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.

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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