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Role of Tauroursodeoxycholic Acid during Expansion and Neural Commitment of Human Induced Pluripotent Stem
Cells under Chemically-defined Conditions
Joana Carolina de Almeida Pinho
Thesis to obtain the Master of Science Degree in
Biomedical Engineering
Supervisors: Professor Maria Margarida Fonseca Rodrigues Diogo
Professor Susana Zeferino Sol Cruz
Examination Committee
Chairperson: Professor Ana Lusa Nobre Fred
Supervisor: Professor Maria Margarida Fonseca Rodrigues Diogo
Member of the Committee: Doctor Sara Alves Xapelli
December 2015
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Acknowledgements
Even if my name is the one appearing as the author of this thesis, it wouldnt have been
possible to complete it without the help (and patience) of a number of other intervenients, inside or
outside the lab.
I would first like to thank my supervisors for always enthusiastically supporting and following
up on my work from the start until the end. Particularly, I am very grateful to Professor Margarida
Diogo for finding such a stimulating project for me and for always being available to discuss, advise
and brainstorm about it. Also, I thank Professor Susana Sol for the opportunity of a partnership with
iMed.ULisboa, for all the guidance, trust and time implemented in making this project more interesting
and valuable. Last but certainly not least, I thank Tiago Fernandes for babysitting me on a daily basis,
teaching me all the skills needed to accomplish my results and for all the time spent helping in more
demanding experiments. It has been a pleasure working with and for all of you.
I thank Professor Joaquim Sampaio Cabral for receiving me in the SCBL team, for the
availability and for being present and contributing to my periodic meetings regarding the project.
Also, I am grateful to Professor Ceclia Rodrigues for the partnership with the fantastic
research team from iMed.ULisboa.
I am most thankful for working aside such great teams as the SCBL and iMed, who created a
great work environment and frequently helped me in moments of despair. I have to emphasize my
Masters colleague Andr, my siamese brother in the first months in the lab; my friend Teresa, for the
company (and laughs) in the last work-loaded months; Carlos for all the help in the work and for
creating such exquisite lunch-hour topics of conversation; Jorge, for the friendly conversations every
morning when sharing a flow shamber and for always being available to help whenever with whatever;
Cludia, for all the advices regarding the experiments and, finally, Maria Ribeiro, for teaching me the
techniques in the Faculty of Farmacy and for spending her valuable time helping me with the tests.
Also, my friends (outside the lab), who dont give up cheering me, even if I couldnt regularly
participate in the weekend party plans. A special thanks goes to Joana, who taught me the basics of
photoshop, necessary for image analysis in this work.
Finally, the most important, my family, my biggest fans and supporters who give me strength
and motivation every day.
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Abstract
The discovery of human induced pluripotent stem cells (hiPSCs) has revolutionized the
research in the stem cell field. In addition to their long-term self-renewal capacity, these cells can be
differentiated towards any cell lineage in the human organism, representing a valuable tool for
regenerative medicine, drug screening and disease modelling. For instance, the commitment of
hiPSCs into the neuronal lineage brings great promise in finding new solutions for neurodegenerative
diseases. Nevertheless, robust, reproducible and efficient culture systems have to emerge in order to
expand and differentiate hiPSCs with high yields, obtaining the quantities necessary to execute those
applications. Previous reports on the role of an endogenous bile acid, tauroursodeoxycholic acid
(TUDCA) as an anti-apoptotic, proliferative and differentiation-modulating agent in neural stem cell
(NSC) cultures have motivated the study of the effect of this bile acid in both hiPSC expansion and
neural commitment into neural progenitor (NP) cells. In this work, different concentrations of TUDCA
were added to a biochemically-defined hiPSC culture media. Although the bile acid did not appear to
influence cell viability and pluripotency, it significantly induced higher fold increases in the number of
cells for concentrations ranging 5 to 10 M, when compared to controls. A hiPSC neural commitment
protocol with addition of TUDCA was implemented, with successful generation of Pax6+/Nestin
+ NPs
within 12 days and posterior generation of rosette-like structures. Notably, quantification of rosettes
showed an increase of 56% and 99% in cultures treated with 5 and 10 M of TUDCA, respectively,
when compared to controls. Here, we demonstrated for the first time the influence of TUDCA in the
proliferation and differentiation of hiPSCs, revealing that this bile acid had a positive influence in both
processes. Importantly, in the future, these findings could be translated to large-scale culture systems.
v
Resumo
A descoberta de clulas estaminais pluripotentes induzidas humanas (hiPSCs) revolucionou a
investigao no campo das clulas estaminais. Para alm da sua capacidade de se auto renovarem a
longo prazo, estas clulas podem ser diferenciadas em qualquer linhagem celular que constitui o
organismo humano, sendo ferramentas valiosas em medicina regenerativa, testes de frmacos e
modulao de doenas. Por exemplo, o comprometimento neural de hiPSCs pode contribur para
encontrar novas solues para doenas neurodegenerativas. No entanto, so necessrios sistemas
de cultura celular robustos, reprodutveis e eficientes para expandir e diferenciar hiPSCs com
rendimentos elevados, obtendo as quantidades necessrias para as aplicaes mencionadas.
Experincias prvias quanto ao cido tauroursodesoxiclico (TUDCA), um cido biliar endgeno,
como agente anti-apopttico, proliferativo e modulador da diferenciao de clulas estaminais neurais
(NSC), motivou o estudo do seu efeito na expanso e comprometimento neural de hiPSCs em
progenitores neurais (NP). Neste trabalho, foram adicionadas diferentes concentraes de TUDCA a
meios de cultura definidos de hiPSCs. Embora a substncia no tenha influenciado a expresso de
marcadores de pluripotncia e viabilidade cellular, observou-se um significativo aumento da
proliferao para as concentraes entre 5 e 10 M, quando comparada com os controlos. Foi
implementado um protocolo de comprometimento neural de hiPSCs com adio de TUDCA, para a
formao de NPs Pax6+/Nestin
+ no prazo de 12 dias e o posterior aparecimento de rosetas neurais. A
quantificao de rosetas mostrou um aumento de 56% e 99% para as concentraes de 5 e 10 M,
respectivamente, quando comparando com o controlo. Este trabalho foi a primeira tentativa de testar
a influncia do TUDCA na proliferao e diferenciao de hiPSCs. Os resultados demonstraram a
influncia positiva que o TUDCA teve em ambos os processos e, no futuro, espera-se que estes
desenvolvimentos possam ser transferidos para sistemas de cultura em larga escala.
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Keywords
Human induced pluripotent stem cells
Tauroursodeoxycholic acid
Expansion
Neural commitment
Neural progenitor cells
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Palavras-chave
Clulas estaminais pluripotentes induzidas humanas
cido tauroursodesoxiclico
Expanso
Comprometimento neural
Progenitores neurais
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List of contents
Acknowledgements ............................................................................................................ iii
Abstract ............................................................................................................................. iv
Resumo ............................................................................................................................... v
Keywords ........................................................................................................................... vi
Palavras-chave .................................................................................................................. vii
List of contents ................................................................................................................. viii
List of figures...................................................................................................................... xi
List of tables ..................................................................................................................... xiii
List of abbreviations ......................................................................................................... xiv
I.INTRODUCTION ................................................................................................................. 1
I.1 Stem Cells ........................................................................................................................................ 1
I.1.1 Human Pluripotent Stem Cells (hPSCs) .................................................................................... 2
I.1.1.1 Human Induced Pluripotent Stem Cell
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