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  • Burn-Derived Stem Cells - A Promising New Cell Source For Skin Regeneration

    Cellules souches issues de peau brûlée

    - Une source prometteuse de cellules pour la régénération de la peau

    by

    Reinhard Dolp

    A thesis submitted in conformity with the requirements for the degree of Master of Science

    Institute of Medical Science University of Toronto\

  • ii

    Burn-Derived Stem Cells - A Promising New Cell Source For Skin Regeneration

    Reinhard Dolp

    Doctor of Medicine

    Institute of Medical Science University of Toronto

    2017

    Abstract

    Introduction: Burns affect millions of people worldwide. Available wound coverage materials

    are insufficient due to a lack of cells. Mesenchymal stem cells (MSCs) promote wound healing

    but are limited by lack of availability. We hypothesize that burned skin contains functioning MSCs

    (burn derived MSCs; BD-MSCs) that promote wound healing.

    Methods: BD-MSCs were compared to umbilical cord MSCs in terms of key biological

    characteristics. Then, skin-scaffolds were cellularized with BD-MSCs, applied onto excisional

    porcine wounds and observed over 30d.

    Results: We found no difference between BD- and UC-MSCs in mitochondrial function,

    proliferation, colony formation, cell cycle stage distribution, reactive oxygen species, and MHC

    I/II expression. BD-MSCs are safe and improved wound healing in mice and pigs.

    Conclusion: Burned skin contains healthy MSCs that promote wound healing. Key biological

    functions are not altered by burn trauma. Further studies are needed to evaluate the role of BD-

    MSCs in wound healing.

  • iii

    Acknowledgments

    First and foremost, I would like to thank my supervisor Professor Dr. Marc Jeschke and Dr.

    Amini-Nik for welcoming me into the team and enabling me to achieve my dream to become an

    academic clinician in Canada.

    Next, I would like to thank my committee members Prof. Post and Prof. Morshead for their

    valuable feedback.

    I am very grateful for the people that I have worked with - in particular, the fantastic lab-

    technicians Alexa Parousis, Andrea-Kaye Datu, and Nazihah Bakhtyar.

    Finally, I would like to thank my family - Robert, Monika, and Maximilian Dolp - as well as my

    partner Jonathan Ausman.

  • iv

    Contributors

    The following people/organizations have been instrumental in the collection of materials and

    data for this thesis:

    1. The obstetrical and gynecological department of Sunnybrook, Dr. Herer, and Dr. Zaltz,

    for the collection of umbilical cords.

    2. The Ross Tilley Burn Center with all their members and patients for providing the burned

    skin.

    3. The Integra LifeScience Corporation for providing Integra®.

    I want to thank Toronto Hydro for their generous donations that enabled our research.

  • v

    Table of Contents

    Acknowledgments.......................................................................................................................... iii

    Contributors ................................................................................................................................... iv

    Table of Contents ............................................................................................................................ v

    List of Tables ................................................................................................................................. ix

    List of Figures ................................................................................................................................. x

    List of Abbreviations .................................................................................................................... xii

    Chapter 1 Introduction .................................................................................................................... 1

    Introduction ............................................................................................................................. 1

    1.1 Burn injury ...................................................................................................................... 1

    1.1.1 Definition and current treatment ............................................................................. 1

    1.1.2 Challenges in modern burn care ............................................................................. 4

    1.2 Skin substitutes ............................................................................................................. 12

    1.3 Physiology of wound healing and its meaning for cell therapy .................................... 16

    1.4 The role of stem cells in skin regeneration and wound healing .................................... 22

    1.4.1 Overview - classifications and characteristics of stem cells ................................ 22

    1.4.2 Mesenchymal stem cells as wound therapy .......................................................... 31

    1.5 Preliminary data ............................................................................................................ 34

    Chapter 2 Rationale, Hypothesis, and Aim ................................................................................... 37

    Rationale, Hypothesis, and Aim ........................................................................................... 37

    2.1 Rationale ....................................................................................................................... 37

    2.2 Hypothesis..................................................................................................................... 37

  • vi

    2.3 Aim ............................................................................................................................... 38

    Chapter 3 Material and Methods................................................................................................... 39

    Material and Methods ........................................................................................................... 39

    3.1 Stem Cell extraction and culturing ............................................................................... 39

    3.1.1 Conventional Stem Cell Extraction Method ......................................................... 41

    3.1.2 Enzymatic Stem Cell Extraction Method ............................................................. 41

    3.1.3 Cell culturing ........................................................................................................ 42

    3.1.4 Quantification of cell yield ................................................................................... 42

    3.2 Determination of mesenchymal stem cell character ..................................................... 43

    3.2.1 Flow Cytometry for stem cell surface markers ..................................................... 43

    3.2.2 Differentiation Assay ............................................................................................ 44

    3.3 Determination of key biological characteristics ........................................................... 45

    3.3.1 Population doubling time ...................................................................................... 45

    3.3.2 Colony forming assay ........................................................................................... 46

    3.3.3 Proliferation via bromodeoxyuridine (BrdU) staining .......................................... 46

    3.3.4 Cell cycle analysis................................................................................................. 47

    3.3.5 Reactive oxygen species (ROS) expression .......................................................... 47

    3.3.6 Apoptosis .............................................................................................................. 48

    3.3.7 Glycolytic and Mitochondrial function ................................................................. 49

    3.4 Tumorigenicity .............................................................................................................. 50

    3.4.1 In Vitro .................................................................................................................. 50

    3.4.2 In Vivo .................................................................................................................. 51

    3.5 Immunogenicity and -reactivity .................................................................................... 52

  • vii

    3.5.1 Flow cytometry for major histocompatibility complex (MHC) I and II and toll-

    like receptor (TLR) 4 ............................................................................................................ 52

    3.5.2 QPCR for toll-like receptor (TLR) 1-10 ............................................................... 53

    3.5.3 Secretion Profile.................................................................................................... 56

    3.6 Stem cell integration into Integra® ............................................................................... 57

    3.7 Porcine Model .......................................................................................