DIFFERENTIATION OF RECOMBINANT MYOBLASTS IN ALGINATE MICROCAPSULES By KELLY MACMILLAN BOWIE, B.Sc. A Thesis Submitted to the school of Graduate Studies in Partial Fulfillment ofthe Requirements for the Degree Master ofScience McMaster University 1997 © Copyright by Kelly MacMillan Bowie, June, 1997 MASTER OF SCIENCE MCMASTER UNIVERSITY 1997 Hamilton, Ontario TITLE: Differentiation of Recombinant Myoblasts in Alginate Microcapsules AUTHOR: Kelly MacMillan Bowie, B.Sc. (University of Western Ontario) SUPERVISOR: Dr. P.L. Chang EXAMINING COMMITTEE: Dr. M.A. Rudnicki Dr. C. Nurse NUMBER OF PAGES: xii,l75 11 ABSTRACT A cost effective approach to the delivery of therapeutic gene products in vivo is to immunoprotect genetically-engineered, universal, non-autologous cells in biocompatible microcapsules before implantation. Myoblasts may be an ideal cell type for encapsulation due to their inherent ability to differentiate into myotubes, thereby eliminating the problem of cell overgrowth within the capsular space. To evaluate the interaction between the differentiation program and the secretory activity of the myoblasts within the microcapsule environment, we transfected C2C 12 myoblasts to express human growth hormone and followed their expression of muscle differentiation markers, such as creatine phosphate kinase (CPK) protein and up-regulation of muscle-specific genes (ie. myosin light chains 2 & 1/3, Troponin I slow, Troponin T, myogenin and MyoD1). As the transfected myoblasts were induced to differentiate for up to two weeks, their myogenic index (i.e. the percentage of multinucleate myoblasts) increased from 0 to -50%. Concomitantly, up-regulation of differentiation marker RNA levels, and as much as a 23-fold increase in CPK activity, were observed. After encapsulation and the induction of differentiation, the myoblasts showed a lag phase of -3 days before an increase in CPK was observed, although the level of CPK activity increased by as much as 63-fold. The myogenic index of the encapsulated cells remained at zero. The rate of human growth hormone secretion was relatively constant throughout the two-week differentiation period, at an average of 7.78 x 1o-2 ng hGH per hour per )lg protein, 111 however, human growth hormone secretion was slightly decreased by about twofold during the differentiation of encapsulated myoblasts. In conclusion, the differentiation of myoblasts into myotubes is retarded after encapsulation while the secretion of a recombinant product is slightly reduced. Further studies are necessary to elucidate the cause of this atypical differentiation pattern such that the proliferation and differentiation of the encapsulated myoblasts may be optimized to provide a stable vehicle for gene delivery. IV ACKNOWLEDGEMENTS I would like to thank Dr. P.L. Chang for supervising my Master's thesis and for the opportunity to work in her laboratory. Many thanks, as well, to Dr. M.A. Rudnicki, Dr. R. Jacobs and Dr. C. Nurse for serving on my advisory and/or examining committees, and for their helpful suggestions along the way. My appreciation to Dr. M.A. Rudnicki for supplying the plasmid vectors containing the muscle-specific gene inserts used as probes, the C2C 12 myoblasts used in this study and the useful advice given. I would like to acknowledge Rob Perry with respect to his helpful suggestions and protocols, as well as Karen Howie for her assistance with the cytocentrifugation apparatus. Thanks also to Dr. Ron Carter in Cytogenetics for the use of their fluo.tescence microscope. It is with the utmost appreciation that I acknowledge all of my friends and colleagues in 3H31 who have been a continuous source of guidance and support. I would like to mention Dr. Gonzalo Hortelano, Michael Peirone, Kelly Robinson, Andrea Brunsting and Tracy Stockley in particular, who have not only offered their technical expertise on occasion, but who, in their friendship, have also provided a great deal of advice and encouragement along the way. A special thank-you, as well, to Scott Bowie for his assistance and the use ofhis computer. Lastly, I wish to mention my parents, Charlie and Maureen MacMillan, for their endless love and support over the years. To my husband, Jim..... words cannot express how grateful I am to you for your constant belief in my abilities, your love and your friendship ..... you have been a true blessing in my life. v TABLE OF CONTENTS ABSTRACT................................................................................................................... 111 ACKNOWLEDGEMENTS............................................................................................ v LIST OF FIGURES......................................................................................................... IX LIST OF TABLES........................................................................................................... X ABBREVIATIONS......................................................................................................... Xl 1.0 INTRODUCTION..................................................................................................... 1 1.1 Autologous Cell-Mediated Gene Therapy......................................... 3 1.1.1 In Vivo Gene Therapy........................................................ 3 1.1.1.1 Retroviral Vectors................................................ 3 1.1.1.2 Adenoviral Vectors.............................................. 5 1.1.1.3 Direct DNA Injection........................................... 8 1.1.1.4 Liposome-Mediated DNA Transfer...................... 9 1.1.2 Ex Vivo Gene Therapy ........................................................ 11 1.1.2.1 Viral Vectors ......................................................... 13 1.1.2.2 Recombinant Cell Grafts ...................................... 14 1.2 Non-Autologous Cell-Mediated Gene Therapy .................................. 17 1.2.1 Biocompatible Encapsulation Devices ................................ 17 1.2.1.1 Alginate Polylysine Microcapsules ...................... 20 1.2.1.1.1 Chemical Composition .......................... 20 1.2.1.1.2 AP A Encapsulation Studies ................... 22 1.3 Myoblast Cell Lines ............................................................................ 25 1.3 .1 Myogenesis.......................................................................... 25 1.3.2 Myoblast Differentiation ..................................................... 27 1.3.2.1 Muscle-Specific Gene Expression ........................ 29 1.3.3 Myoblast Fusion .................................................................. 33 1.3.4 Regulation of Myoblast Proliferation/Differentiation bybFGF.............................................................................. 36 1.4 Rationale and Goals for Thesis .......................................................... 39 2.0 MATERIALS AND METHODS.............................................................................. 41 2.1 Cell Lines ........................................................................................... 41 2.2 Preparation of Competent DH5a Cells.............................................. 41 2.3 Heat Shock Transformation ofDH5a Cell........................................ 42 2.4 Small Scale Preparation of Plasmid DNA. ........................................ 43 2.5 Large Scale Preparation of Plasmid DNA ......................................... 43 2.6 Transfection ofC2C12 Cells .............................................................. 44 Vl 2.7 Determination of hGH Secretion Rate ............................................... .46 2.8 RNA Isolation ..................................................................................... 47 2.9 Northern Blot. ..................................................................................... 48 2.10 Dot Blot. ........................................................................................... 50 2.11 Probe Preparation ............................................................................. 51 2.12 Myoblast Differentiation Determination - CPK Assay ..................... 52 2.13 Protein Determination ...................................................................... 53 2.14 hGH Immunofluorescence ................................................................ 53 2.15 Myogenic Index Determination ........................................................ 54 2.16 Microencapsulation of Cells............................................................. 55 2.17 Characterization of Encapsulated Cells ............................................ 57 2.17.1 Cell Viability ..................................................................... 57 2.17.2 Cell Release ....................................................................... 57 2.17.3 Determination of Cell Number per Capsule ...................... 58 2.18 Data Analysis ................................................................................... 59 3.0 RESULTS................................................................................................................... 60 3.1 Expression ofHuman Growth Hormone ............................................ 60 3.1.1 hGH Secretion Rates: Comparison ofEncapsulated and Unencapsulated Myoblasts .................................................. 61 3.1.2 Immunofluorescent Staining of hGH................................... 67 3.2 Qualitative Analysis of Myoblast Differentiation ............................... 74 3.2.1 Myogenix Index ..................................................................