Regulation of the anti-inflammatory properties of MSC spheroids by matrix metalloproteinases A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Biology, Medicine and Health 2018 Thomas J Bosworth School of Medical Sciences Table of Contents 1. Introduction .......................................................................... 16 1.1. Cardiac remodelling after myocardial infarction .......................... 16 1.1.1. Inflammatory response after MI .................................................. 16 1.1.2. MMPs in cardiac remodelling ...................................................... 22 1.2. Anti-inflammatory properties of mesenchymal stromal cells ..... 29 1.2.1. MSCs contribute to innate immunity ........................................... 29 1.2.2. MSCs contribute to adaptive immunity ........................................ 33 1.2.3. MSCs improve outcomes after MI through secretion of specific anti-inflammatory cytokines and MMPs ................................................ 35 1.3. Microenvironment regulation of MSCs for cardiac repair ............ 37 1.3.1. Regulation of cytokine and MMP expression by MSCs............... 37 1.3.2. The ECM as a potent regulator of MSC function ........................ 38 1.3.3. Are spheroids a new paradigm in MSC research? ...................... 40 1.4. Summary .......................................................................................... 44 1.5. Hypothesis ....................................................................................... 46 1.6. Aims.................................................................................................. 46 2. Materials and Methods ......................................................... 48 2.1. Cell culture ....................................................................................... 48 2.1.1. Adherent cultures ........................................................................ 48 2.1.2. Spheroid cultures ........................................................................ 49 2.2. MSC characterisation ...................................................................... 49 2.2.1. Flow cytometry ............................................................................ 49 2.2.2. Osteogenic differentiation ........................................................... 50 2.2.3. Adipogenic differentiation ............................................................ 51 2.2.4. Chondrogenic differentiation ....................................................... 51 2.3. Quantitative reverse transcription polymerase chain reaction ... 52 2.3.1. Isolation of RNA .......................................................................... 52 2.3.2. cDNA synthesis .......................................................................... 53 2.3.3. qPCR .......................................................................................... 53 2.4. Western blot ..................................................................................... 57 2.4.1. Protein isolation .......................................................................... 57 2.4.2. SDS-PAGE ................................................................................. 58 2.4.3. Western blotting .......................................................................... 58 2.4.4. Densitometry ............................................................................... 59 2.5. MMP Antibody Array ....................................................................... 61 2.6. Immunofluorescence ...................................................................... 62 2 2.6.1. Adherent MSCs .......................................................................... 62 2.6.2. Spheroids .................................................................................... 63 2.7. MMP activity assay .......................................................................... 64 2.8. siRNA knockdown ........................................................................... 65 2.9. Cytokine, conditioned medium, and MMP stimulation ................. 66 2.9.1. Cytokine stimulation .................................................................... 66 2.9.2. Conditioned medium stimulation ................................................. 67 2.9.3. MMP stimulation ......................................................................... 67 2.10. Macrophage culture ...................................................................... 68 2.10.1. Macrophage polarisation ........................................................... 68 2.11. Zymosan-induced peritonitis ....................................................... 68 2.12. ELISA .............................................................................................. 69 2.13. In vivo imaging .............................................................................. 70 2.14. Statistical analysis ........................................................................ 70 3. Spheroid culture conditions are determinants of the MSC spheroid microenvironment .................................................... 72 3.1. Characterisation of MSCs ............................................................... 73 3.1.1. Surface marker expression by MSCs .......................................... 73 3.1.2. Differentiation potential of MSCs ................................................ 74 3.1.3 Summary ..................................................................................... 80 3.2. MSC spheroids express increased levels of MMPs compared with adherent cultures ................................................................................... 80 3.2.1. mRNA expression levels of MMPs .............................................. 80 3.2.2. Protein expression levels of MMPs ............................................. 81 3.2.3. Secreted levels of MMPs ............................................................ 82 3.2.3. Summary .................................................................................... 83 3.3. MMP expression and extracellular matrix deposition is altered by spheroid culture conditions .................................................................. 85 3.3.1. Spheroid size .............................................................................. 85 3.3.2. Spheroid culture time .................................................................. 87 3.3.3. Spheroid formation method ......................................................... 90 3.3.4. Summary .................................................................................... 92 3.5. MSC spheroids express increased levels of specific cytokines compared with adherent cultures ......................................................... 93 3.5.1. Gene expression levels of inflammatory cytokines ..................... 93 3.5.2. Protein expression levels of cytokines ........................................ 94 3.6. Summary .......................................................................................... 96 4. Regulation of the spheroid microenvironment by MMPs 100 3 4.1. The spheroid microenvironment is regulated by secreted factors ............................................................................................................... 100 4.2. Regulation of MSC inflammatory secretome by selected cytokines ............................................................................................... 104 4.2.1. Regulation of p65 and STAT-3 signalling .................................. 104 4.2.2. Regulation of TSG-6 and COX-2 expression ............................ 104 4.2.3. Regulation of MMP-1 and MMP-3 expression .......................... 107 4.2.4. Summary .................................................................................. 107 4.3. Spheroid derived MMP expression is regulated by IL-1RI and TSG-6 ..................................................................................................... 109 4.3.1. IL-1RI and TSG-6 regulate cytokine expression by MSC spheroids ............................................................................................................ 109 4.3.2. IL-1RI and TSG-6 regulate MMP expression by MSC spheroids ............................................................................................................ 111 4.3.3. IL-1RI and TSG-6 regulate p65 and STAT-3 signalling activation ............................................................................................................ 113 4.3.4. Summary .................................................................................. 113 4.4. The inflammatory secretome of MSC spheroids is regulated by MMPs ..................................................................................................... 115 4.4.1. MMP-1 and MMP-9 regulate expression of one another ........... 115 4.4.2. MMP-1 and MMP-9 drive anti-inflammatory TSG-6 and COX-2 expression whereas MMP-9 inhibits TSG-6 expression ..................... 118 4.4.3. Knockdown of MMP-1 and MMP-3 alter p65 and STAT-3 signalling pathway activation .............................................................. 120 4.4.4. Summary .................................................................................. 120 4.5. Effect of recombinant MMPs on MSC spheroids ........................ 122 4.6. Summary ........................................................................................ 124 5. Optimising conditions for assessing the in