Studies relating to the differentiation of human embryonic stem cells A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2015 Georgios Anyfantis List of Figures .................................................................................................... 10 List of Tables ...................................................................................................... 13 Declaration ........................................................................................................ 15 Copyright statement .......................................................................................... 15 Acknowledgements ........................................................................................... 16 Abbreviations .................................................................................................... 17 Chapter 1: Introduction ...................................................................................... 23 1.1 Diabetes Mellitus ......................................................................................... 23 1.2 Pancreatic function and Organogenesis ........................................................ 24 1.2.1 Pancreatic Function .................................................................................... 24 1.2.2 Pancreatic Organogenesis ........................................................................... 26 1.3 Signalling Pathways associated with pancreas development ........................ 26 1.3.1 Signals from Endothelial Cells ..................................................................... 26 1.3.2 Fgf Signalling .............................................................................................. 28 1.3.3 TGFβ signalling ........................................................................................... 28 1.3.4 Transcription Factors .................................................................................. 29 1.4 Markers of Differentiation ............................................................................ 30 1.4.1 Using Animal Models in Developmental Studies .......................................... 34 1.4.2 Differences between human and animal models ......................................... 35 1.5 The Use of hESC ............................................................................................ 36 2 1.5.1 Embryonic Stem Cells ................................................................................. 36 1.5.1.1 Historical Perspective ....................................................................................... 36 1.5.2 Derivation of human embryonic stem cells.................................................. 37 1.5.3 Characteristics of ESC ................................................................................. 37 1.6 Self-renewal and pluripotency ...................................................................... 38 1.6.1 The LIF Pathway ......................................................................................... 39 1.6.2 The FGF Pathway ........................................................................................ 40 1.6.3 The TGFβ family ......................................................................................... 41 1.6.4 The PI3K/Akt Signalling Pathway ................................................................ 41 1.6.5 Transcription Factors .................................................................................. 41 1.6.6 Telomerase Activity .................................................................................... 42 1.6.7 Epigenetic Mechanisms .............................................................................. 43 1.6.8 Cell Cycle Regulation in hESC ...................................................................... 43 1.6.9 The Study of hESC - Potential ...................................................................... 44 1.6.10 Definitive Endoderm (DE) – Insulin producing cells .................................... 45 1.7. hESC for the production of insulin producing cells ........................................ 47 1.7.1 The first step – Insulin producing cells from mESC ....................................... 47 1.7.2 Genetic manipulation of human embryonic stem cells ................................. 49 1.7.3 Potential problems with the generation of insulin producing cells from hESC 50 1.7.4 ATP and Purinergic Receptors ..................................................................... 50 1.7.5 Identification of functional purinergic receptors ......................................... 53 3 1.7.6 The Missing Links - Role of Neural Signalling in Pancreatic Development ..... 54 1.7 Aims and Objectives ..................................................................................... 55 Chapter 2: Materials and Methods .................................................................... 57 2.1 Tissue culture ............................................................................................... 57 2.1.1 Human embryonic stem cell culture ............................................................ 57 2.1.2 MEF cell culture .......................................................................................... 57 2.1.3 Mitotic inactivation of MEFs ....................................................................... 60 2.1.4 Generation and collection of hESC conditioned medium .............................. 60 2.1.5 Freezing down MEFs and hESCs .................................................................. 61 2.1.6 Thawing hESCs onto mitotically inactivated MEFs ....................................... 61 2.1.7 Enzymatically passaging the HUES1 hESCs with trypsin ................................ 62 2.1.8 Enzymatically passaging the HUES1 and H7 hESCs with collagenase IV ......... 63 2.1.9 Mechanically passaging the HUES1 and H7 hESCs ........................................ 63 2.1.10 Removing the differentiated parts of a hESC colony between passages ...... 64 2.1.11 Differentiation of HUES1 and H7 hESCs to definitive endoderm (DE) ......... 65 2.1.12 Total RNA extraction from HUES1 and H7 hESC......................................... 65 2.1.13 DNase I digestion treatment of the extracted RNA samples ...................... 66 2.1.14 Assessing RNA Integrity ........................................................................... 67 2.1.15 Conversion of Total RNA to cDNA for use in QPCR analysis ....................... 67 2.1.16 PCR Amplification of cDNA........................................................................ 69 2.1.17 TAE agarose gel electrophoresis of DNA .................................................... 70 4 2.1.18 Real-time qPCR analysis of cDNA .............................................................. 71 2.1.19 Choice of Housekeeping Genes ................................................................. 71 2.1.20 Primer Design for PCR Amplification ......................................................... 72 2.2 Calcium fluorescence imaging and microfluorimetry ..................................... 73 2.2.1 Plating HUES1 hESC on coverslips................................................................ 76 2.2.2 Loading the colonies with Fura-2/pluronic acid and mounting of coverslips on the experimental platform .................................................................................. 77 2.2.3 Test solutions ............................................................................................. 78 2.2.4 Fitting of the dose response curve .............................................................. 79 2.2.5 P2Y Receptors – Study of Functional Profile ................................................ 79 2.2.5.1 Stimulation with ATP in the absence of extracellular Ca2+ .............................. 79 2.2.5.2 Stimulation with ATP in the presence of cyclopiazonic acid (CPA).................... 80 2.2.5.3 Stimulation with ATP in the absence of extracellular calcium, with the addition of MRS2578. ........................................................................................................................... 80 2.2.6 Immunostaining of coverslips ..................................................................... 80 2.2.7 Confocal Microscopy .................................................................................. 82 2.3. Alkaline Phosphatase Pluripotency Assays ................................................... 82 2.4. Creating a Lentiviral Vector containing the GFP under the influence of the Pdx1 gene promoter. ......................................................................................... 83 2.5 Lentiviral production and testing .................................................................. 85 2.5.1 The pTiger-mPdx1 I-II-III-RFP/rIns2-eGFP vector .......................................... 88 5 Chapter 3: Facilitating attachment of HUES1 human embryonic stem cells to glass coverslips .......................................................................................................... 90 3.1 Introduction ................................................................................................. 90 3.2 Aims ............................................................................................................ 92 3.3 Materials and Methods ................................................................................ 92 3.3.1 Preparation of Matrigel coated glass coverslips in 24 well plates ................