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Immunomodulatory and Regenerative Effects of Mesenchymal Stromal Cells in Trauma

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Immunomodulatory and Regenerative Effects of Mesenchymal Stromal Cells in Trauma Ulm University Medical Center Institute of Transfusion Medicine Prof. Dr. med. Hubert Schrezenmeier Immunomodulatory and regenerative effects of mesenchymal stromal cells in trauma Dissertation to obtain the Doctoral Degree of Human Biology at the Medical Faculty of Ulm University Elisa Maria Amann Juliaca (Peru) 2018 Present Dean: Prof. Dr. rer. nat. Thomas Wirth 1st reviewer: Prof. Dr. med. Hubert Schrezenmeier 2nd reviewer: Prof. Dr. med. Rolf Brenner Date of graduation: November 9th, 2018 Parts of this thesis were already published in Amann EM, Rojewski MT, Rodi S, Fürst D, Fiedler J, Palmer A, Braumüller S, Huber-Lang M, Schrezenmeier H, Brenner RE (2018) Systemic recovery and therapeutic effects of transplanted allogenic and xenogenic mesenchymal stromal cells in a rat blunt chest trauma model. Cytotherapy 20: 218-231. DOI: 10.1016/j.jcyt.2017.11.005. Epub 2017 Dec 7. CC BY-NC-ND 4.0 https://creativecommons.org/licenses/by-nc-nd/4.0/ Amann EM, Groß A, Rojewski MT, Kestler HA, Kalbitz M, Brenner RE, Huber- Lang M, Schrezenmeier H (2019) Inflammatory response of mesenchymal stromal cells after in vivo exposure with selected trauma-related factors and polytrauma serum. PLoS ONE 14(5):e0216862. DOI: 10.1371/journal.pone.0216862. eCollection 2019 CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ List of contents List of contents LIST OF ABBREVIATIONS III 1. INTRODUCTION 1 1.1. Polytrauma 1 1.2. Blunt chest trauma and acute lung injury 2 1.3. Human multipotent mesenchymal stromal cells 5 1.4. Aims of the study 14 2. MATERIAL AND METHODS 16 2.1. Tools, machines, consumables and software 16 2.2. Chemicals and kits 19 2.3. Antibodies 22 2.4. Cells 23 2.5. Cultivation of mesenchymal stromal cells, NR8383 cells and C2C12 cells 23 2.6. Mesenchymal stromal cells and polytrauma 26 2.7. Effects of manipulating MSC for in vivo experiments 32 2.8. Animal experiments 40 2.9. Statistics 51 3. RESULTS 52 3.1. Mesenchymal stromal cells and polytrauma 52 3.2. Characterization of rat mesenchymal stromal cells 72 3.3. Effects of manipulating MSC for in vivo experiments 73 3.4. Animal experiments 96 I List of contents 4. DISCUSSION 135 4.1. The role of interleukin 1 beta in polytrauma 135 4.2. Characterization of rat mesenchymal stromal cells 142 4.3. Effects of manipulating MSC for in vivo experiments 143 4.4. Mesenchymal stromal cells in blunt chest trauma 150 4.5. Conclusion 160 5. SUMMARY 162 6. REFERENCE LIST 164 APPENDIX 186 ACKNOWLEDGEMENTS 198 CURRICULUM VITAE: ELISA MARIA AMANN 199 II List of abbreviations List of abbreviations AIS Abbreviated Injury Scale ALI Acute lung injury Alpha MEM Minimal Essential Medium Eagle, Alpha modification ANOVA Analysis of variance APC Allophycocyanin APC-Cy7 Allophycocyanin coupled with the cyanine dye Cy7 ARDS Acute respiratory distress syndrome ASC Adipose-derived mesenchymal stromal cells BAL Broncho-alveolar lavage fluid BM-MSC Bone marrow derived mesenchymal stromal cell(s) BSA Bovine serum albumin C3a Complement component 3a C5a Complement component 5a CARS Compensatory anti-inflammatory response syndrome CCL C-C motif chemokine ligand CCR C-C motif chemokine receptor CD Cluster of differentiation CFSE Carboxyfluorescein diacetate succinimidyl ester CFU Colony forming units cm Centimetre cm2 Square centimetre CSF Colony stimulating factor CXCL C-X-C motif chemokine ligand CXCR C-X-C motif chemokine receptor d Days DAMP Damage-associated molecular pattern DGU Deutsche Gesellschaft für Unfallchirurgie DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DPBS Dulbecco´s phosphate buffered saline e.g. Exempli gratia EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay FACS Fluorescence activated cell sorting III List of abbreviations FBS Fetal bovine serum FGF Fibroblast growth factor FITC Fluorescein isothiocyanate FSC-H Forward scatter height g Gram g Standard gravity (9.8 m/s2) GMP Good manufacturing practice Gy Gray [absorbed radiation dose] h Hours HLA Human leucocyte antigen HMGB1 High mobility group box 1 hMSC Human multipotent mesenchymal stromal cell(s) hMSC (IL1B) IL1B pre-stimulated hMSC HSA Human serum albumin ICAM Intercellular adhesion molecule ICAM1 Synonym: CD54 IDO Indoleamine 2,3-dioxygenase IFNG Interferon gamma Ig Immunoglobulin IGF1 Insulin-like growth factor 1 IL Interleukin IL1A Interleukin 1 alpha IL1B Interleukin 1 beta IL1RN Interleukin 1 receptor antagonist ISCT International Society for Cellular Therapy ISS Injury Severity Score IU International units kg Kilogram l Litre LPS Lipopolysaccharide M Molar, mol/l MAPK Mitogen-activated protein kinases MEM Minimum essential media mg Milligram µg Microgram IV List of abbreviations min Minutes µl Microlitre ml Millilitre µm Micrometre mM Millimolar, mmol/l mmHg Millimetre of mercury MMP Matrix metallopeptidase MODS Multiple organ dysfunction syndrome MOF Multiple organ failure MPO Myeloperoxidase MSC Mesenchymal stromal cells NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells ng Nanogram nm Nanometre P Probability PCR Polymerase chain reaction PDGF-BB Homodimer of platelet-derived growth factor PE Phycoerythrin PerCP Peridinin-chlorophyll-protein PGE2 Prostaglandin E2 PKH+ Events in flow cytometry analysis being in PKH26 gate, PKH26- fluorescent, PKH26-stained PKH- Events in flow cytometry analysis not being in PKH26 gate PL Platelet lysate PML Polymorphonuclear leucocytes PTCH Polytrauma cocktail high PTCL Polytrauma cocktail low PTGS2 Prostaglandin-endoperoxide synthase 2 RFU Relative fluorescence units rMSC Rat multipotent mesenchymal stromal cell(s) rMSC (IL1B) IL1B pre-stimulated rMSC RNA Ribonucleic acid s Seconds SD Standard deviation SIRS Systemic inflammatory response syndrome SSC-H Sideward scatter height V List of abbreviations TGFB1 Transforming growth factor beta 1 THBD Thrombomodulin THPO Thrombopoietin TLR Toll-like receptor TMB 3,3′,5,5′-Tetramethylbenzidine TNF Tumor necrosis factor (alpha) TNFAIP6 Tumor necrosis factor alpha inducible protein 6 TSG-6 TNFAIP6 TxT Blunt chest trauma TxT+hMSC Blunt chest trauma and treatment with human MSC TxT+hMSC (IL1B) Blunt chest trauma and treatment with IL1B-primed human MSC TxT+rMSC Blunt chest trauma and treatment with rat MSC TxT+rMSC (IL1B) Blunt chest trauma and treatment with IL1B-primed rat MSC U Units UC-MSC Umbilical cord-derived mesenchymal stromal cells VCAM1 Vascular cell adhesion molecule 1, synonym: CD106 VEGFA Vascular endothelial growth factor A VI Introduction 1. Introduction 1.1. Polytrauma Road injuries are a leading cause of death worldwide [96]. The Trauma Register DGU® (Deutsche Gesellschaft für Unfallchirurgie) published in their Annual Report 2017 that the most common causes of trauma are traffic accidents (51 %) and falls from various heights (42 %) [85]. Polytrauma is a life-threatening disease with a hospital mortality of 11 % (Trauma Register DGU® 2016: 3507 patients) [85]. Furthermore, 30 % of surviving patients have disabilities at time of discharge (Trauma Register DGU® 2016: 9588 patients; [85]). After discharge from hospital life expectancy of severely injured patients is significantly lower than that of normal population [178]. The current definition of polytrauma is that at least two body regions must be significantly injured and at least one physiological problem (high age, unconsciousness, hypotension, acidosis or coagulopathy) must exist [188]. To define polytrauma and to assess the risk of death the Abbreviated Injury Scale (AIS) and the Injury Severity Score (ISS) are used. AIS and ISS indicate the number and severity of injuries [92]. The AIS divides the body in seven regions (external, head, neck, thorax, abdomen, spine and extremities) and evaluates the severity of the injuries in the body regions with a scale from one to six [3,92]. The ISS is calculated by summing up the squares of the highest AIS score in each of the three most severely injured areas [9]. In polytrauma the ISS is usually greater than 15 [84,186]. Mortality of polytrauma patients is composed of early mortality (within 24 hours (h): 5.1 %; for example due to haemorrhagic shock) and late mortality (within 30 days (d): 10.8 %; due to brain injuries or host defence failure) [85,118]. Fractures and soft tissue injuries (first hits) induce local and systemic inflammation. Second hits like metabolic acidosis, ischaemia/reperfusion injuries, infections or massive transfusions increase this systemic inflammatory reaction and distant organs and tissues are affected [84,118,215]. Polytrauma is often accompanied by a systemic inflammatory response syndrome (SIRS) and a compensatory anti-inflammatory response syndrome (CARS). The imbalance of these immune responses, epithelial cell damage, parenchymal cell damage and cell death lead to sepsis, multiple organ dysfunction syndrome (MODS) or multiple organ failure (MOF) [84,118]. At the development of SIRS, CARS, sepsis, MODS 1 Introduction and MOF numerous cells, especially the innate immune system with phagocytic activity (monocytes, macrophages and neutrophils), protection mechanisms (coagulation cascade, kallikrein–kinin system, complement system, acute phase reaction) and mediators (damage-associated molecular pattern (DAMP), pathogen-associated molecular pattern, cytokine, chemokine, neuromediators, heat shock proteins and oxygen radicals) are involved [84,118]. Until today, there is an effort to define markers that have been linked to an increased disease severity and poorer clinical outcomes in severely injured patients [54,218,236]. The spectrum of biomarkers includes inflammatory
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