Patient specific stem cell models: focus on glioblastoma and neuronal differentiation aspects of chronic granulomatous disease

Thesis

Reference

Patient specific stem cell models: focus on glioblastoma and neuronal differentiation aspects of chronic granulomatous disease

NAYERNIA, Zeynab

Abstract

Pluripotent stem cells can in vitro replicate key pathophysiological aspects of central nervous system. In the first project, we have developed a 3D co-culture system using cancer stem cells isolated from glioblastoma patients and a human stem cell-derived engineered neural tissue, which allowed us to reproduce numerous hallmarks of glioblastoma in vivo, including invasion and formation of secondary foci. Transcriptomic analysis identified that IFN response genes was induced specifically in the co-culture system that was significantly correlating with patient survival. In a second project, we have focused on the role of ROS generating NOX2 enzyme during neural differentiation. Our study demonstrates that during early stages of neurogenesis there is a regulatory role of NOX2, which is conserved from mouse to human.

This suggests a contribution of redox mechanisms in the maintenance of neural stem/progenitor. In conclusion, this study exemplifies the potential of stem cells for the study of human pathophysiology.

NAYERNIA, Zeynab. Patient specific stem cell models: focus on glioblastoma and neuronal differentiation aspects of chronic granulomatous disease. Thèse de doctorat : Univ. Genève, 2014, no. Sc. 4657

URN : urn:nbn:ch:unige-383307

DOI : 10.13097/archive-ouverte/unige:38330

Available at:

http://archive-ouverte.unige.ch/unige:38330

Disclaimer: layout of this document may differ from the published version.

UNIVERSITE DE GENEVE

Département de génétique et évolution FACULTE DES SCIENCES Professor Ivan Rodriguez

Département de pathologie et immunologie FACULTE DE MEDECINE

Professor Karl-Heinz Krause

___________________________________________________________________

Patient specific stem cell models: focus on

glioblastoma and neuronal differentiation aspects of chronic granulomatous disease

THESE

Présentée à la faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Zeynab Nayernia de

Shiraz (IR)

Thèse n° 4657 Genève

2014

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ACKNOWLEDGMENT

This Dissertation would have not been possible without the support of multiple wonderful people around me who have helped continuous support and questioned me throughout my PhD.

First of all I would like to thank my supervisor Karl-Heinz Krause for hosting me in his lab and allowing me to work on an exciting topic of stem cell research and for his advice throughout my PhD. I would like to thank my co-supervisor Ivan Rodriguez and the committee members Marie José Stasia and Nicolas Demaurex for reading my thesis and for their advice throughout my PhD. I would like to thank my PhD godfathers Dr. Alexandre Lobrinus and Prof. Pierre-Yves Dietrich for monitoring and sponsoring my thesis.

I’d like to also acknowledge all my lab colleagues and friends who have helped me along my PhD work either through advising, listening, or teaching: Tamara Seredenin,! Stéphanie Julien, Olivier Plastre, Lasta Kocjancic Curty, Ophélie Cherpin, Julien Cachat, Aleksandra Filippova, Mathurin Baquie, Marilena Colaianna, Maxime Feyeux, Hedi Peterson, Sabrina Villy, Vannary Tieng Caulet, Michel Dubois-Dauphin, Christophe Delgado, Hadrien Soldati and Véronique Pruvost.

And of course Vincent Jaquet, thank you for challenging and advising me from the start, I appreciate your support and confidence in me.

I would like to thank other past members of our laboratory: Silvia Sorce, Renier Myburgh, Christine Deffert, Michela Schäppi, Olivier Basset, Lone Roesingh and my

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colleagues at the hospital of Geneva (HUG) Olivier Preynat-Seauve, Erika Cosset and Diderik Tirefort for their advice during the first project of this PhD work.

A big thank you is owed to the people in several facilities for their invaluable help.

First, I want to thank the genomic platform of the Faculty of Medicine of Geneva and in particular Didier Chollet and Christelle Barraclough for their help in Q-PCR experiments. I’d like to thank the all the members of the bioimaging platform, especial Sergei Startchik for image analysis, Olivier Brun! and François Prodon! for assisting me in using confocal microscopy. I’d like to thank also the flow cytometry platform, Cécile Gameiro and Jean- Pierre Aubry–Lachainay for assisting me in using flow cytometry and data analysis. I am also grateful to Frédérique Sloan Béna of the genomic sequencing facility for her informative discussion and guidelines for array-CGH sequencing data analysis and Danielle Ben Nasr and Marie Ebrahim Male for their help at the histology platform.

On a personal note, I’d like to thank my best friends Alireza, Moe Kaji, Rezwane, Shahrzad, Noemi and Evelyn Erni for being my support system whenever I needed them during these years. Last, I’d like to thank my family, my two lovely brothers Hossein and Hamed and in particularly my parents, Mehran and Karim who have always supported me in whatever I do and understand me even when I’m 5000 miles away. They allowed me to always be who I want to be and encouraged me to follow my dreams.

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INDEX

RESUME………...……….8

ABSTRACT……….………10

INTRODUCTION………..……….11

1.THE CONCEPT OF STEM CELL………11

1.1. EMBRYONIC STEM CELLS………..………11

1.2. REPROGRAMMING AND INDUCED POTENCY ………..………13

1.2.1. NUCLEAR TRANSFER………14

1.2.2. DIRECT REPROGRAMMING……….………15

1.2.2.1. IPSC IN MODELLING CNS………..………16

1.2.2.2.IPSC TO STUDY REDOX PATHWAYS IN CNS DISEASE …...…19

1.3 ADULT STEM CELLS………..………21

1.3.1.ADULT NEUROGENESIS……….………21

1.3.1.1.PHYSIOLOGICAL ROLE OF ROS: FOCUS ON NEUROGENESIS.. ………..………22

1.3.1.2 USING IPSC FOR UNDERSTANDING CNS PHYSIOLOGY….….24 1.4. CANCER STEM CELLS………..………26

1.4.1. GLIOBLASTOMA BRAIN TUMOR………28

2. HYPOTHESES AND AIMS OF THE PROJECT ………...………33

FIRST PART ……….……….………35

RESEARCH ARTICLE1……….………...…...………39

SUPPLEMENTARY FIGURES ………...…...………52

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SECOND PART………...…...………57

RESEARCH ARTICLE 1………...………...………86

ABSTRACT………...………...………88

INTRODUCTION………...……..……...………89

MATERIAL &METHODS…………..………...………..……92

RESULTS………...……….………...………101

DISCUSSION………...………...………...………109

FIGURES & LEGENDS………..……...………116

SUPPLEMENTARY FIGURES………..…………...………134

REFERENCES………...………..………...………154

DISCUSSION AND FUTURE PROSPECTIVES………...………161

REFERENCES ………...………...………...………173

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LIST OF ABBREVIATION

AD Alzheimer’s disease

ALS Amyotrophic Lateral Sclerosis

BDNF Brain Derived Neurotrophic Factor

CNS Central Nervous System

CSC Cancer Stem Cells

cDNA Complementary Deoxyribonucleic acid

CO2 Carbon dioxide

DG Dentate Gyrus

DNA Deoxyribonucleic acid

ES Stem Cells

ENT Engineered human Neural Tissue

EGF Epidermal Growth Factor

ECM Extracellular Matrix

FGF2 Fibroblast Growth Factor 2

HESCs Human Pluripotent Stem cells

H2O2 Hydrogen Peroxide

HHV-6 Human Herpes Virus 6

HD Huntington’s disease

IFN Interferon

IPSC Induced pluripotent stem cells

KLF-4 Kruppel-like factor 4

kDa Kilodalton

MRI Magnetic Resonance Imaging

NOX NADPH Oxidase

NPCs Neural Precursor Cells

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NSCs Neural Stem Cells

NRG Neuregulin

NT Nuclear Transfer

O2 Oxygen

OCT4 Octamer-binding transcription factor 4

PSC Pluripotent Stem Cells

PD Parkinson’s disease

Q-PCR Quantitative Polymerase Chain Reaction

RA Retionic Acid

ROS Reactive Oxygen Species

RNA Ribonucleic acid

SVZ Subventricular zone

SCNT Somatic Cell Nuclear Transfer

SOX2 sex determining region Y-box 2

STAT-1 Signal Transducers and Activators of Transcription

SV40 simian vacuolating virus 40

TMZ Temozolomide

TFP Tomato Fluorescent Protein

3-D Three- Dimension

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RESUME

De par leurs capacités de renouvellement et de différenciation en un tissu donné, les cellules souches embryonnaires humaines permettent de reproduire in vitro certains aspects critiques des maladies du système nerveux central. Dans ce travail, nous avons utilisé des cellules souches pluripotentes humaines d’une part pour créer un modèle in vitro de glioblastome et d’autre part pour étudier le rôle de l’enzyme NOX2 dans la différentiation neuronale. Pour le premier projet, nous avons développé un système de co-culture en 3-D en utilisant des cellules souches cancéreuses de glioblastome isolées à partir de patients et un tissu neural dérivé de cellules souches humaines. Ce système nous a permis de reproduire in vitro de nombreuses caractéristiques des glioblastomes in vivo telles l’invasion et la formation de foci secondaires. Nous avons identifié par analyse transcriptomique qu’un groupe de gènes de la voie de signalisation de STAT-1 était spécifiquement induit dans le système de co-culture et qu’il y avait une bonne corrélation entre les profils d’expression génique in vitro et la survie des patients. Dans un second projet, nous avons utilisé des cellules embryonnaires pluripotentes induites (CEPI) dérivées d’un patient atteint de granulomatose septique (GS) causée par une mutation dans le gène codant pour NOX2, une enzyme qui génère des formes réactives de l’oxygène. Nous avons montré que NOX2 est exprimée transitoirement à un stade précoce de différenciation neuronale. Toutefois morphologiquement la différenciation neuronale entre les cellules déficientes en NOX2 et les cellules normales n’est pas différente, mais certains marqueurs précoces de neurogenèse tels que la nestine et le BDNF sont diminués chez les cellules déficientes en NOX2. Nous avons confirmé la diminution de la nestine et du BDNF dans les régions neurogéniques de cerveau de souris génétiquement modifiées (NOX2 knockout). L’absence de NOX2 induit également une diminution du statut oxydatif et de la prolifération des précurseurs neuraux, suggérant un rôle clef des oxidants

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générés par NOX2 dans la différenciation et la prolifération des cellules souches neurales. En conclusion, cette étude exemplifie quelques-unes des possibilités immenses de l’utilisation des cellules souches humaines pour l’étude de la physiopathologie humaine.

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ABSTRACT

Because of their ability to self-renew and differentiate into various tissue types, human embryonic stem cells can in vitro replicate key aspects of central nervous system pathology and physiology. In this study, we have used human pluripotent stem cells to generate an in vitro model of glioblastoma and to study the role of NOX2 during neuronal differentiation. In the first project, we have developed a three-dimensional co-culture system using cancer stem cells isolated from glioblastoma patients and a human stem cell-derived engineered neural tissue. This system allowed us to reproduce numerous hallmarks of glioblastoma in vivo, such as invasion and formation of secondary foci. Transcriptomic analysis identified that a group of genes of the IFN/STAT-1 signaling pathway was induced specifically in the co-culture system. Moreover, significant correlation between the profile of specific gene expression and patient survival was detected. In a second project, we have used induced pluripotent stem cells (iPSC) derived from a patient with chronic granulomatous disease, a genetic disease caused by a mutation in the gene coding for the reactive oxygen species generating enzyme NOX2. We have shown that NOX2 is transiently expressed at an early stage of neural differentiation. Although no morphological differences were seen between normal and NOX2-deficient cells, early markers of neurogenesis such as nestin and BDNF were decreased in NOX2-deficient cells. Furthermore, we confirmed that nestin and BDNF are decreased in vivo in the neurogenic regions of NOX2 knockout mice. NOX2 deficiency also induces a decrease of the oxidative status and proliferation of neural precursor, suggesting a key role of NOX2-derived oxidants in differentiation and proliferation of neural adult stem cells. In conclusion, this study exemplifies a subset of the tremendous potential of human stem cells for the study of human pathophysiology.

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INTRODUCTION

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1. THE CONCEPT OF STEM CELLS

Stem cells are undifferentiated cells that have the capacity to divide (self-renew) and differentiate towards all specialized cell; endoderm, mesoderm and ectoderm. There are two general types of stem cells existing in mammals; i) embryonic stem cells, which can be isolated from the inner cell mass of blastocyst embryonic stage and ii) adult stem cells, which are tissue specific stem cells existing during the postnatal life. These cells are capable to divide into parent stem cells (self renew) and differentiate into progenitors, which is essential for development (embryonic stem cells) and for normal maintenance (adult stem cells) of regenerative organs such as skin, blood and brain tissue.

However, stem cells can also be found in some tumors. These so-called cancer stem cells exhibit self-renewal and differentiation capacity, and are thereby essential components of tumor propagation and differentiation. Unlike normal stem cells, cancer stem cells exhibit a dysregulated and aberrant proliferation. These different types of stem cells and their application are discussed more in details in the following sections of introduction.

1.1.EMBRYONIC STEM CELLS

Stem cells are “undifferentiated” cells with two unique characteristics: unlimited cell division to make more stem cells (self-renewal) and the capacity to differentiate into mature specialized cell type of the body (potency). During fertilization, the cell that directly results from the union of egg and sperm results in a unicellular zygote, which has the highest potency and can give rise to 220 cell types in mammalian organism. These are the only cells that can give rise to the entire cells of the whole embryo (totipotent), extra embryonic cells and

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placenta. During the embryonic development, the stem cells from the inner cell mass are able to differentiate into all somatic cells of the multicellular organism (pluripotent) (Figure 1.A).

Throughout the embryonic development, cells progressively lose their pluripotency potential and become multipotent and specialized into three main germ layer of the body: endoderm, ectoderm and mesoderm and eventually give rise to mature cells of the body (unipotent).

In 1981, scientific groups successfully isolated for the first time mouse embryonic stem cells from the embryonic blastocyst stage^1,2. These cells can be cultured in vitro for an unlimited time under specific condition (Figure1.B).

Figure 1. Schematic representation of stem cell plasticity and differentiation. A) Totipotent cells are the first cells generated by the fusion of a single sperm with an egg cell. During early stage of embryonic differentiation (blastocyst), pluripotent stem cells are formed in the inner cell mass (3-5 days old embryo). These cells can be cultured in vitro and similarly to the in vivo situation, they are able to differentiate into all cell types of the entire organism. B) During the postnatal life, multipotent stem cells are present in the adult body, and can replace dead differentiated cells. Some mature adult cells (e.g. fibroblast) can be genetically modified by transfection with defined pluripotency related transcription factors, which lead to de-differentiation into a stem cell state. These cells are termed induced pluripotent stem cell and can be cultured in vitro.

Zygote Totipotent

Pluripotent Embryonic

stem cell

Multipotent Adult

stem cell

Unipotent Mature

cells

Differentiation

Embryonic stem cell

Induced pluripotent stem cells in vitro

1"

2" 3"

4"

reprogramming

A" B"

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1.2. REPROGRAMMING AND INDUCED POTENCY

During embryonic development, each cell presents different epigenetic characteristics.

Thus, these differences in epigenetic pattern correlate with cell fate decision and differentiation potential. For example, what makes a neuron different from a fibroblast is based on its specific gene expression profile. The transcriptome is regulated by epigenetic modification during development to determine the fate of a cell in a specific tissue.

In 1957, the developmental biologist, Conrad Hal Waddington invented a developmental concept that he called “epigenetic landscape” (Figure 2). This landscape consists in hills and valleys, where the different cell populations roll like marble balls. The fate of a cell population would reside where the balls would stop rolling, i.e. at the bottom of the valleys. By analogy with a ball which takes the path of a valley cannot move to another one, the more the cells will be differentiated and the more they lose potency. By moving the balls to the top of the hills, it could be possible to reprogram the cells by changing their gene expression profile to convert them to stem cell and eventually differentiate into a new cell population (at the bottom of another valley)³. During the last decades, scientist have confirmed Waddington intuition by demonstrating that under certain experimental conditions, cell differentiation can be converted and reprogrammed back into a less differentiated state.

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Figure 2. Waddington's epigenetic landscape model (modified). According to this model, cells are like marble balls rolling from the top of hills to reach their final differentiation fate when they stop rolling at the bottom of the different valleys. A differentiated cell type cannot therefore change fate and differentiate into another cell type. The only possibility is to carry the ball back to the top of the hill.

1.2.1. NUCLEAR TRANSFER

Nuclear transfer (NT) was the first strategy developed to assess whether the nuclear environment of embryonic state is different that the one of differentiated state. Nuclear transfer was developed in 1952 in a study by Briggs and King who described a nuclear reprogramming of adult somatic cells by nuclear transfer experiment with frog eggs Brigg 1952. Subsequently in 2004, studies demonstrated successful generation of mammalian pluripotent embryonic stem cells by somatic cell nuclear transfer (SCNT) from unipotent neurons and B cells^4-7.

These studies, demonstrated that the oocyte must contain environmental factors and nuclear proteins that can change gene expression pattern of adult somatic cells back to pluripotent stem cell state.

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1.2.2. DIRECT REPROGRAMMING

Since the first nuclear transfer and reprogramming experiment in 1952, remarkable progresses were made during the last decade in the field of somatic cell reprogramming. In the past years, a major breakthrough in the stem cell field consisted in the discovery of a new technology using genetic manipulation. Based on the observation that embryonic cells have specific transcription factors, which are potent inducers of cell fate towards an embryonic stage, Kazutoshi Takahashi and Shinya Yamanaka described in 2006 an artificial reprogramming strategy. They selected four transcription factors Oct4, SOX2, Klf4 and c- Myc and expressed them by retroviral transfection into somatic cells. These four genes were sufficient to reprogram adult mouse fibroblast cells into an ES stage. This technique allows the generation of artificial pluripotent stem cells from adult somatic cells^8,9. The generated cells were named “induced pluripotent stem cells (iPSC)” and can be cultured in vitro under specific condition (Figure 1.B).

During the past years the process of iPSC cells generation was challenged. The first generated iPSCs were similar but not identical to ES cells and therefore called iPSC-like ES.

For example, in generated iPSCs the transcription and epigenetic pattern was reset partially when compared to ES cell state and were therefore partially reprogrammed. Moreover, since overexpression of c-Myc can cause tumorigenesis it was shown that because of the reactivation of this transcription factor, many iPS cells developed into tumors. These observations raised the question of changing the transcription factors to improve the efficiency of reprogramming. Eventually, several combinations of transcription factors were tested which revealed that not all four transcription factors are required for reprogramming of somatic cells in to ES cells. Several investigations revealed that the transcription factors Klf4 and c-Myc, can be replaced by NANOG and the RNA-binding protein LIN28 to increase the efficiency and safety of human somatic cells reprogramming^10-12. After this reprogramming,

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somatic cells lose their phenotype and show pluripotency characteristics just like human embryonic stem cells, namely self-renewal and the ability to differentiate into all three germ layers: ectoderm, endoderm and mesoderm as they can induce the formation of teratoma after injection into immunodeficient mice¹³. This has been a main breakthrough allowing the study in particular of human genetic disease at the cellular levels after differentiation in vitro in the desired cell type^8,9.

Since the discovery of human iPSCs, they have been extensively used to study a variety of human disease and used for therapeutic approaches. Since 2007, iPSCs have been produced from various cell types including liver¹⁴, stomach¹⁵, keratinocyte¹⁶, blood cells¹⁷ and brain^18,19.

1.2.2.1. IPSC IN MODELLING CNS

ES-like properties of iPSCs such as self-renewal and their differentiation capacity represent a precious tool for the study of basic applied human biology. Although animal models have been traditionally useful to understand certain aspects of human disease, they have several shortcomings such as species-specific differences and other issues, such as non- physiological levels of human transgene expression. Such issues are partially solved by modeling human disease by using stem cells in vitro²⁰. Reprogramming of human somatic cells with transcription factors offers a tremendous potential to study patient specific iPSC.

Unique characteristics of iPSC allow the study of pathological pathways at the level of a single cell population rather than the complete organism and represent an unlimited source of cells to either model specific diseases or to be used for regenerative medicine.

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In 2008, a first feasibility study of the approach was reported for iPSC lines generated from skin fibroblast of patients affected with inherited genetic disorders including Parkinson’s disease (PD), Huntington’s the disease (HD), Duchenne (DMD) and muscular dystrophy (BMD), adenosine deaminase deficiency-related severe combined immunodeficiency (ADA- SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease type III, type 1 diabetes mellitus, Down syndrome (DS/trisomy 21), juvenile-onset and Lesch-Nyhan syndrome. However, in this study, differentiation of these disease specific iPSCs lines into neuronal subtypes has not been examined¹³.

Since then, induced pluripotent stem cells (iPSC) were differentiated in vitro into specific neuronal types for modeling neurological disorders using somatic cell reprogramming from patients suffering from Huntington disease (HD)²¹, Parkinson disease (PD)²², Alzheimer disease (AD)^23,24, Amyotrophic Lateral Sclerosis (ALS)²⁵, Spinal Muscular Atrophy (SMA)²⁶, Familial Dysautonomia²⁷ and Timothy Syndrome²⁸. Eventually autologous cell- transplantation based therapy could be performed with specific induced pluripotent stem cells from patients. For example in a neurological disorder like Parkinson’s disease, iPSCs could be generated from the patient, differentiated into dopaminergic neurons, and be transplanted back into the brain to replace affected neurons²⁹.

However the main application of disease specific iPSCs generation is to differentiate them into the specific cell type that is affected in the disease also in order to identify the cellular phenotype related to the pathology. In some instances, disease specific iPSCs are not only generated to study the phenotype but also to be used to replace damaged cells in the disease: dopaminergic neurons for Parkinson’s disease (PD), motor neurons for amyotrophic lateral sclerosis (ALS), cholinergic neurons for Alzheimer’s disease (AD) and median spiny neurons in Huntington’s disease (HD). However, using iPSC for disease modeling in late- onset neurological diseases such as HD, PD and ALS have been shown to be challenging³⁰.

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Great advances have been achieved in all these diseases by using neurological disease specific iPSC lines.

IPSCs have shown great potential also to model early-onset neurodevelopmental diseases including schizophrenia^31,32, Rett syndrome^33,34 and Down syndrome/trisomy 21. In such neurodevelopmental disorders, abnormal and/or delayed neuronal differentiation is observable during the process of in vitro neuronal differentiation. Thus, it allowsstudying the molecular mechanisms underlying the defective neuronal development³⁵. There is a wide range of protocols which have been developed to generate a different differentiated cell types of the central nervous system including neuronal progenitor cells, neurons, astrocytes and oligodendrocytes (Figure 3). Disease specific iPSCs offer an obvious and valuable approach as a platform for drug testing and screening for potential therapeutic agents. Furthermore, the potential of disease specific iPSC to differentiate into different cell types derived from the same donor (e.g. neurons and cardiomyocyte) benefit the field of therapeutic and toxicological response in different cellular types. Generation of disease specific human iPSCs benefits also population studies and personalized medicine.

In addition to differentiation into desired neuronal subtype in culture, neuronal functionality and connectivity patterns can be evaluated by measurement of synapse plasticity, electrophysiological analysis and single cell expression level of synapse related proteins. For example, in a comparison between Down syndrome iPSCs and his monozygotic non-affected twin brother defects in neuronal architecture and density (e.g. measured by neuronal differentiation stage markers) and neuronal functionality (e.g. by quantification of synapse related marker such as synaptophysin) were identified³⁶.

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Figure 3. Modeling CNS diseases in vitro by using iPSCs. Adult somatic cells can be reprogrammed to exhibit stem cell properties. These reprogrammed pluripotent cells can be differentiated into neural progenitors and their derivative cells such as mature neurons and glial cells such as oligodendrocytes and astrocytes. Figure taken from³⁴

1.2.2.2.IPSC TO STUDY REDOX PATHWAYS IN CNS DISEASE Oxidative stress and altered ROS metabolism are almost invariably observed during disease progression of various neurodegenerative disorders (e.g. ALS, AD and PD) and neurodevelopmental disease (e.g. schizophrenia) to^37,38. However, the direct mechanisms correlating deleterious ROS levels with impairment of neural cells are unknown. Thus, to find efficient antioxidant therapeutic approaches, it is important to understand precisely regulatory mechanism of ROS generation during progression of disease, and how ROS can induce cellular modifications such as DNA and RNA damage, mitochondrial and protein dysfunction and lipid peroxidation.

A few studies have used human iPSCs to study the role of ROS in neurological disease³⁹. Increasing evidence describes schizophrenia as a neurodevelopmental disease characterized by alteration in neuronal circuits and neurocognitive deficits⁴⁰. Oxidative stress is strongly linked to pathological aspects of schizophrenia including aberrant mitochondrial function, inflammatory response, abnormalities of oligodendrocyte, and the impairment of fast-spiking interneurons^37,38.

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The scientific group of Paulsen generated human iPSCs from patients affected with schizophrenia and studied the role of ROS metabolism during in vitro neuronal differentiation. A twofold higher oxygen consumption was measured in the neural precursor cells (NPC) derived from schizophrenic patients compared to controls. This increased oxygen consumption was associated with increased ROS generation which could be reverted by the mood stabilizer valproic acid⁴¹.

Taken together, human iPSC allow studying metabolic changes such as ROS associated to different pathological stages of neurological disease in various neural cell types.

Thus, these investigations lead to a better understanding of disease development and emphasizing potential therapeutic targets for treatment and preventive medicine.

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1.3 ADULT STEM CELLS

A proportion of pluripotent stem cells exist during the adult life. After birth, some tissues retain multipotent stem cells, which are called “adult stem cells”. These cells are tissue-restricted progenitors that maintain the function of adult organs by replacing the cells that are lost due to normal cellular function such as injury or apoptosis. Adult stem cells have been discovered in several postnatal organs including the brain (neural stem cells) and the blood (hematopoetic stem cells)⁴².

1.3.1. ADULT NEUROGENESIS

Adult brain harbors two restricted regions of neurogenic zone; the subventricular zone (SVZ) and the dentate gyrus (DG). These regions contain populations of quiescent neural stem cells (NCSs) that differentiate into neural progenitors upon cellular stimulation.

However, a proportion of neural stem cells undergo differentiation through asymmetric cell division and a proportion within the neurogenic zone remains quiescent. The NPC cells in the dentate gyrus generate dentate granule neurons in the hippocampus and the progenitors from subventricular zone are projected to olfactory bulb to differentiate into mature neurons (Figure 4).

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Figure 4. Schematic representation of adult neurogenesis. A) Adult neurogenesis occurs in two restricted areas of the brain; the dentate gyrus of the hippocampus and the subventricular zone. B) During the path of neurogenesis and neural differentiation, quiescent neural stem cells (NSCs) which can give rise to amplifying neural stem cells which then differentiate into neural precursors and eventually new born neurons.

1.3.1.1. PHYSIOLOGICAL ROLE OF ROS: FOCUS ON NEUROGENESIS

Oxygen (O2) is a developmental morphogen during neural differentiation, which may regulate diverse pathways controlling cell fate decision. During early stage of neuronal development, low oxygen level regulates diverse pathways of neural differentiation⁴³. Thus, the oxygen availability is extremely important since it regulates genes that control physiological conditions. In addition to the levels of O2, ROS are also key players of

Dentate Gyrus sub-ventricular zone

quiescent

NCSs amplifying

NSCs neural

precursor cells new born neurons

Adult neurogenesis

Cell types

A"

B"

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physiological condition of the CNS. However the exact mechanism relating oxygen level to ROS generation is not quite clear, as it has been demonstrated that low oxygen level stimulates ROS generation⁴⁴. This is rather paradoxal since ROS directly derive from oxygen, but it is generally accepted that this is due to a shunt in normal oxygen metabolism towards ROS generation.

Recent evidence indicates that a certain level of ROS generation is a key regulator of different pathways during neurodevelopment^45-48. Neural maturation-related factors like neuregulin (NRG), nerve growth factor (NGF), fibroblast growth factor 2 (FGF2) and retinoic acid (RA) increase ROS generation^49-52.

In postnatal brain neurogenic niches, quiescence and differentiation of neural stem cells are regulated by partial pressure of oxygen (pO2) and ROS generation. In vivo studies have detected higher ROS redox status in the SVZ zone of adult rodent brain, which could be related to the proliferation properties of neural stem/precursor cells within the neurogenic niche. Moreover, administration of the specific growth factor brain derived neurotrophic factor (BDNF) leads to higher ROS generation in NPCs. Controversially, a lower proliferation rate of NPC is observed with the withdrawal of two main growth factors epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2)⁴⁶.

Recently, Walton and colleagues described an oxidative stress status in vivo of dividing proliferating cells within both SVZ and DG neurogenic regions. They measured a high ROS generation during a transient stage of exercise-induced neurogenesis. They also identified an elevation in oxidized DNA and peroxidation of lipids as well as induction of oxidation-responsive genes in exercise-induced model of hyperactive hippocampal

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neurogenesis. These observations strongly suggest a neurogenesis-associated oxidative stress, which is not deleterious to the cells but rather regulates self-renewal and proliferative properties! of neural precursor cells⁵³.! In vitro neurodevelopmental models using neuroblastoma cell line have highlighted ROS as a key factor for neuronal differentiation and decreasing ROS generation restrains neural differentiation⁵⁴.

Taken together, these recent observations reveal the important role of ROS in neural differentiation and even more precisely at the early stage of neural differentiation when the cells exhibit higher proliferation rate. The exact source of these regulatory ROS and their targets remain to be identified.

1.3.1.2. USING IPSC FOR UNDERSTANDING CNS PHYSIOLOGY

As stated earlier, a main focus of stem cell biology is to generate human cellular systems to recapitulate specific diseases in vitro. However, in order to successfully mimic the in vivo condition it is essential to understand normal tissue physiology, such as for example the metabolic environmental factors, which are necessary for normal neurogenesis and neural differentiation. The studies described above have shown that stem cells can be used to study not only disease specific pathological aspects of oxidative stress-mediated metabolic changes but also how low oxygen and certain level of ROS generation represent physiological factors associated with neurogenesis.

! Identification of cellular environmental factors to improve neuronal differentiation and maintenance of neural stem/progenitor cells in vitro is key for human stem cells biology. To this extent, several studies were designed to modulate levels of O2 and ROS in stem cells culture and assess their neural differentiation.

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Stacpoole and colleagues, have studied differentiation culture of neural precursor cells derived from human embryonic stem cells using either 3% O2 (hypoxia) or 20% O2

(hyperoxia). This study revealed an enhanced survival rate of NPCs in hypoxic conditions in comparison to 20% environmental oxygen. In addition, the neural differentiation condition with 3% O2 resulted in a twofold increase of motor neuron precursors defined by the marker OLIG2 (encoding oligodendrocyte lineage transcription factor 2). These finding represent a step towards recapitulation of appropriate environmental factors impacting neural differentiation, and thus it may represent a significant advancement in disease modeling and cell-based therapies⁵⁵.

Additionally, the group of Le Belle and colleagues described an in vitro neural differentiation study in which NPCs derived from human embryonic stem cells were cultured under different concentration of exogenously ROS (H2O2). They defined two NPCs populations with low level of ROS (ROS ^lo) and high level of ROS (ROS ^hi) in which they demonstrate a higher NPCs proliferation and self-renewal rate in ROS ^hi in comparison to ROS ^lo 46.

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1.4. CANCER STEM CELLS

Cancer is normally initiated by a mutation in regulatory mechanisms that control cell division and proliferation, signal transduction pathway and DNA damage (oncogenic mutations). Under normal conditions, cells with abnormal mutations are eliminated from the cell replication cycle, however mutations may accumulate at some low incidence and initiate cancer. Malignant tumors contain cells with functional heterogeneity with different proliferation and differentiation abilities. A defined subset of cancer cells display stem cell characteristics including self-renewal and differentiation into all types of cells found within cancers. These cells are called cancer stem cells (CSC). They are characterized by self- renewal and differentiation into malignant progenitors. These unique properties allow the preservation of cancer stem cell pool within the tumor and causes malignant spreading of the tumor, invasion into surrounding healthy tissue and new tumor site formation. For this reason, various therapeutic approaches hold hope for cancer treatment by targeting proliferative cancer stem cells⁵⁶.

Cancer stem cells have been reported in most of the human tumors and can be identified and isolated from the patient biopsy by using approaches such as fluorescence activated cell sorting (FACS) for specific surface markers^57,58 and can be cultured in vitro.

The isolation of cancer stem cells and in vitro culture for various cancers, including brain tumors, opened novel perspectives in the field of cancer biology and related mechanism.

During the 80s, the group of Mina Bissell was one of the first to underline the importance of developing three-dimensional based models in vitro in order to more closely recapitulate the multicellular physiological tumor environment. This was defined as “tumor engineering”⁵⁹. Her rationale was that “cancer is not a disease of single cells but rather a problem of the organs". Accordingly, three-dimensional-based in vitro models have been

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developed to study a variety of solid tumors, including glioblastomas. This type of models provides important insights into tumor biology such as cell-cell interaction, cellular differentiation and tissue re-organization, synthesis of extracellular matrix (ECM) and cell- matrix interaction⁶⁰. These are the so called “multicellular tumor spheroids” which consist of a three-dimensional arrangement of mixed tumor cells and extracellular matrix. Once cancer stem cells are grown above a certain size, a characteristic cellular layer structure is formed due to gradient supply of oxygen and nutrients. This results in a characteristic cytoarchitecture which is composed of an outer layer of proliferating cells, a middle layer of quiescent viable cells and a center of dead necrotic cells (Figure 5)⁶¹.

This method to recapitulate the tumor in vitro has been extensively used for diverse tumors, including glioblastoma, which is discussed in the next section.

Figure 5. Multicellular spheroid growth. Tumor stem cells can be grown in culture due to the high proliferation rate. Cell growth in spheroids results in formation of different layers due to gradient supply of necessary factors for cell proliferation such as oxygen (O₂) and nutrient. This results in an outer layer of proliferation cell, a middle layer of viable cells and eventually a necrotic center.

O₂ and nutrients CO2 and waste

quiescent viable cells proliferating cells

necrotic cells Cancer stem cell

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1.4.1. GLIOBLASTOMA BRAIN TUMOR

FACTS ABOUT GLIOBLASTOMA

Many brain tumors develop from glial cells in the brain and are therefore termed “gliomas”.

According to their severity, brain tumors are categorized into different grades. Grade I (pilocytic astrocytoma) and grade II (astrocytoma) are relatively sensitive to treatment whereas grade III (anaplastic astrocytoma) and grade IV (glioblastoma) tumors are more resistant to therapy and more lethal^62,63. Glioblastoma are the most common form of primary brain tumors with low survival rate after diagnosis and extremely rare recovery after treatment. Glioblastoma belong to one of the severe tumors of the central nervous system (CNS). It is the second leading cause of cancer death of adolescents and for unknown reasons it is more common in males than in females^64,65. According to the world health organization, the incidence rate of glioblastomas is 3-4/100,000 person-years, which varies between different world regions. The incidence rate is higher in developing and industrial countries such as Europe and the United States (3.3/100,000 person-years) than in Asia and Africa (1.6/100,000 person-years) (Figure 6). In children, brain tumors are the reason for one fourth of all cancer deaths^66,67.

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Figure 6. World map of glioblastomas incidence. Map representing the glioblastomas incidence for males in different regions of the world (incidence rate per 100000). Map adapted from human cancer database http://www.pubcan.org/index.php.

The exact reason why such brain tumors are more common in certain countries and among specific ethnic groups is not clear. However, it has been suggested that the reason is in fact due to the fact that diagnosis is more efficient in developed countries such as Northern Europe and United State in comparison to countries such as India and African countries. This may lead more reported cases for brain tumor in developed countries. However, there are also evidences that some differences among ethnic groups play a role in brain tumor for example, in the United States Caucasians have a higher rate of glioma than Americans of African origin. However, the exact etiology underlying these differences remains unknown.

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Different types of brain tumor have been reported according to age. The average age for diagnosis with primary brain tumor is 53 years while this moves to 62 years for high-grade brain tumor such as glioblastoma. As for other types of cancer, increased incidence in elderly people might be due to vulnerable immune system leading to lower efficacy in protection against the disease⁶⁸.

Most gliomas are sporadic. Glioblastoma have been associated with several environmental factors such as viruses including simian vacuolating virus 40 (SV40)⁶⁹, human herpesvirus 6 (HHV-6)⁷⁰ and cytomegalovirus⁷¹. There is a connection between glioblastoma and malaria. It has been suggested that Anopheles mosquitoes the carriers of malaria can transmit some virus favoring glioblastoma development⁷². There are also connections between glioblastoma and alcohol consumption⁷³ as well as ionizing radiation⁷⁴.

DIAGNOSIS AND TREATMENT

The environment in which these tumors grow and the interaction they have with the host environment makes current treatments for brain tumors challenging. The diagnosis is usually performed by magnetic resonance imaging (MRI) for tumor detection upon appearance of brain tumor symptoms such as chronic headache, seizure, loss of consciousness and motor sensory function. Following diagnosis, surgical resection of the tumor is performed by a skilled neurosurgeon. The tumor is generally dissected with extra tissue surrounding the primary tumor side in order to remove all glioblastoma tumor cells⁷⁵. In order to remove the entire tumor, surgical resection is coupled with chemo-and radio-therapy⁵⁸.

After surgery, patients are treated with chemotherapeutic agents locally at the resection site such as Carmustine® ⁷⁶ or by orally applied systematic chemotherapy such as temozolomide (TMZ)⁷⁷. Post-surgical chemotherapeutic treatments have significantly

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increased the survival time of glioblastoma patients. Another frequently used therapeutic approach is radiotherapy, which is used in almost 50% of all cancers. In case of glioblastoma, gamma rays are directed to the side of tumor to generate direct DNA damage. However this is not without risk as further damage can occur due to free radicals formation⁷⁸. During and after the treatments, glioblastoma patient are regularly monitored for recurrence. Although improvements have been made in glioblastomas monitoring, no major therapeutic advances have been made in the case of high-grade glioblastoma patient survival. This is due to malignant tumorigenic properties of glioblastoma cells during the interaction with normal brain tissue, which affects the implementation of therapeutics^79,80. Due to difficulties in treatment, the average survival rate for glioblastoma after diagnosis is much lower (35%) than the survival rate of non-central nervous system cancers such as breast tumor (89%) and prostate (100%)⁸¹.

RECAPITULATING GLIOBLASTOMA USING CANCER STEM CELLS

Similar to other solid tumors, glioblastoma is multicellular and heterogeneous tumors composed of malignant tumor cells and a small proportion of cancer stem cells which are critical therapeutic targets since they are highly resistant to radiotherapy and chemotherapy⁷⁶.

Important pathological aspects of glioblastomas involve the interaction of tumor cells with their environment such as invasion, formation of secondary foci, inflammation and angiogenesis. Alteration of extracellular matrix properties, interaction with stromal cells, hypoxia and angiogenesis (for blood supply) are hallmarks of glioblastoma tumors, which all play roles in the malignant progression of the disease^82-85. These unique characteristics lead to diffuse growth and invasion of tumor cells into the surrounding brain tissue⁸⁶.

Various in vitro and in vivo models have been developed to study malignant aspects of

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glioblastoma pathogenesis. However, one major challenge of existing models used to study glioblastomas tumorigenesis in vitro and in vivo is how closely they are able to mimic major aspects of tumor growth in patients. Diverse in vivo and in vitro models have been generated to study malignant aspects of glioblastomas such as invasion. In the in vivo models, xenografts of human tumor cells are implanted or injected into immunocompromised animals^87-89. Recent models of glioblastomas involve genetically alterations in mice models leading to initiation glioblastomas tumor development⁹⁰. Even though these models have generated important information regarding glioblatoma and have been extensively used, they have species-specific limitations, affecting the experimental outcome. Apart from animal models, one commonly used experimental setting in vitro is monolayer culture of glioblastoma cell lines. This cellular model is mostly used to study the invasion of glioblastomas and related molecular mechanisms. Two dimensional monolayer cultures of cell lines have also been developed, however these cells are usually adherent leading to a lack of intact cell connection in the three dimensional microenvironment. Thus, although much has been learned from these models, they have limitations, as they do not mimic the patient environment, as the tumor-host tissue interactions are not recapitulated.

As stated in the previous section, the main aim advantage of three-dimensional culture models is to recapitulate the pathological cellular composition of cancer in vitro. Over the last years glioblastoma patient biopsy were dissected and primary glioblastomas tissue can be kept in culture where they form 3-D spheroids, which freely float in the medium without adhering to the surface^91-93. The advantages of using such a model in glioblastoma research is that the native genotype and karyotype is better preserved in isolated glioma stem cells in comparison to cell lines which accumulate aberrations during several passages⁹⁴. Moreover, by using patient specific cancer cells, different therapeutic approaches and drug response in an individual patient could be assessed, which is a key step forwards personalized medicine⁹⁵.

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2. HYPOTHESES AND AIMS OF THE PROJECT

Most of the neurological studies in humans are based on post mortem tissue or brain imaging. This leads to numerous biases in the analysis: postmortem tissue represents most of the time the final stage of the disease, collected tissue is extremely heterogeneous in term of treatment, age and delay in collection after death among others^96,97. Post mortem tissue has limited use to study neurodevelopmental diseases and the molecular mechanisms underlying pathological progression of disease⁹⁸. To overcome these limitations, new relevant models are needed to study specific cell types affected by disease.

The main objective in this study was to use stem cells from different origins in order to study specific critical aspects of human CNS pathology and physiology in vitro. More precisely, we used pluripotent stem cells in two different projects: (i) generate a model of glioblastoma development and (ii) generate a model to study the oxidant-generating enzyme NOX2 during neuronal differentiation:

Project 1. In the first project, we used two types of stem cells to develop an in vitro model for glioblastoma development. For this, we used an in vitro co-culture system with glioblastoma brain tumor stem cells isolated from patients and embryonic stem cell derived-engineered neural tissue. Using this co-culture system, we studied the following specific aspects:

• Molecular identification of specific genes induced during tumor-host tissue interaction in vitro.

• Comparison of main tumorigenic and malignant pathological aspects of glioblastomas multiform in a three-dimensional co-culture system with human engineered neural tissue.

• Correlation of the gene expression profile during in vitro glioblastoma tumorigenesis and patient survival.

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Project 2. As discussed in the introduction, ROS has an important role in various CNS pathologies but also in physiological conditions. In the second part of the project we focused on the physiological role of ROS by specifically studying the ROS generating NOX2 enzyme during neural differentiation.

For this we used specific-iPSC from a NOX2-deficient patient with chronic granulomatous disease, a genetic disease due to a mutation in the CYBB gene coding for the oxidant generating NOX2 enzyme. We used these iPSC to study the role of NOX2 during neuronal differentiation. During this project we focused on the following aspects:

• Neuronal differentiation of iPSC lines in vitro.

• Comparing of disease specific iPSC lines (NOX2-/-) with control at different stages of in vitro neuronal differentiation.

• Address the potential role of NOX2-derived oxidants in vivo by focusing on adult neural stem cells in neurogenic rich brain regions of normal and genetically modified mice (NOX2-/-).

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FIRST PART

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Eram Garden, Shiraz

“The knowledge of anything, since all things have causes, is not acquired or complete unless it is known by its causes”

Avicenna (980 – 1037)

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DESCRIPTION OF THE STUDY; GENE EXPRESSION PROFILE ANALYSIS OF GLIOBLASTOMA SPHERES CO-CULTURED WITH ENGINNERED NEURAL TISSUE

In the field of glioblastoma research, in vitro models are extensively used to explore tumor biology. However the great challenge in developing such models is to determine how close they resemble the human glioblastoma. This is particularly important for tumor development and responses to chemotherapy and radiotherapy. For example the discovery of tumor stem cells⁹⁹ has allowed challenging specific aspects of tumor treatment. The in vitro tumor models based on gliobtastoma stem cells have shown that they are resistant to various tumor treatment strategies including the chemotherapy and radiotherapy used in glioblastoma patients¹⁰⁰. In addition these studies have revealed a direct correlation between glioblastoma stem cell growth in tumor lesion in vitro and poor clinical outcome and patient disease progression¹⁰¹.

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In the course of this thesis, we have used human stem cells to develop a three- dimensional in vitro model based on air-liquid interface culture method to study some of the aspect of glioblastoma development. For this, we have co-cultured glioblastoma spheroids derived from patients with an engineered human neural tissue (ENT) developed previously in our laboratory¹⁰². Glioblastoma spheroid cells were transiently transfected with tomato fluorescent protein (TFP) in order to track the glioblastomas cell development during the co- culture with ENT.

In the first part of this study, we aimed at the validation of the in vitro model and especially the development and survival of the tumor cells during the period of co-culture.

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Glioblastoma spheres were co-cultured with ENT for two weeks in vitro. During this period of co-culture, glioblastoma were developing within engineered neural tissue as evidenced by immunostaining for glioma markers and presence of TFP fluorescence. Histological sections at different time points during co-culture revealed an invasion phenotype of glioblastoma tumor cells into the ENT as well as secondary tumor side formation and presence of peritumoral necrotic cells. Our result showed that our glioblastoma co-culture in vitro model displays important histopathological hallmarks of glioblastoma attributed to therapeutic and disease progression!failure of therapies in the clinic^82,103-105.

In glioblastoma patients, malignant progression of tumor into healthy adjacent tissue involves molecular changes¹⁰⁶. Once the in vitro glioblastoma model was validated, we sought to investigate molecular changes occurring during the co-culture system using comparative cDNA microarray. For this we used the following conditions; glioblastoma spheres before and after differentiation on air-liquid interface, ENT alone and glioblastoma spheres co-culture with ENT for two weeks. This analysis revealed important transcriptional changes between the different conditions. Notably, the gene expression profile of the condition glioblastoma spheres co-culture with ENT was different from that of ENT alone or glioblastoma spheres alone suggesting transcriptional changes due to tumor-host interaction.

Fold change and functional analysis revealed a significant induction and up-regulation of regulatory genes belonging to type I interferon-signaling cascade specifically in the co-culture of glioblastoma spheres with ENT. These genes are involved in host defense and known to be downstream genes of the interferon-STAT-1 signaling cascade¹⁰⁷.

In order to confirm that the observed changes in gene expression is specific for glioblastoma development into ENT, the same culture conditions were used for breast carcinoma tumor cell co-culture on ENTs. A panel of induced and up-regulated genes was

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selected and their expression was assessed by Q-PCR for glioblastoma co-culture with ENT and breast carcinoma co-culture with ENT. This analysis confirmed that the identified profile in gene expression is restricted to glioblastoma sphere interaction with host tissue in vitro and is not induced by another type of solid tumor. Interestingly, this panel of interferon genes signaling has been identified as predictors of glioblastoma resistance to radiotherapy^108,109 and poor patient survival¹¹⁰.

The gene expression changes observed in glioblastoma-ENT co-culture were compared to data from cancer genomic atlas dataset. The significantly up-regulated genes fell into three main functional groups; i) interferon response related genes, ii) genes related to extracellular matrix and iii) cholesterol-related genes. Notably, genes related to interferon and cholesterol-related genes showed significant correlation with glioblastoma patient survival.

Taken together, development of glioblastoma within engineered neural tissue in vitro, could be used as a relevant model to study gene expression mechanism related to tumor-host interaction. This study is described in detail in a research article published in Biomaterial¹¹¹ and reported hereafter.

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RESEARCH ARTICLE 1

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