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Comparison of Schwann Cells Derived from Peripheral Nerve with Schwann Cells Differentiated from Skin-Derived Precursors

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Comparison of Schwann Cells Derived from Peripheral Nerve with Schwann Cells Differentiated from Skin-Derived Precursors Comparison of Schwann Cells Derived From Peripheral Nerve With Schwann Cells Differentiated From Skin-derived Precursors by Shaalee Dworski A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto © Copyright by Shaalee Dworski 2011 Comparison of Schwann Cells Derived From Peripheral Nerve With Schwann Cells Differentiated From Skin-derived Precursors Shaalee Dworski Master of Science Institute of Medical Science University of Toronto 2011 Abstract Schwann cells are the glial cells of the peripheral nervous system. When transplanted into the injured central or peripheral nervous systems they promote repair. Traditionally Schwann cells have been isolated from the sciatic nerve, creating nerve-SC. An alternative Schwann cell source is from the differentiation of skin-derived precursors (SKPs), stem cells found in the skin, to Schwann cells (SKP-SC). SKP-SC have shown enhanced regenerative ability compared to nerve- SC. This study compares nerve-SC with SKP-SC at the functional and gene expression level to determine their degree of similarity and find their sources of variance. The functional ability of both Schwann cell types appeared similar. Their gene expression, as assessed by microarray, was similar but not identical. Genes that differed between nerve-SC and SKP-SC may represent differences important to regeneration. The similarity of SKP-SC to nerve-SC supports the use of SKP-SC for repair, and reasons for enhanced regeneration by SKP-SC are suggested. ii Acknowledgements I would like to thank my supervisor, Dr. Freda Miller, for the opportunity that she has given me, for her ongoing support throughout my journey, and for being an exemplary role model. Thank you to my committee members Dr. David Kaplan and Dr. Jane Aubin for their guidance, intellectual input, and support. My sincere appreciation goes out to the past and present members of the laboratories of Dr. Miller and Dr. Kaplan for their guidance, technical help, and friendship, especially Dr. Jeffrey Biernaskie, Dr. Konstantin Feinberg, Karen Jones, Asli Dedeagac, Hiroyuki Jinno, and Smitha Paul. A heartfelt thank you to my family and friends for their constant encouragement. Dedicated to Dr. Peter B. Moens. iii Table of Contents Abstract ........................................................................................................................................... ii Acknowledgements ........................................................................................................................ iii Table of Contents ........................................................................................................................... iv Abbreviations ................................................................................................................................ vii List of Tables .................................................................................................................................. x List of Figures ................................................................................................................................ xi List of Appendices ........................................................................................................................ xii Chapter 1 Introduction .................................................................................................................... 1 1.1 Schwann cells ................................................................................................................... 1 1.1.1 Endogenous roles ...................................................................................................... 1 1.1.2 Developmental origin................................................................................................ 2 1.1.3 Markers of Schwann cell developmental stage ......................................................... 3 1.1.3.2 Genes expressed selectively at different stages of Schwann cell development ..... 6 1.1.3.3 Genes expressed during myelination by Schwann cells ........................................ 7 1.1.3.4 Genes expressed during de-differentiation of Schwann cells ................................ 8 1.1.4 Isolation................................................................................................................... 10 1.2 Peripheral nervous system injury and disease ................................................................ 11 1.2.1 Demyelinating diseases ........................................................................................... 11 1.2.2 Peripheral nerve injury ............................................................................................ 11 1.2.3 Role of Schwann cells in repair .............................................................................. 12 1.2.4 Schwann cell transplantation intervention .............................................................. 14 1.3 Central nervous system injury and disease .................................................................... 14 1.3.1 Demyelinating diseases ........................................................................................... 14 1.3.2 Spinal cord injury .................................................................................................... 14 1.3.3 Role of Schwann cells in repair .............................................................................. 15 1.3.4 Schwann cell transplantation intervention .............................................................. 16 1.4 Skin-derived precursors.................................................................................................. 16 1.4.1 Endogenous roles .................................................................................................... 16 1.4.2 Developmental origin.............................................................................................. 17 1.4.3 Isolation................................................................................................................... 17 1.4.4 Multipotency ........................................................................................................... 18 1.4.5 Differentiation to Schwann cells ............................................................................. 19 1.5 Skin-derived precursors for peripheral and central nervous system repair ................... 19 1.5.1 Peripheral nerve injury ............................................................................................ 19 1.5.2 Spinal cord injury .................................................................................................... 20 1.6 Comparison of nerve-derived Schwann cells with Schwann cells differentiated from skin-derived precursors ............................................................................................................. 21 1.6.1 Neurotrophin production ......................................................................................... 21 1.6.2 Peripheral nerve injury reparative ability ............................................................... 21 1.6.3 Spinal cord injury reparative ability ....................................................................... 22 1.7 Aims and hypothesis ...................................................................................................... 22 Chapter 2 Methods ........................................................................................................................ 24 iv 2.1 Cell isolation .................................................................................................................. 24 2.1.1 Nerve-SC isolation .................................................................................................. 24 2.1.2 Trunk SKPs isolation .............................................................................................. 25 2.1.3 SKP-SC isolation .................................................................................................... 25 2.2 Schwann cell verification ............................................................................................... 26 2.2.1 Immunocytochemistry ............................................................................................ 26 2.2.2 SCG axon association ............................................................................................. 27 2.2.3 DRG myelination .................................................................................................... 28 2.2.4 Sciatic nerve crush and cell transplantation ............................................................ 29 2.2.5 Microscopy ............................................................................................................. 29 2.3 Microarray analysis ........................................................................................................ 29 2.3.1 RNA isolation, purification, and quality analysis ................................................... 29 2.3.2 Array hybridization ................................................................................................. 32 2.3.3 Data pre-processing and quality analysis ................................................................ 32 2.3.4 Data analysis ........................................................................................................... 33 2.3.5 Visualization of the data ......................................................................................... 34 2.3.6
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