UNIVERSITY OF MANCHESTER Transdifferentiation to pancreatic progenitors A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2019 Stephen John Wearne School of Medical Sciences Division of Diabetes Endocrinology and Gastroenterology Contents Contents..................................................................................................................................................................................... 1 List of tables ............................................................................................................................................................................... 3 List of figures.............................................................................................................................................................................. 4 List of abbreviations ................................................................................................................................................................... 5 List of publications ..................................................................................................................................................................... 8 Abstract ...................................................................................................................................................................................... 9 Declaration ............................................................................................................................................................................... 10 Copyright statement ................................................................................................................................................................. 10 Acknowledgements .................................................................................................................................................................. 11 1. Introduction .......................................................................................................................................................................... 12 1.1. Overview of the pancreas ............................................................................................................................................ 12 1.2. Development towards pancreatic progenitors .............................................................................................................. 14 1.2.1. Endoderm to early pancreas signalling network ................................................................................................... 14 1.2.2. Transcription factor network of the early pancreas .............................................................................................. 17 1.2.3. Late pancreas signalling network ......................................................................................................................... 40 1.2.4. Transcription factor expression in human and mouse pancreas development .................................................... 42 1.3. Pancreatic cell production ............................................................................................................................................ 45 1.3.1. Transdifferentiation............................................................................................................................................... 45 1.3.2. In vitro differentiation ............................................................................................................................................ 50 1.3.3. Organoid culture ................................................................................................................................................... 54 1.4. Project goal and methods ............................................................................................................................................ 57 2. Materials and Methods ......................................................................................................................................................... 61 2.1. Materials....................................................................................................................................................................... 61 2.1.1. Solutions............................................................................................................................................................... 61 2.1.2. Primers ................................................................................................................................................................. 62 2.1.3. Plasmids ............................................................................................................................................................... 65 2.1.4. Antibodies............................................................................................................................................................. 65 2.2. Methods ....................................................................................................................................................................... 66 2.2.1. Bioinformatic analysis........................................................................................................................................... 66 2.2.2. Molecular cloning ................................................................................................................................................. 67 2.2.3. AAV production and testing .................................................................................................................................. 69 2.2.4. qPCR .................................................................................................................................................................... 71 2.2.5. Cell culture ........................................................................................................................................................... 73 2.2.6. Western blot ......................................................................................................................................................... 76 2.2.7. Immunofluorescence ............................................................................................................................................ 77 2.2.8. RNAscope ............................................................................................................................................................ 77 2.2.9. Immunohistochemistry ......................................................................................................................................... 77 3. Results ................................................................................................................................................................................. 79 3.1. LgPCA and Mogrify identify a shared cohort of genes active in specifying the pancreas ............................................ 79 1 3.2. In vitro differentiation allows the derivation of the crucial factors for transdifferentiation ............................................. 81 3.3 The generation and validation of a reliable AAV production protocol ........................................................................... 85 3.4. PDX1 AAV is able to repress hepatic genes in the HepG2 cell line............................................................................. 89 3.5. Combined exposure of key transcription factors via AAV delivery can induce endogenous expression of tissue specific genes ..................................................................................................................................................................... 91 3.6. The spatial expression profile of PTF1A, RBPJL and NR5A2 overlap during late embryogenesis ............................. 97 3.7. LgPCA and Mogrify determined transcription factors are able to alter expression of pancreatic genes in FLFs ......... 99 3.8. PDX1, MNX1 and FOXA2 AAVs are able to induce endogenous expression of multiple markers of pancreatic progenitors ........................................................................................................................................................................ 108 3.9. LgPCA but not Mogrify unveils further genes specifying for the early pancreas ........................................................ 119 4. Discussion .......................................................................................................................................................................... 122 4.1. Bioinformatic analysis of potential transcription factors able to transdifferentiate to the pancreas ............................ 122 4.2. The description of temporal gene expression during in vitro differentiation to pancreatic progenitors ...................... 126 4.3. An improved method for AAV generation and purification ......................................................................................... 132 4.4. AAVs are effective vectors for gene delivery and expression .................................................................................... 134 4.5. AAVs are able to induce transdifferentiation .............................................................................................................. 135 4.6. The characterisation