanuele Cocucci Robert J Coffey Anabela Cordeiro-da-Silva Yvonne Couch Frank Aw Coumans Beth Coyle Rossella Crescitelli Miria Ferreira Criado Crislyn D'Souza-Schorey Saumya Das Amrita Datta Chaudhuri Paola de Candia Eliezer F De Santana Olivier De Wever Hernando A Del Portillo Tanguy Demaret Sarah Deville Andrew Devitt Bert Dhondt Dolores Di Vizio Lothar C Dieterich Vincenza Dolo Ana Paula Dominguez Rubio Massimo Dominici Mauricio R Dourado Tom Ap Driedonks Filipe V Duarte Heather M Duncan Ramon M Eichenberger Karin Ekström Samir El Andaloussi Celine Elie-Caille Uta Erdbrügger Juan M Falcón-Pérez Farah Fatima Jason E Fish Miguel Flores-Bellver András Försönits Annie Frelet-Barrand Fabia Fricke Gregor Fuhrmann Susanne Gabrielsson Ana Gámez-Valero Chris Gardiner Kathrin Gärtner Raphael Gaudin Yong Song Gho Bernd Giebel Caroline Gilbert Mario Gimona Ilaria Giusti Deborah Ci Goberdhan André Görgens Sharon M Gorski David W Greening Julia Christina Gross Alice Gualerzi Gopal N Gupta Dakota Gustafson Aase Handberg Reka A Haraszti Paul Harrison Hargita Hegyesi An Hendrix Andrew F Hill Fred H Hochberg Karl F Hoffmann Beth Holder Harry Holthofer Baharak Hosseinkhani Guoku Hu Yiyao Huang Veronica Huber Stuart Hunt Ahmed Gamal-Eldin Ibrahim Tsuneya Ikezu Jameel M Inal Mustafa Isin Alena Ivanova Hannah K Jackson Soren Jacobsen Steven M Jay Muthuvel Jayachandran Guido Jenster Lanzhou Jiang Suzanne M Johnson Jennifer C Jones Ambrose Jong Tijana Jovanovic-Talisman Stephanie Jung Raghu Kalluri Shin-Ichi Kano Sukhbir Kaur Yumi Kawamura Evan T Keller Delaram Khamari Elena Khomyakova Anastasia Khvorova Peter Kierulf Kwang Pyo Kim Thomas Kislinger Mikael Klingeborn David J Klinke Miroslaw Kornek Maja M Kosanović Árpád Ferenc Kovács Eva-Maria Krämer-Albers Susanne Krasemann Mirja Krause Igor V Kurochkin Gina D Kusuma Sören Kuypers Saara Laitinen Scott M Langevin Lucia R Languino Joanne Lannigan Cecilia Lässer Louise C Laurent Gregory Lavieu Elisa Lázaro-Ibáñez Soazig Le Lay Myung-Shin Lee Yi Xin Fiona Lee Debora S Lemos Metka Lenassi Aleksandra Leszczynska Isaac Ts Li Ke Liao Sten F Libregts Erzsebet Ligeti Rebecca Lim Sai Kiang Lim Aija Linē Karen Linnemannstöns Alicia Llorente Catherine A Lombard Magdalena J Lorenowicz Ákos M Lörincz Jan Lötvall Jason Lovett Michelle C Lowry Xavier Loyer Quan Lu Barbara Lukomska Taral R Lunavat Sybren Ln Maas Harmeet Malhi Antonio Marcilla Jacopo Mariani Javier Mariscal Elena S Martens-Uzunova Lorena Martin-Jaular M Carmen Martinez Vilma Regina Martins Mathilde Mathieu Suresh Mathivanan Marco Maugeri Lynda K McGinnis Mark J McVey David G Meckes Katie L Meehan Inge Mertens Valentina R Minciacchi Andreas Möller Malene Møller Jørgensen Aizea Morales-Kastresana Jess Morhayim François Mullier Maurizio Muraca Luca Musante Veronika Mussack Dillon C Muth Kathryn H Myburgh Tanbir Najrana Muhammad Nawaz Irina Nazarenko Peter Nejsum Christian Neri Tommaso Neri Rienk Nieuwland Leonardo Nimrichter John P Nolan Esther Nm Nolte-'t Hoen Nicole Noren Hooten Lorraine O'Driscoll Tina O'Grady Ana O'Loghlen Takahiro Ochiya Martin Olivier Alberto Ortiz Luis A Ortiz Xabier Osteikoetxea Ole Østergaard Matias Ostrowski Jaesung Park D Michiel Pegtel Hector Peinado Francesca Perut Michael W Pfaffl Donald G Phinney Bartijn Ch Pieters Ryan C Pink David S Pisetsky Elke Pogge von Strandmann Iva Polakovicova Ivan Kh Poon Bonita H Powell Ilaria Prada Lynn Pulliam Peter Quesenberry Annalisa Radeghieri Robert L Raffai Stefania Raimondo Janusz Rak Marcel I Ramirez Graça Raposo Morsi S Rayyan Neta Regev-Rudzki Franz L Ricklefs Paul D Robbins David D Roberts Silvia C Rodrigues Eva Rohde Sophie Rome Kasper Ma Rouschop Aurelia Rughetti Ashley E Russell Paula Saá Susmita Sahoo Edison Salas-Huenuleo Catherine Sánchez Julie A Saugstad Meike J Saul Raymond M Schiffelers Raphael Schneider Tine Hiorth Schøyen Aaron Scott Eriomina Shahaj Shivani Sharma Olga Shatnyeva Faezeh Shekari Ganesh Vilas Shelke Ashok K Shetty Kiyotaka Shiba Pia R-M Siljander Andreia M Silva Agata Skowronek Orman L Snyder Rodrigo Pedro Soares Barbara W Sódar Carolina Soekmadji Javier Sotillo Philip D Stahl Willem Stoorvogel Shannon L Stott Erwin F Strasser Simon Swift Hidetoshi Tahara Muneesh Tewari Kate Timms Swasti Tiwari Rochelle Tixeira Mercedes Tkach Wei Seong Toh Richard Tomasini Ana Claudia Torrecilhas Juan Pablo Tosar Vasilis Toxavidis Lorena Urbanelli Pieter Vader Bas Wm van Balkom Susanne G van der Grein Jan Van Deun Martijn Jc van Herwijnen Kendall Van Keuren-Jensen Guillaume van Niel Martin E van Royen Andre J van Wijnen M Helena Vasconcelos Ivan J Vechetti Tiago D Veit Laura J Vella Émilie Velot Frederik J Verweij Beate Vestad Jose L Viñas Tamás Visnovitz Krisztina V Vukman Jessica Wahlgren Dionysios C Watson Marca Hm Wauben Alissa Weaver Jason P Webber Viktoria Weber Ann M Wehman Daniel J Weiss Joshua A Welsh Sebastian Wendt Asa M Wheelock Zoltán Wiener Leonie Witte Joy Wolfram Angeliki Xagorari Patricia Xander Jing Xu Xiaomei Yan María Yáñez-Mó Hang Yin Yuana Yuana Valentina Zappulli Jana Zarubova Vytautas Žėkas Jian-Ye Zhang Zezhou Zhao Lei Zheng Alexander R Zheutlin Antje M Zickler Pascale Zimmermann Angela M Zivkovic Davide Zocco Ewa K Zuba-Surma

J Extracell Vesicles 2018 23;7(1):1535750. Epub 2018 Nov 23.

Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Kraków, Poland.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
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http://dx.doi.org/10.1080/20013078.2018.1535750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322352PMC
November 2018

Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles.

Stem Cells Transl Med 2017 08 17;6(8):1730-1739. Epub 2017 Jul 17.

Institute of Medical Biology, A*STAR, Singapore.

Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell-related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore convened a Workshop on this topic to discuss the opportunities and challenges associated with development of EV-based therapeutics at the preclinical and clinical levels. This review outlines topic-specific action items that, if addressed, will enhance the development of best-practice models for EV therapies. Stem Cells Translational Medicine 2017;6:1730-1739.
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http://dx.doi.org/10.1002/sctm.17-0055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5689784PMC
August 2017

An In Vitro Potency Assay for Monitoring the Immunomodulatory Potential of Stromal Cell-Derived Extracellular Vesicles.

Int J Mol Sci 2017 Jul 1;18(7). Epub 2017 Jul 1.

GMP Unit, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Strubergasse 22, 5020 Salzburg, Austria.

The regenerative and immunomodulatory activity of mesenchymal stromal cells (MSCs) is partially mediated by secreted vesicular factors. Extracellular vesicles (EVs) exocytosed by MSCs are gaining increased attention as prospective non-cellular therapeutics for a variety of diseases. However, the lack of suitable in vitro assays to monitor the therapeutic potential of EVs currently restricts their application in clinical studies. We have evaluated a dual in vitro immunomodulation potency assay that reproducibly reports the inhibitory effect of MSCs on induced T-cell proliferation and the alloantigen-driven mixed leukocyte reaction of pooled peripheral blood mononuclear cells in a dose-dependent manner. Phytohemagglutinin-stimulated T-cell proliferation was inhibited by MSC-derived EVs in a dose-dependent manner comparable to MSCs. In contrast, inhibition of alloantigen-driven mixed leukocyte reaction was only observed for MSCs, but not for EVs. Our results support the application of a cell-based in vitro potency assay for reproducibly determining the immunomodulatory potential of EVs. Validation of this assay can help establish reliable release criteria for EVs for future clinical studies.
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http://dx.doi.org/10.3390/ijms18071413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535905PMC
July 2017

Manufacturing of Human Extracellular Vesicle-Based Therapeutics for Clinical Use.

Int J Mol Sci 2017 Jun 3;18(6). Epub 2017 Jun 3.

GMP Unit, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria.

Extracellular vesicles (EVs) derived from stem and progenitor cells may have therapeutic effects comparable to their parental cells and are considered promising agents for the treatment of a variety of diseases. To this end, strategies must be designed to successfully translate EV research and to develop safe and efficacious therapies, whilst taking into account the applicable regulations. Here, we discuss the requirements for manufacturing, safety, and efficacy testing of EVs along their path from the laboratory to the patient. Development of EV-therapeutics is influenced by the source cell types and the target diseases. In this article, we express our view based on our experience in manufacturing biological therapeutics for routine use or clinical testing, and focus on strategies for advancing mesenchymal stromal cell (MSC)-derived EV-based therapies. We also discuss the rationale for testing MSC-EVs in selected diseases with an unmet clinical need such as critical size bone defects, epidermolysis bullosa and spinal cord injury. While the scientific community, pharmaceutical companies and clinicians are at the point of entering into clinical trials for testing the therapeutic potential of various EV-based products, the identification of the mode of action underlying the suggested potency in each therapeutic approach remains a major challenge to the translational path.
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http://dx.doi.org/10.3390/ijms18061190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5486013PMC
June 2017

A Good Manufacturing Practice-grade standard protocol for exclusively human mesenchymal stromal cell-derived extracellular vesicles.

Cytotherapy 2017 04 7;19(4):458-472. Epub 2017 Feb 7.

Microscopy Facility, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria; GMP Laboratory, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria; University Clinic for Blood Group Serology and Transfusion Medicine, Paracelsus Medical University (PMU), Salzburg, Austria.

Background Aims: Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) may contribute to biological processes such as tissue regeneration, immunomodulation and neuroprotection. Evaluation of their therapeutic potential and application in future clinical trials demands thorough characterization of EV content and production under defined medium conditions, devoid of xenogenic substances and serum-derived vesicles. Addressing the apparent need for such a growth medium, we have developed a medium formulation based on pooled human platelet lysate (pHPL), free from animal-derived xenogenic additives and depleted of EVs.

Methods: Depletion of EVs from complete growth medium was achieved by centrifugation at 120 000 g for 3 h, which reduced RNA-containing pHPL EVs to below the detection limit.

Results: Bone marrow (BM)-derived MSCs propagated in this medium retained the characteristic surface marker expression, cell morphology, viability and in vitro osteogenic and adipogenic differentiation potential. The proliferation rate was not significantly affected after 48 h but was decreased by 13% after 96 h. EVs collected from BM-MSCs cultured in EV-depleted medium revealed a similar RNA pattern as EVs generated in standard pHPL EV-containing medium but displayed a more clearly defined pattern of proteins characteristic for EVs. Reduction of pHPL content from 10% to 2% or serum-/pHPL-free conditions strongly altered MSC characteristics and RNA content of released EV.

Conclusions: The 10% pHPL-based EV-depleted medium is appropriate for purification of exclusively human MSC-derived EVs. With this Good Manufacturing Practice-grade protocol, characterization and establishment of protein and RNA profiles from MSC-derived EVs can now be achieved to identify active components in therapeutic EVs for future clinical application.
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http://dx.doi.org/10.1016/j.jcyt.2017.01.001DOI Listing
April 2017

Metabolomic profiling identifies potential pathways involved in the interaction of iron homeostasis with glucose metabolism.

Mol Metab 2017 01 31;6(1):38-47. Epub 2016 Oct 31.

First Department of Medicine, Paracelsus Medical University, Müllner Hauptstrasse 48, 5020 Salzburg, Austria; Obesity Research Unit, Paracelsus Medical University, Müllner Hauptstrasse 48, 5020 Salzburg, Austria. Electronic address:

Objective: Elevated serum ferritin has been linked to type 2 diabetes (T2D) and adverse health outcomes in subjects with the Metabolic Syndrome (MetS). As the mechanisms underlying the negative impact of excess iron have so far remained elusive, we aimed to identify potential links between iron homeostasis and metabolic pathways.

Methods: In a cross-sectional study, data were obtained from 163 patients, allocated to one of three groups: (1) lean, healthy controls (n = 53), (2) MetS without hyperferritinemia (n = 54) and (3) MetS with hyperferritinemia (n = 56). An additional phlebotomy study included 29 patients with biopsy-proven iron overload before and after iron removal. A detailed clinical and biochemical characterization was obtained and metabolomic profiling was performed via a targeted metabolomics approach.

Results: Subjects with MetS and elevated ferritin had higher fasting glucose (p < 0.001), HbA1c (p = 0.035) and 1 h glucose in oral glucose tolerance test (p = 0.002) compared to MetS subjects without iron overload, whereas other clinical and biochemical features of the MetS were not different. The metabolomic study revealed significant differences between MetS with high and low ferritin in the serum concentrations of sarcosine, citrulline and particularly long-chain phosphatidylcholines. Methionine, glutamate, and long-chain phosphatidylcholines were significantly different before and after phlebotomy (p < 0.05 for all metabolites).

Conclusions: Our data suggest that high serum ferritin concentrations are linked to impaired glucose homeostasis in subjects with the MetS. Iron excess is associated to distinct changes in the serum concentrations of phosphatidylcholine subsets. A pathway involving sarcosine and citrulline also may be involved in iron-induced impairment of glucose metabolism.
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http://dx.doi.org/10.1016/j.molmet.2016.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5220278PMC
January 2017

Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine.

ACS Nano 2016 04 15;10(4):3886-99. Epub 2016 Mar 15.

Institute for Transfusion Medicine, University Hospital Essen, University Duisburg-Essen , 45147 Essen, Germany.

Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.
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http://dx.doi.org/10.1021/acsnano.5b08015DOI Listing
April 2016

An alternative mini buffy coat preparation method for adult patients with extracorporeal photopheresis contraindications.

J Clin Apher 2017 Feb 4;32(1):12-15. Epub 2016 Mar 4.

Department for Blood Group Serology and Transfusion Medicine, SALK - Paracelsus Medical University (PMU), Salzburg, Austria.

Background: Extracorporeal photopheresis (ECP) is an important cell-based therapy for various diseases but is limited to patients eligible for apheresis. We developed an alternative mini buffy coat (BC) preparation method using the Spectra Optia® apheresis system and compared its efficacy of white blood cell (WBC) recovery with the standard mini BC preparation method already established for pediatric patients.

Methods: Whole blood (450 ± 45 mL) samples were collected from 30 randomly selected healthy volunteer blood donors and divided into two groups. In the first group, WBCs were separated with a fully automated separator device (Compomat G4 ). In the second group, BCs were separated with the bone marrow processing program of the Spectra Optia apheresis system.

Results: There were no significant differences in total leukocyte counts per product between the two groups. In contrast, lymphocyte counts per product were significantly higher (P < 0.001) in BCs separated from apheresis.

Conclusion: Our novel technique resulted in similar WBC yields but higher lymphocyte yields than the standard mini BC preparation method. This method can serve as an alternative to WBC collection in conventional ECP for adult patients with apheresis contraindications. J. Clin. Apheresis 32:12-15, 2017. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jca.21455DOI Listing
February 2017

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.

J Extracell Vesicles 2015 31;4:30087. Epub 2015 Dec 31.

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany;

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4698466PMC
http://dx.doi.org/10.3402/jev.v4.30087DOI Listing
January 2016

Mechanical fibrinogen-depletion supports heparin-free mesenchymal stem cell propagation in human platelet lysate.

J Transl Med 2015 Nov 10;13:354. Epub 2015 Nov 10.

Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria.

Background: Pooled human platelet lysate (pHPL) is an efficient alternative to xenogenic supplements for ex vivo expansion of mesenchymal stem cells (MSCs) in clinical studies. Currently, porcine heparin is used in pHPL-supplemented medium to prevent clotting due to plasmatic coagulation factors. We therefore searched for an efficient and reproducible medium preparation method that avoids clot formation while omitting animal-derived heparin.

Methods: We established a protocol to deplete fibrinogen by clotting of pHPL in medium, subsequent mechanical hydrogel disruption and removal of the fibrin pellet. After primary culture, bone-marrow and umbilical cord derived MSCs were tested for surface markers by flow cytometry and for trilineage differentiation capacity. Proliferation and clonogenicity were analyzed for three passages.

Results: The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained. All MSC types were propagated in standard and fibrinogen-depleted medium. Flow cytometric phenotype profiles and adipogenic, osteogenic and chondrogenic differentiation potential in vitro were independent of MSC-source or medium type. Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium. Interestingly, this proliferative response to heparin was not detected after an initial contact with fibrinogen during the isolation procedure.

Conclusions: Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies.
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http://dx.doi.org/10.1186/s12967-015-0717-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641400PMC
November 2015

Biological properties of extracellular vesicles and their physiological functions.

J Extracell Vesicles 2015 14;4:27066. Epub 2015 May 14.

Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433489PMC
http://dx.doi.org/10.3402/jev.v4.27066DOI Listing
May 2015

Lesion-induced accumulation of platelets promotes survival of adult neural stem / progenitor cells.

Exp Neurol 2015 Jul 24;269:75-89. Epub 2015 Mar 24.

Wellcome Trust and MRC Cambridge Stem Cell Institute & Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom; Institute of Molecular Regenerative Medicine, Paracelsus Medical University Salzburg, Salzburg, Austria; Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University Salzburg, Salzburg, Austria. Electronic address:

The presence of neural stem/progenitor cells (NSPCs) in specific areas of the central nervous system (CNS) supports tissue maintenance as well as regeneration. The subependymal zone (SEZ), located at the lateral ventricle's wall, represents a niche for NSPCs and in response to stroke or demyelination becomes activated with progenitors migrating towards the lesion and differentiating into neurons and glia. The mechanisms that underlie this phenomenon remain largely unknown. The vascular niche and in particular blood-derived elements such as platelets, has been shown to contribute to CNS regeneration in different pathological conditions. Indeed, intracerebroventricularly administrated platelet lysate (PL) stimulates angiogenesis, neurogenesis and neuroprotection in the damaged CNS. Here, we explored the presence of platelets in the activated SEZ after a focal demyelinating lesion in the corpus callosum of mice and we studied the effects of PL on proliferating SEZ-derived NSPCs in vitro. We showed that the lesion-induced increase in the size of the SEZ and in the number of proliferating SEZ-resident NSPCs correlates with the accumulation of platelets specifically along the activated SEZ vasculature. Expanding on this finding, we demonstrated that exposure of NSPCs to PL in vitro led to increased numbers of cells by enhanced cell survival and reduced apoptosis without differences in proliferation and in the differentiation potential of NSPCs. Finally, we demonstrate that the accumulation of platelets within the SEZ is spatially correlated with reduced numbers of apoptotic cells when compared to other periventricular areas. In conclusion, our results show that platelet-derived compounds specifically promote SEZ-derived NSPC survival and suggest that platelets might contribute to the enlargement of the pool of SEZ NSPCs that are available for CNS repair in response to injury.
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http://dx.doi.org/10.1016/j.expneurol.2015.03.018DOI Listing
July 2015

Iron depletion with a novel apheresis system in patients with hemochromatosis.

Transfusion 2015 May 21;55(5):996-1000. Epub 2014 Nov 21.

Blood Group Serology and Transfusion Medicine, Salzburg University Hospital (SALK), Salzburg, Austria.

Background: Phlebotomy represents the standard treatment option for iron overload in hemochromatosis (HC). Recently, red blood cell (RBC) apheresis has increasingly been used to remove iron. In this study we evaluated the depletion program of the newly developed Spectra Optia device.

Study Design And Methods: Adult male patients (n = 11) with HC were RBC depleted with the Spectra Optia device (Terumo BCT). In total, 24 procedures were performed. A volume of 300 to 550 mL of RBCs was withdrawn per single treatment.

Results: No significant adverse events were recorded. A median blood volume of 857.3 ± 23.3 mL was processed. The median procedure time was 12.0 ± 0.4 minutes. The mean reduction of Hct value in each procedure was approximately 6% (Hct pre 42.6 ± 0.5% vs. Hct post 36.6 ± 0.6%) and iron removed per procedure was 405.2 ± 23.3 mg.

Conclusion: The Spectra Optia device proved to be highly efficient in depleting RBCs in HC patients and allows for short procedure time. The Optia device can be safely used in this clinical setting. We recommend its use in case of severe iron overload if rapid iron depletion needs to be achieved and in case of cardiac compromise due to less blood volume removed.
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http://dx.doi.org/10.1111/trf.12949DOI Listing
May 2015

Identification of an effective early signaling signature during neo-vasculogenesis in vivo by ex vivo proteomic profiling.

PLoS One 2013 24;8(6):e66909. Epub 2013 Jun 24.

Stem Cell Research Unit, Medical University of Graz, Graz, Austria.

Therapeutic neo-vasculogenesis in vivo can be achieved by the co-transplantation of human endothelial colony-forming progenitor cells (ECFCs) with mesenchymal stem/progenitor cells (MSPCs). The underlying mechanism is not completely understood thus hampering the development of novel stem cell therapies. We hypothesized that proteomic profiling could be used to retrieve the in vivo signaling signature during the initial phase of human neo-vasculogenesis. ECFCs and MSPCs were therefore either transplanted alone or co-transplanted subcutaneously into immune deficient mice. Early cell signaling, occurring within the first 24 hours in vivo, was analyzed using antibody microarray proteomic profiling. Vessel formation and persistence were verified in parallel transplants for up to 24 weeks. Proteomic analysis revealed significant alteration of regulatory components including caspases, calcium/calmodulin-dependent protein kinase, DNA protein kinase, human ErbB2 receptor-tyrosine kinase as well as mitogen-activated protein kinases. Caspase-4 was selected from array results as one therapeutic candidate for targeting vascular network formation in vitro as well as modulating therapeutic vasculogenesis in vivo. As a proof-of-principle, caspase-4 and general caspase-blocking led to diminished endothelial network formation in vitro and significantly decreased vasculogenesis in vivo. Proteomic profiling ex vivo thus unraveled a signaling signature which can be used for target selection to modulate neo-vasculogenesis in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066909PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3691264PMC
February 2014

Brain pericyte plasticity as a potential drug target in CNS repair.

Drug Discov Today 2013 May 22;18(9-10):456-63. Epub 2012 Dec 22.

Institute of Molecular Regenerative Medicine, Paracelsus Medical University Salzburg, Austria.

Brain pericytes (BrPCs) are essential cellular components of the central nervous system neurovascular unit involved in the regulation of blood flow, blood-brain barrier function, as well as in the stabilization of the vessel architecture. More recently, it became evident that BrPCs, besides their regulatory activities in brain vessel function and homeostasis, have pleiotropic functions in the adult CNS ranging from stromal and regeneration promoting activities to stem cell properties. This special characteristic confers BrPC cell plasticity, being able to display features of other cells within the organism. BrPCs might also be causally involved in certain brain diseases. Due to these properties BrPCs might be potential drug targets for future therapies of neurological disorders. This review summarizes BrPC properties, disorders in which this cell type might be involved, and provides suggestions for future therapeutic developments targeting BrPCs.
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http://dx.doi.org/10.1016/j.drudis.2012.12.007DOI Listing
May 2013

Tri-lineage potential of intraoral tissue-derived mesenchymal stromal cells.

J Craniomaxillofac Surg 2013 Mar 13;41(2):110-8. Epub 2012 Aug 13.

Department of Orthopedic Surgery, Medical University of Graz, Graz, Austria.

The purpose of this study was to analyse the potential of intraoral tissues as a source of mesenchymal stromal and progenitor cells (MSPCs) for usage in future cell-based therapy models. Cells were isolated from four different tissues harvested during oral surgery intervention: (1) bone explants from the posterior maxilla, (2) bone explants from the oblique line, (3) from the mandibular periosteum, and (4) from the dental pulp. Donor sites and tissues were evaluated in terms of their accessibility, donor-site morbidity and average time period until appearance of MSPC colonies. Cell characterization was performed by flow cytometry and evaluation of in vitro osteogenic, adipogenic and chondrogenic differentiation potential. Adherent cell colonies were isolated from tissues from all sites after 4-8 days. The cells showed characteristics of MSPCs, so they were expanded up to clinical scales and demonstrated multipotency. The lowest donor-site morbidity was observed in the posterior maxilla harvests, while the highest donor-site morbidity was associated with harvests from mandibular sites. All sites seem to be potential sources of mesenchymal stromal and progenitor cells for tissue engineering approaches. Therefore, harvest morbidity and patient acceptance should affect the choice of the appropriate site.
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http://dx.doi.org/10.1016/j.jcms.2012.06.001DOI Listing
March 2013

Globular domain of adiponectin: promising target molecule for detection of atherosclerotic lesions.

Biologics 2011 7;5:95-105. Epub 2011 Sep 7.

Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz.

Background: Adiponectin, an adipocyte-specific plasma protein, has been shown to accumulate in injured endothelial cells during development of atherosclerotic lesions. In this study, we investigated the potential of different adiponectin subfractions with special emphasis on globular adiponectin (gAd) to recognize and visualize atherosclerotic lesions.

Methods: Recombinant mouse gAd and subfractions of full-length adiponectin (ie, trimeric, hexameric, and oligomeric forms) were fluorescence-labeled. Aortas of wild-type and apoprotein E-deficient mice fed a high cholesterol diet were dissected and incubated with the labeled biomarkers. Imaging was performed using confocal laser scanning microscopy.

Results: Confocal laser scanning microscopic images showed that gAd binds more strongly to atherosclerotic plaques than full-length adiponectin subfractions. Further, we showed that gAd accumulates preferentially in endothelial cells and the fibrous cap area of plaques. Here we demonstrate for the first time that gAd recognizes atherosclerotic plaques on aortic sections of apoprotein E-deficient mice.

Conclusion: These results suggest that gAd, in addition to its physiological properties, is also suitable as a target molecule for prospective diagnostic strategies in imaging atherosclerotic lesions.
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http://dx.doi.org/10.2147/BTT.S22863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3195669PMC
November 2011

Pro-angiogenic induction of myeloid cells for therapeutic angiogenesis can induce mitogen-activated protein kinase p38-dependent foam cell formation.

Cytotherapy 2011 Apr 3;13(4):503-12. Epub 2010 Dec 3.

Stem Cell Research Unit, University of Graz, Graz, Austria.

Background Aims: Clinical trials for therapeutic angiogenesis use blood- or bone marrow-derived hematopoietic cells, endothelial progenitor cells (EPC) and mesenchymal stromal cells (MSC) for vascular regeneration. Recently concerns have emerged that all three cell types could also contribute to atherosclerosis by foam cell formation. Therefore, we asked whether human myelomonocytic cells, EPC or MSC can accumulate lipid droplets (LD) and develop into foam cells.

Methods: LD accumulation was quantified by flow cytometry, confocal microscopy and cholesterol measurement in each of the cell types. The impact of an initial pro-angiogenic induction on subsequent foam cell formation was studied to mimic relevant settings already used in clinical trials. The phosphorylation state of intracellular signaling molecules in response to the pro-angiogenic stimulation was determined to delineate the operative mechanisms and establish a basis for interventional strategies.

Results: Foam cells were formed by monocytes but not by EPC or MSC after pro-angiogenic induction. Mitogen-activated protein kinase (MAPK) p38 phosphorylation was enhanced and kinase inhibition almost abrogated intracellular LD accumulation in monocytes.

Conclusions: These data suggest that hematopoietic cell preparations containing monocytes bear the risk of foam cell formation after pro-angiogenic induction. Instead, EPC and MSC may drive vascular regeneration without atherogenesis aggravation. A thorough understanding of cell biology is necessary to develop new strategies combining pro-angiogenic and anti-atherogenic effects during cell therapy.
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http://dx.doi.org/10.3109/14653249.2010.536214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3173858PMC
April 2011

Replicative senescence-associated gene expression changes in mesenchymal stromal cells are similar under different culture conditions.

Haematologica 2010 Jun 6;95(6):867-74. Epub 2010 Jan 6.

Stem Cell Research Unit Graz, Medical University of Graz, Austria.

Background: Research on mesenchymal stromal cells has created high expectations for a variety of therapeutic applications. Extensive propagation to yield enough mesenchymal stromal cells for therapy may result in replicative senescence and thus hamper long-term functionality in vivo. Highly variable proliferation rates of mesenchymal stromal cells in the course of long-term expansions under varying culture conditions may already indicate different propensity for cellular senescence. We hypothesized that senescence-associated regulated genes differ in mesenchymal stromal cells propagated under different culture conditions.

Design And Methods: Human bone marrow-derived mesenchymal stromal cells were cultured either by serial passaging or by a two-step protocol in three different growth conditions. Culture media were supplemented with either fetal bovine serum in varying concentrations or pooled human platelet lysate.

Results: All mesenchymal stromal cell preparations revealed significant gene expression changes upon long-term culture. Especially genes involved in cell differentiation, apoptosis and cell death were up-regulated, whereas genes involved in mitosis and proliferation were down-regulated. Furthermore, overlapping senescence-associated gene expression changes were found in all mesenchymal stromal cell preparations.

Conclusions: Long-term cell growth induced similar gene expression changes in mesenchymal stromal cells independently of isolation and expansion conditions. In advance of therapeutic application, this panel of genes might offer a feasible approach to assessing mesenchymal stromal cell quality with regard to the state of replicative senescence.
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http://dx.doi.org/10.3324/haematol.2009.011692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2878782PMC
June 2010

Platelet-derived growth factors for GMP-compliant propagation of mesenchymal stromal cells.

Biomed Mater Eng 2009 ;19(4-5):271-6

Stem Cell Research Unit, Medical University of Graz, Austria.

Stem cell-based therapies are a promising prospect for regenerative medicine. Particularly, human multipotent mesenchymal stromal cells (MSCs) are currently in focus regarding their regenerative and immune modulating capacities. An increasing number of clinical trials investigating MSC efficiency and safety are ongoing. Ex vivo propagation of human MSCs is considered to be a prerequisite for MSC therapy. The to date standard use of fetal bovine serum in cell culture bears risks including xenoimmunization and transmission of pathogens. Alternatively, human platelet-derived growth factors have been efficiently implemented into routine MSC expansion protocols. In compliance with good manufacturing practice we established an effective time- and resource-saving procedure for MSC propagation in an animal serum-free system. Bone marrow was seeded without manipulation directly in pooled human platelet lysate (pHPL) and L-glutamine supplemented minimum essential medium without antibiotics. Clinical scale expanded MSCs were harvested already after primary culture. MSC quality, identity, purity and function were assessed according to a defined panel of release criteria and comparative genomic hybridization was used to determine genomic stability. Because various potential risks of MSCs have recently been reported, further research is required to prove efficiency and long-term safety of human MSCs for cell therapy.
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http://dx.doi.org/10.3233/BME-2009-0591DOI Listing
March 2010