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Versao Definitiva Jun2017 UNIVERSIDADE DE LISBOA FACULDADE DE FARMÁCIA Development of three-dimensional umbilical cord-derived mesenchymal stem cell cultures for differentiation into hepatocyte-like cells: a potential breakthrough in toxicological drug screening Madalena Zincke dos Reis Fernandes Cipriano Orientadora: Doutora Joana Paiva Gomes Miranda Co-orientadores: Doutor Jorge Miguel Silva Santos Doutora Katrin Zeilinger Tese especialmente elaborada para a obtenção do grau de Doutor em Farmácia, especialidade de Toxicologia 2017 UNIVERSIDADE DE LISBOA FACULDADE DE FARMÁCIA Development of three-dimensional umbilical cord-derived mesenchymal stem cell cultures for differentiation into hepatocyte-like cells: a potential breakthrough in toxicological drug screening Madalena Zincke dos Reis Fernandes Cipriano Orientadora: Doutora Joana Paiva Gomes Miranda Co-orientadores: Doutor Jorge Miguel Silva Santos Doutora Katrin Zeilinger Tese especialmente elaborada para a obtenção do grau de Doutor em Farmácia, especialidade de Toxicologia Júri: Presidente: Doutora Matilde da Luz dos Santos Duque da Fonseca e Castro, Professora Catedrática e Directora da Faculdade de Farmácia da Universidade de Lisboa Vogais: Doutor Jorge Manuel Lira Gonçalves Ruas, Associate Professor, Karolinska Institutet, Sweden Doutor Félix Dias Carvalho, Professor Catedrático, Faculdade de Farmácia da Universidade do Porto Doutora Diana Esperança dos Santos Nascimento, Investigadora Auxiliar, Instituto de Engenharia Biomédica da Universidade do Porto Doutora Sofia de Azeredo Pereira Costa, Professora Auxiliar, Faculdade de Ciências Médicas da Universidade Nova de Lisboa Doutora Cecília Maria Pereira Rodrigues, Professora Catedrática, Faculdade de Farmácia da Universidade de Lisboa Doutor Nuno Filipe da Rocha Guerreiro de Oliveira, Professor Auxiliar com Agregação, Faculdade de Farmácia da Universidade de Lisboa Doutora Joana Paiva Gomes Miranda, Investigadora Auxiliar, Faculdade de Farmácia da Universidade de Lisboa iMed.ULisboa / Fundação para a Ciência e Tecnologia, Portugal 2017 This work has been performed at: Chemical Biology and Toxicology Group, Research Institute for Medicines (CBT, iMed.ULisboa) in the Faculty of Pharmacy of University of Lisbon, Portugal; ECBio, S.A., Portugal; and Berlin-Brandenburg Center for Regenerative Therapies (BCRT) in the Charité Universitätsmedizin in Berlin, Germany; under the scientific supervision of Joana Paiva Gomes Miranda, PhD, and co-supervision of Miguel Silva Santos, PhD, and Dr. med. vet. Katrin Zeilinger. Madalena Cipriano is a recipient of a PhD fellowship (SFRH/BD/87508/2012) from the Fundação para a Ciência e Tecnologia (FCT), Lisboa, Portugal and this work was developed within the framework of the FCT funded project “Development of 3D culture models for human UCX® cell-derived hepatocytes for predictive toxicology and cell therapy”, reference PTDC/SAU-TOX/110457/2009. ACKNOWLEDGEMENTS First and foremost I would like to express my special appreciation to my advisors. To Joana Miranda, my main advisor, special thanks for believing in me. While I was still an undergraduate student, you gave me the opportunity to start a small research project and patiently teach me all about the basic laboratory and cell culture rules, techniques and tricks. You also lighted up the science bug on me and tought me to run for what I wanted. You always pushed me to grow as a researcher. I always felt constant support and friendship, especially in the most difficult times. Thank you for the opportunity to be part of our team, for creating the conditions of a complete education at several levels, scientifically, socially and humanly. With you I learned how important it is to be part of a team, support other team members and to live in different environments to strength your capacities. A special acknowledgement is also due to Miguel Santos. Thank you for the relentless support and availability, as well as critical thinking and scientific discussions, crucial for the great course of this work. To Katrin Zeillinger my co-advisor, for all the support and trust, as well as for welcoming me into her research group, in the Charité – Universitätsmedizin Berlin (Germany). Without your cooperation and support a significant part of this work would not have been possible. An acknowledgement is also due to the Faculdade de Farmácia, Universidade de Lisboa, in particular to the Departamento de Ciências Toxicológicas e Bromatológicas, and the Research Institute for Medicines (iMed.ULisboa) for the facilities and conditions that allowed me to develop my PhD work, as well as to the Charité – Universitätsmedizin and ECBio, SA,. To the Portuguese government (Fundação para a Ciência e a Tecnologia) and FEDER for the funding, namely my PhD grant (SFRH/BD/87508/2012) and to the project funding (PTDC/SAU-TOX/110457/2009), as well as the strategic projects PEst-OE/SAU/ UI4013/2011 and UID/DTP/04138/2013. To all my colleagues from the Chemical Biology and Toxicology Group, special thanks for all the help and constant support. I would like to thank Professor Matilde Castro, the CBT coordinator at the time I joined CBT, for all support during this thesis. To my first lab colleagues Ana Sofia Coelho and Joana Marques. Joana, you are much more than a CBT colleague but a friend for life in and outside of science. I would also acknowledge Elysse Filipe, Patrícia Guerreiro, Pedro Pinheiro, Ana and João. To Inês Ferreira for receiving me in her home in Berlin, and for being always there for scientific and political discussions. I vii am also especially thankful to Sérgio, Inês, Bernardo and Joana Rodrigues, present in this last period of this thesis, for growing together as a team. To Professor Nuno Oliveira for his support, scientific discussions and for being always present. To ECBio, SA, in particular to Miguel and also to Rita Barcia, Pedro Cruz and Hélder Cruz, for supporting my work, for providing the stem cells, for the scientific discussions and contribution for paper manuscripts. I am also grateful for being always welcome to run experiments there. To AG Zeillinger, Bioreactor Group, the Berlin-Brandenburg Center for Regenerative Therapies (BCRT) in the Charité Universitätsmedizin in Berlin, Germany. Words cannot express how thankful I am for the warm welcome and the way you took me into your group and made me feel at home (even when home was quite far away). To Fanny, for being always there to solve bioreactor problems and to arrange social activities. A special acknowledgement to Nora Freyer, for being always there for scientific, political and cultural discussions. You were my support during a new chapter of my life that made me grow so much in those short 6 months. I would also acknowledge the support of Christine, Marco, Thomas and Jasmina that were also part of these great times. To Ruas Jorge Group, Molecular and Cellular Exercise Physiology, Department of Physiology and Pharmacology, Karolinska Institutet in Stockholm, Sweden. In particular to Jorge Ruas and Jorge Correia for welcoming me in their lab and for the scientific input on a crucial moment of the work here presented. To Sofia Pereira and her CEDOC team, namely Catarina, Nádia and Nuno for the precious collaboration and support in the last year of this project in the HPLC methods and data analysis as well as for being so kind and helpful to me. Finally, I need to acknowledge my always supporting family, my friends and my team for being there and believing in me. viii ABSTRACT A standard truly predictive in vitro hepatic model has been sought for reducing animal testing, increasing drug development efficiency and improving prediction of adverse drug reactions. The liver is the main organ responsible for xenobiotic metabolism, being particularly exposed to chemicals and its metabolites. The currently available hepatic in vitro models are insufficient. Those present low biotransformation activity, quickly lose its differentiated phenotype in vitro and/or have relevant interspecies differences. Therefore, the differentiation of stem cells (SC) into hepatocyte-like cells (HLC) has been suggested as an alternative approach to provide a representative human hepatic model. However, a functional hepatocyte-like phenotype has not yet been achieved. In this thesis, we intended to demonstrate that by (i) using a human neonatal mesenchymal stem cell (hnMSC) source, UCX®, (ii) designing a protocol to differentiate this hnMSC into HLC using the cytokines and growth factors present during liver embryogenesis and (iii) resorting to 3D culture conditions would provide an environment closer to the in vivo, and allow a more representative cellular morphology, gene expression and biological behaviour of the hepatocyte. Umbilical cord matrix hnMSC were selected for the hepatic differentiation due to its high availability, expansion capacity and genomic stability upon expansion, and, due to its more primordial origin, its higher differentiation capacity relative to other MSC sources. The hepatic differentiation protocol was step-wise developed under 2D cell culture conditions. FGF-2 at 4 ng/mL and the absence of FGF-4 resulted in an improved endoderm commitment and foregut induction during the first step of the differentiation protocol. The epigenetic modifiers trichostatin A (TSA), 5-azacytidine (5-AZA) and dimethyl sulfoxide (DMSO) were tested for an improved hepatoblast formation and maturation to HLC. The 21 day-long protocol was extended up to day 34, when a global gene expression analysis was performed. Transcriptomic analyses
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