Differentiation of Human Mesenchymal Stem/Stromal Cells into Myogenic Cells for Urethral Sphincter Muscle Engineering Sara Ferreira Martins Gomes Thesis to obtain the Master of Science Degree in Biotechnology Supervisors: Professor Cláudia Alexandra Martins Lobato da Silva Professor Joaquim Manuel Sampaio Cabral Examination Committee Chairperson: Professor Arsénio do Carmo Sales Mendes Fialho Supervisor: Professor Cláudia Alexandra Martins Lobato da Silva Member of the Committee: Professor Gabriel António Amaro Monteiro December 2015 ii I dedicate this thesis to the memory of my father, Henrique Martins Gomes (November 4th, 1946 - May 11th, 2015) iii iv Acknowledgments First, I would like to thank Professor Joaquim Cabral and Professor Cláudia Silva for making it possible for me to develop my dissertation work at the Stem Cell Bioengineering and Regenerative Medicine Laboratory (SCBL-RM), at IBB-BERG-IST, and for accepting to be my Supervisors. I am truly grateful for the opportunity to work in this field of study, which I have been passionate about since I was in high school. I also want to thank Professor Cláudia for all the guidance and encouragement and for all the trust put into my work and my ideas. Second, I owe a big thank you to Irina Simões, who guided me since the beginning, taught me everything I needed to know about this project and was always accessible to help me even at thousands of kilometres away in Zurich. I could not have achieved the results I present here without her knowledge and direction, and I could not have been attributed a better mentor. Third, I want to thank my colleagues at the SCBL-RM laboratory, who were always available to help me and give me guidance when I needed: Ana Fernandes, Márcia Mata, Francisco Moreira, Diogo Pinto, Raquel Cunha, Marta Costa, João Silva, Cláudia Miranda, Tiago Dias and Carlos Rodrigues. You all had an important part in the development of this project and of my laboratory skills. I want to give a warm thank you to Alexandra Salvado, Ângela Neves and Mafalda Cavalheiro for being my partners in this journey, and for all the support and laughter that we shared over the last two years. Without you, group assignments would have been extremely dull and definitely not as productive as they were. Last but definitely not least, I want to thank my family, especially the three most important gentlemen in my life: my boyfriend/best friend, my brother, and my father. Rui, you are my rock: you were there for me through the toughest times, holding me together and giving me a reason to fight for. My dear Mano, I want to thank you for all the wise words, support and guidance, especially over the last 6 months, and for encouraging me to always do my best. Dad, thank you for telling me how proud of me you were; those words are what keep me going in the hardest moments. I hope I keep making you proud. I deeply miss you. v vi Abstract Stress urinary incontinence (SUI) is a medical condition that requires novel alternative therapies aiming to restore and maintain the integrity and function of the urethral sphincter, the muscle layer responsible for the normal continence mechanism. This MSc project targeted the establishment of effective myogenic differentiation protocols for Mesenchymal Stem/Stromal Cells (MSCs) for urethral sphincter engineering, with particular focus on exploring the myogenic potential of the Stromal Vascular Fraction (SVF) of the adipose tissue. The ability of MSCs to differentiate into smooth muscle cells (SMCs) and skeletal myofibers, the main constituents of the sphincter, has already been demonstrated in few in vivo and in vitro studies, but with little translation of this knowledge into clinical settings. The effects of 5-aza-2’-deoxycytidine (5-AZAd) and PD98059, chemical inducers of skeletal muscle and smooth muscle differentiation in MSCs, respectively, were tested herein. MSC differentiation into both cell types was evaluated by the detection of smooth and skeletal muscle lineage-specific markers by flow cytometry and immunofluorescence techniques at different timepoints in early cell passages (P<3). The expression of skeletal muscle markers was assessed in magnetically sorted and unsorted SVF cells coated on distinct substrates. Myogenesis-committed cells were identified in uncultured SVF, but no myoblast-like cells were isolated. Still, SVF-derived cells were shown to possess intrinsic myogenic potential that was enhanced when combined with culture substrates (in particular, gelatin coating), thus holding great potential for skeletal muscle engineering applications. Conversely, 5-AZAd supplementation failed to induce myogenesis and triggered severe cytotoxic effects, while PD98059 did not provide enough stimuli to sustain smooth muscle differentiation, which likely requires the use of 3-D culture conditions and/or biomechanical stimulation. Keywords Stress Urinary Incontinence Mesenchymal Stem/Stromal Cells Stromal Vascular Fraction Myogenic Differentiation Smooth Muscle Cells Skeletal muscle Cells vii viii Resumo A incontinência urinária de esforço é uma condição que requer novas terapias que visem restaurar e manter a integridade e função do esfíncter uretral, a camada de músculo responsável pelo mecanismo de continência normal. Este Projeto de Mestrado teve como objectivo o desenvolvimento de protocolos que permitam a diferenciação eficaz de células estaminais/estromais mesenquimais (ou mesenquimatosas) - CEMs - em linhagens miogénicas para reconstituição do esfíncter uretral, com especial foco no estudo do potencial miogénico da fracção vascular estromal (FVE) do tecido adiposo. Neste sentido, foi testado o efeito dos compostos 5-aza-2’-desoxicitidina (5-AZAd) e PD98059, indutores químicos de diferenciação em músculo esquelético e liso, respectivamente, em CEMs. A diferenciação de CEMs em ambos os tipos de músculo foi avaliada através da detecção de marcadores específicos de cada linhagem, por técnicas de citometria de fluxo e imunofluorescência em tempos de cultura distintos e em passagens celulares baixas (P<3). A expressão de marcadores de músculo esquelético foi avaliada também em células da FVE minimamente processada e isoladas magneticamente, plaqueadas sob superfícies revestidas com componentes da matriz extracelular (gelatina e fibronectina). Foram detectadas células miogénicas na FVE minimamente processada, no entanto não foram isoladas células com morfologia mioblástica. Ainda assim, foi mostrado que as células obtidas a partir da FVE são dotadas de um potencial miogénico intrínseco, reforçado na presença de substratos, sendo potencialmente exequível a aplicação destas células em estratégias de reconstituição de músculo esquelético. Por outro lado, a adição de 5-AZAd não levou à indução de miogénese e conduziu a efeitos citotóxicos, enquanto que a adição de PD98059 não foi suficiente para sustentar a diferenciação de CEMs em músculo liso, que provavelmente requer a implementação de condições de cultura em 3-D e/ou estimulação biomecânica. Palavras-Chave Incontinência Urinária de Esforço Células Estaminais/Estromais Mesenquimais (ou Mesenquimatosas) Fração Vascular Estromal Diferenciação Miogénica Células do Músculo Liso Células do Músculo Esquelético ix x Index ACKNOWLEDGMENTS ......................................................................................................................... V ABSTRACT ........................................................................................................................................... VII RESUMO ................................................................................................................................................ IX LIST OF FIGURES ............................................................................................................................... XIII LIST OF TABLES ............................................................................................................................... XVI ABBREVIATIONS LIST ..................................................................................................................... XVII I. INTRODUCTION .............................................................................................................................. 1 I.1 BACKGROUND ............................................................................................................................... 1 I.1.1 Urinary Incontinence ........................................................................................................... 1 I.1.2 Regenerative Medicine ........................................................................................................ 3 I.1.3 Muscle Cells ........................................................................................................................ 8 I.1.4 Mesenchymal Stem/Stromal Cells .................................................................................... 11 I.1.5 Tissue Engineering-derived Urethral Constructs in Clinical Trials .................................... 17 I.2 MYOGENIC DIFFERENTIATION OF MSCS: CURRENT STATUS ......................................................... 18 I.2.1 Differentiation of MSCs into Skeletal Muscle Cells ........................................................... 18 I.2.2 Differentiation of MSCs into SMCs .................................................................................... 22 I.3 AIM OF STUDIES.........................................................................................................................