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Université de Montréal

Dérivation de cellules souches pluripotentes induites autologues à partir du clonage somatique équin

Par OLIVIA EILERS SMITH

Département de biomédecine vétérinaire Faculté de médecine vétérinaire

Mémoire présenté à la Faculté de médecine vétérinaire en vue de l’obtention du grade de

maître ès sciences (M.Sc.)

en sciences vétérinaires option Reproduction

Décembre 2013

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Résumé

Le recours aux cellules souches pour améliorer la réparation et guérison des blessures et maladies musculosquelettiques chez le cheval est de plus en plus fréquent. Les développements récents dans la reprogrammation cellulaire ont permis le développement de nouvelles sources de cellules souches pour ces thérapies régénératives. Des cellules souches pluripotentes induites (iPS) autologues peuvent être dérivées de cellules adultes par la reprogrammation directe à travers l'expression induite des gènes de pluripotence. Le clonage par transfert nucléaire (SCNT) suivi de la dérivation de cellules souches embryonnaires (ES) permet la reprogrammation indirecte des cellules adultes. Cependant, l’efficacité de ces deux méthodes pour la dérivation de cellules pluripotentes génétiquement stables est faible. Nous avons donc combiné les techniques SCNT et iPS dans le but de développer un protocole efficace de dérivation de cellules iPS autologues à partir de fibroblastes de la peau équine. Quatre facteurs de reprogrammation ont été introduits dans les cellules fibroblastes de fœtus clonés (ntFF) ainsi que les cellules ES provenant d’embryons clonés (ntES) pour induire leur reprogrammation en cellules iPS autologues. Les cellules ntFF-iPS et ntES-iPS ont des capacités prolifératives avancées et expriment des marqueurs de pluripotence importants. Par contre, les cellules ntES ont une efficacité de reprogrammation significativement supérieure aux cellules nt-FF et forment des colonies trois fois plus rapidement. Contrairement aux cellules ntES, les cellules ntES-iPS démontrent une augmentation de l’expression des marqueurs de pluripotence et survivent à la culture cellulaire prolongée. Les résultats présentés dans ce mémoire attestent que l’utilisation de la reprogrammation secondaire de cellules FF et ES clonées permet la production de cellules souches pluripotentes autologues stables chez le cheval.

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Abstract

For veterinarians, regenerative medicine in horses has focused mainly in the use of stem cells for arthritis, tendon and ligament repair, indicating a need for treating musculoskeletal injuries. The recent developments in cell reprogramming have paved the way for alternative cell sources for stem cell therapies. Autologous pluripotent stem cell lines can be derived from adult cells either by direct reprogramming through induced expression of pluripotency genes (iPS) or indirectly by reprogramming through somatic cell nuclear transfer (SCNT) followed by the derivation of embryonic stem cells (ESC). However, outcome efficiencies of SCNT and iPS protocols are invariably low, indicating that alone neither of these reprogramming routes is sufficient for deriving genetically and epigenetically stable pluripotent stem cells. We hereby report on the production of autologous equine iPS cells by combining SCNT and iPS reprogramming protocols. Adult skin fibroblasts were used for SCNT, and the resulting cloned embryos were either used to obtain cloned fetal fibroblast cells (ntFF), or used for ESC culture (ntES). Cells were then transfected with reprogramming factors to derive autologous iPS cells. Both ntFF-iPS and ntES-iPS cells are capable of extensive proliferation and express important pluripotency factors. However, ntES reprogramming efficiency is significantly higher than ntFF cells, with ntES-iPS colonies forming three times faster. Additionally, ntES-iPS cells showed improved pluripotency marker expression when compared with ntES cells. The results presented in this memoir indicate that stable equine iPS cell lines may be readily obtained from secondary reprogramming of cloned ntFF and ntES cells, opening novel avenues for developing autologous pluripotent stem cell therapies.

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Table des matières

Résumé ...………... i

Abstract ...………. ii

Table des matières ………... iii

Liste des tableaux ………. v

Liste des figures ……….. vi

Liste des sigles et abréviations ………... vii

Remerciements ……… x

Introduction …….………..………. 1

Chapitre 1 : Recension de la littérature ………...……… 4

1.1 Article 1 ……… 4

1.2 Technique de reprogrammation par induction à la pluripotence (iPS) ……….. 16

1.2.1 Sélection du type de cellule à reprogrammer ……… 16

1.2.2 Sélection des facteurs de reprogrammation ………... 17

1.2.3 Sélection de la méthode de livraison ………. 18

1.2.4. Sélection des facteurs à ajouter au milieu de culture ………... 18

1.3 Phases de reprogrammation ……….. 19

1.4 Reprogrammation secondaire ……… 20

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Chapitre 2 : Hypothèse et objectifs ………...……….. 23

2.1 Hypothèse ……… 23 2.2 Objectifs ……….. 23 2.3 Conception expérimentale ……….. 23 Chapitre 3 : Article 2 ………..………. 25 ABSTRACT ………. 27 INTRODUCTION ……… 28

MATERIAL AND METHODS ………... 30

RESULTS ……… 37 DISCUSSION ……….. 41 ACKNOWLEDGEMENT ………... 46 REFERENCES ……… 47 FIGURE LEGENDS ……… 52 TABLES ………... 54 FIGURES ………. 55 Discussion ……..…...………..……... 61 Conclusion. ……..…….………..……... 67 Bibliographie. ………..…….. 68

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  v  

Liste des tableaux

Chapitre 3

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  vi  

Liste des figures Chapitre 1

Figure 1: Overview of the pluripotent stem cells used in equine regenerative médicine. …….. 7 Figure 2: Comparison of embryonic stem (ES) and induced pluripotent stem (iPS) cell

production for regenerative medicine in horses. ……… 11

Chapitre 2

Figure 1 : Dérivation de cellules souches pluripotentes induites (iPS) autologues de cheval... 24

Chapitre 3

Figure 1: Morphology of equine fetal fibroblasts and their iPS colonies. ……… 55 Figure 2: Comparative mRNA expression by qRT-PCR of Control ntFF and ntFF-eiPS cells for

the endogenous pluripotent genes OCT-4, Klf-4, NANOG and Sox-2. ……… 56

Figure 3: Morphology of equine ntES-eiPS colonies. ……….. 57 Figure 4: ntES-eiPS colony expression for pluripotent proteins NANOG, SSEA-1, SSEA-3,

SSEA-4, TRA-1-60 and Rex-1 by immunohistochemical staining. ……….. 58

Figure 5: Comparative qRT-PCR analysis of ntES and ES-eiPS cell mRNA abundance of the

endogenous pluripotent genes OCT4, KLF4, NANOG and SOX2. ……….. 59

Figure 6. OCT4 methylation pattern in control ntES and ES-eiPS cells. ……….. 60

Discussion

Figure 2 : Estimation du temps pour l’obtention de cellules souches pluripotentes induites (iPS)

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Liste des abréviations

2eRS Secondary reprogramming system Système de reprogrammation secondaire

AF Adult fibroblast

Fibroblaste adulte

ALK Anaplastic lymphoma kinase

Voie de la kinase du lymphome anaplastique

AP Alkaline phosphatase

Phosphatase alcaline

AT Adipose tissue

Tissu adipeux

AT-MSC Adipose tissue mesenchymal stem cell

cellules souches mesenchymateuses du tissu adipeux bFGF basic fibroblast growth factor

Facteur de croissance de fibroblaste de base

BM Bone marrow

Moelle osseuse

BM-MSC Bone marrow mesenchymal stem cell

Cellules souches mesenchymateuses de la moelle osseuse

DNA Deoxyribonucleic acid

Acide désoxyribonucléique

mtDNA Mitochondrial DNA

ADN mitochondriale

EB Embryoid body

Corps embryonnaire ESC Embryonic stem cell

Cellule souche embryonnaire

ntES nuclear transfer embryonic stem cell

Cellules souche embryonnaire de clonage par transfert nucléaire

FF Fetal fibroblast

Fibroblastes fœtales

ntFF nuclear transfer fetal fibroblast

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GFP Green fluorescent protein Protéine de fluorescence verte GSK Glycogen synthase kinase

Voie de glycogène synthase kinase HAT Histone acétyltransférase

ICM Inner cell mass

Masse cellulaire interne iPSC induced pluripotent stem cell

Cellule souche pluripotente induite

Jak Janus kinase

kinase Janus

Klf Krupple-like factor

Facteur Krupple-like LIF Leukemia inhibitor factor

Facteur inhibiteur de leucémie

MKOS Facteurs de reprogramation Yamanaka (c-Myc, Klf4, Oct4 et Sox2) MEK Mitogen-activated extracellular kinase

kinase activée par des mitogènes extracellulaires MEKK Mitogen-activated protein kinase kinase

protéine kinase kinase activée mitogéniquement MEF Mouse embryonic fibroblast

Cellules fibroblastes embryonnaires de souris MET Mesenchymal-to-epithelial transition

Transition mésenchymale-à-épitheliale MSC Mesenchymal stem cell

Cellule souche mésenchymateuse c-Myc Facteur de transcription c-myc

Oct 4 Facteur de transcription liant l’octamere 4

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PB Transposon piggyBac

PG Parthénogénèse

RNA Ribonucleic acid

Acide ribonucléique

REX Reduced expression protein Protéine d’expression réduite SCNT Somatic cell nuclear transfer

Clonage par transfert nucléaire d’une cellule somatique Sox2 (sex determining region Y)-box 2

Facteur de transcription de la région de determination Y, boîte2 SSEA Stage specific embryonic antigen

Antigène embryonnaire de stade spécifique Stat3 Signal transducer and activator of transcription 3

Transducteur de signal et activateur de transcription 3 SV40LT Simian Vacuolating Virus 40 Large TAg

Antigène Simian Vacuolating Virus 40 Large TAg SVF Stromal vascular fraction

Fraction des cellules vasculaires stromales hTERT human telomerase reverse transcriptase

Télomérase reverse transcriptase humain TRA Tumoral rejection antigen

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Remerciements

Ces travaux ont été réalisés grâce à l’aide, au support et à l’encouragement de plusieurs personnes qui ont marqué la durée de cette maîtrise.

Tout d’abord, un remerciement distingué au personnel du laboratoire du Dr. Smith et du Dr. Murphy, un groupe d’étudiants et de professionnels exceptionnels. Votre aide, votre générosité et vos connaissances m’ont été indispensables.

Au Dr. Murphy, je vous suis reconnaissante pour la liberté que vous m’avez accordée durant le développement de mon projet. Merci de m’avoir si bien accueilli dans votre équipe, de m’avoir enseigné et de m’avoir ouvert les yeux aux multiples possibilités de la science.

Je tiens à souligner le support et la sagesse qui me sont venus du Dr. Smith, un chercheur inspirant grâce à qui j’ai pu développer ma pensée critique et mon raisonnement scientifique.

J’aimerais aussi remercier Dr. Laverty et son équipe qui sont de merveilleux collaborateurs avec qui l’entraide et l’agrément étaient toujours présents.

De plus, je remercie l’organisme subventionnaire Morris Animal Foundation pour sa généreuse contribution au projet.

 

Enfin, je tiens aussi à remercier ma sœur Julia pour son aide indispensable lors de la correction de ce mémoire et ma mère Aparecida pour son support moral et sa capacité de toujours me garder motivée.

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Introduction

Les blessures et maladies musculosquelettiques, fréquentes chez les chevaux de course et de ferme, ont des conséquences importantes sur leurs carrières et leur qualité de vie. Ces problèmes ont donc un impact financier et sentimental non négligeable. La guérison de certains tissus musculosquelettiques, tels que les ligaments, tendons et le cartilage, est lente et peu efficace puisqu’ils perdent leur organisation structurelle et sont donc fonctionnellement déficients par rapport aux tissus originaux. Cette situation, qui est commune chez le cheval, prédispose l’animal à une récidive de la lésion initiale (Denoix and Pourcelot, 1997; Crevier-Denoix, et al., 1997). Les caractéristiques de cette guérison lente et incomplète font du cheval un excellent modèle animal pour étudier les problèmes de réparation musculosquelettique ainsi que pour expérimenter des nouvelles méthodes de thérapie chez l’homme (Frisbee and Stewart, 2011).

Jusqu’à récemment, les mesures pour le traitement de ces problèmes musculosquelettiques chez le cheval étaient palliatives plutôt que curatives. Par contre, depuis quelques années, de nouvelles approches thérapeutiques ont été développées, la majorité provenant de la médecine régénérative. Le principe de ces traitements vise à obtenir une réparation efficace du tissu en réalisant un stimulus apporté par des facteurs de croissance, des cellules souches ou d'autres substances. Ainsi, on espère obtenir une fonctionnalité tu tissu similaire à celle qui existait avant l'accident.

Chez le cheval, les études ont principalement été réalisées sur des cellules souches d'origine mésenchymateuse provenant de la moelle osseuse, du tissu adipeux ou de la veine

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ombilicale d’un fœtus à la naissance. L'injection de ces cellules souches dans les sites de blessures a démontré des améliorations significatives de la cicatrisation et du taux de récidive (Schnabel et al., 2008). Toutefois, l’accessibilité difficile à ces cellules, leur faible potentiel mitogénique et de différentiation lié à l'âge des donneurs ainsi que leur tendance à la dédifférenciation lors de cultures prolongées pour avoir le nombre de cellules ciblé limitent considérablement la capacité des cellules souches mésenchymateuses (MSC) à être utilisées comme thérapie de guérison fiable.

Contrairement au MSC, les cellules souches embryonnaires (ESC) sont une source de cellules souches pluripotentes, capables de prolifération illimitée et de différentiation en cellules des trois feuillets embryonnaires (Kingham and Oreffo, 2013). Même si des applications cliniques multiples sont développées chez les humains, les ESC des ongulés tendent malheureusement à se différencier spontanément après quelques passages in vitro, ce qui limite leur potentiel comme outil thérapeutique vétérinaire (Gandolfi et al., 2012).

En 2006, le groupe de recherche de Yamanaka au Japon a démontré la possibilité de reprogrammer des cellules somatiques différenciées en cellules souches pluripotentes induites (iPS) (Takahashi et al, 2006). Chez le cheval, les iPS ont été dérivées avec succès à partir de cellules fibroblastes adultes et fœtales, mais aucune étude n’a démontré la production de cellules iPS autologues (Nagy et al., 2011; Breton et al, 2012).

Ce mémoire est composé de deux parties : le chapitre premier présente une revue de la littérature portant sur les cellules souches chez les chevaux et les aspects techniques et cliniques de la thérapie cellulaire. Ensuite, le deuxième chapitre fait état des études portées sur la reprogrammation des cellules autologues de chevaux adultes, réalisée soit directement ou à l’aide

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du clonage par transfert nucléaire (SCNT). L’objectif principal de ce projet était de développer un protocole efficace de production de cellules souches pluripotentes induites autologues chez le cheval et d’étudier leurs propriétés pluripotentes.

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Chapitre 1. Recension de la littérature

1.1. Article 1 : Derivation and Potential Applications of Pluripotent Stem Cells for

Regenerative Medicine in Horses.

Publié en 2011

Revue Acta Scientiae Veterinariae Volume 39(Suppl 1) : s273-s283

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O.E. Smith, B.D. Murphy & L.C. Smith. 2011. O.E. Smith, B.D. Murphy & L.C. Smith. 2011. O.E. Smith, B.D. Murphy & L.C. Smith. 2011. O.E. Smith, B.D. Murphy & L.C. Smith. 2011.

O.E. Smith, B.D. Murphy & L.C. Smith. 2011. Derivation and Potential Applications of Pluripotent Stem Cells for Regenerative Medicine in Horses. ssssssssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s273 - s283.

I. INTRODUCTION II. ADULT STEM CELLS

2.1 Adult adipose tissue-derived stem cells 2.2 Bone marrow-derived stem cells III. EXTRA-EMBRYONIC STEM CELLS 3.1 Umbilical cord blood

3.2 Umbilical cord matrix

IV. EMBRYO-DERIVED STEM CELLS

V. INDUCED REPROGRAMMING OF ADULT CELLS VI. CONCLUSION

I. INTRODUCTION

Regenerative medicine is an emerging field of medicine focused on repairing and replacing damaged cells and tissues either by tissue engineering (producing organs in vitro and implanting them in a living organism), or by cell therapy (injecting undifferentiated cells to help stimulate restoration of diseased organs). In the last 20 years the new science consisting of molecular imaging and biotechnology have led to an explosion in the knowledge of the biological processes of the human body. The field of regenerative medicine has grown tremendously. Often, this involves harnessing the properties of stem cells, which are capable of self-renewal and differentiation into many other cell types. Stem cell research provides the basis for the development of future medical procedures in a broad range of human diseases enabling the regeneration of many tissues and organs, including muscles, bone, heart and nerve. When compared to human applications, the progress of regenerative medicine in veterinary medicine is in its infancy. For instance, one of the chief current therapies of human stem cell based regenerative medicine is to treat leukemia and other types of blood related cancer, requiring myeloablative chemotherapy followed by hematopoietic stem cell induced recovery of the immune system [26]. As mentioned above, these applications rarely apply to animals and they usually remain untreated for the sake of the animal’s welfare, as well as monetary reasons. However, being a relatively young and emerging field, stem cell research for human and veterinary medicine remain fundamentally attached to one another. For instance, animal models are used to study the properties and potential of stem cells for future human medicine therapies. Moreover, various stem cell treatments for animal patients are currently being developed and some, like the treatment of

equi-ne tendinopathies with mesenchymal stem cells (MSC), have successfully entered the market for this purpose [32].

Stem cells are generically defined as undif-ferentiated cells that are capable of self-renewal through replication as well as differentiation into specific cell lineages from at least one of the three germ layers [36]. Depending on the developmental stage and tissue from which they are obtained, they can be classified as embryonic, extra-embryonic or adult. There are three measures of potency used to describe levels of plasticity associated with the various kinds of stem cells. Totipotency is used for cells that can form all cells or tissues that contribute to the formation of an organism (ex: the fertilized egg or zygote). Pluripotency is for cells that can differentiate into most but not all cells lines of an organism (ex: embryonic stem cells). Lastly, multipotency can form a small number of cells/tissues that are usually restricted to a particular germ layer (e.g. hematopoietic or mesenchymal stem cells) [29].

Many sources of stem cells exist and when choosing the appropriate one for the effective, stable and long-lasting repair of damaged tissue, a few common criteria should be considered. First a sufficient number of cells must be generated in order to fulfill the treatment. Such cells must also be capable of differentiation towards the right phenotype, and remain in that state. Also, they should be structurally and mechanically compliant with the native tissue and successfully avoid immunological rejection. Finally, they should adopt the appropriate cellular organization with extracellular matrix production, with or without the presence of structural support, and be able to integrate completely with the damaged tissue.

Adult-derived stem cells are somewhat easily accessible, are found in various tissues of the living organism, and when used autologously do not require treatment to lower the risks of graft rejection. However, MSC have a low proliferative potential, making the production of a large number of cells difficult, and their differentiation is often restricted to a specific cell lineage. Embryonic stem (ES) cells, on the other hand, have unlimited self-renewing abilities as well as multilineage differentiation potential but their derivation is more complicated, requiring the production and destruction of embryos. Their clinical use is also risky since high plasticity may lead to

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O.E. Smith, B.D. Murphy & L.C. Smith. 2011. O.E. Smith, B.D. Murphy & L.C. Smith. 2011. O.E. Smith, B.D. Murphy & L.C. Smith. 2011. O.E. Smith, B.D. Murphy & L.C. Smith. 2011.

O.E. Smith, B.D. Murphy & L.C. Smith. 2011. Derivation and Potential Applications of Pluripotent Stem Cells for Regenerative Medicine in Horses. ssssssssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s273 - s283.

uncontrolled teratoma formation. Currently, adult-derived stem cells are the most commonly used cells in the clinical field, and scientists are still conducting experimental transplantation therapies in animal models to assess the safety and long-term stable functioning of transplanted cells.

Horses are pioneering the application of regenerative medicine in several veterinary fields. Not only do horses hold enormous potential as a model for a various of medical conditions found in humans, such as injuries or diseases related to muscles, tendons, ligaments and joints, they also represent substantial commercial value in sport and recreational fields. One of the most common injuries in these large animals involves the musculoskeletal system causing serious consequences due to poor response to stan-dard treatment used successfully in other species. In the case of bone fracture, casting and long-term

immobilization is either impossible or accompanied by high risks of devastating secondary complications, such as damaged cartilage, tendons and ligaments that have a low capacity to heal. Similar complications are commonly observed in other species, including humans. Successful grafting therapies have recently been developed in the horse using autologous MSCs [10]. Although equine MSCs show improvement in the early healing response of articular cartilage lesions, they do not enhance long-term tissue repair and clinical treatments have yet to do so.

The objective of this review is to highlight our current understanding of stem cell biology, with particular emphasis on equine studies, by comparing the various sources of stem cells (Figure 1). Current and potential applications of equine regenerative medicine will also be reviewed.

Figure 1. Overview of the pluripotent stem cells used in equine regenerative medicine. Examples of adult

(bone marrow), extra-embryonic (allantois), embryonic (inner cell mass-derived) and induced pluripotent stem (iPS) cells show specific morphology in vitro and have multilineage differentiation potential for healing several injuries in horses.

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O.E. Smith, B.D. Murphy & L.C. Smith. 2011. Derivation and Potential Applications of Pluripotent Stem Cells for Regenerative Medicine in Horses. ssssssssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s273 - s283.

II. ADULT STEM CELLS

Mesenchymal stem cells (MSCs) are an adult-derived stem cell population that can be isolated from multiple body tissues. These multipotent cells have the capacity to differentiate into lineages of mesenchymal origins, including osteoblasts (bone), chondrocytes (cartilage) and adipocytes (adipose tissue) [34]. Some prefer to refer to MSCs as multipotent stromal cells or mesenchymal progeni-tor cells, observing that the term “stem” might attribute more biological properties than the MSCs actually hold [9]. Only cells that have shown self-renewal ability, in-vivo long-term survival, and tissue repopulation with multilineage differentiation should be identified as MSCs [16].

Equine MSCs are of particular interest both for basic research and for the therapeutic approach to musculoskeletal diseases in the horse. Their multilineage differentiation potential gives them the capability to contribute to the repair of tendon, ligament and bone damage. Yet, enthusiasm for the use of MSCs for therapeutic use is tempered by their age-dependent decline in absolute numbers and the invasive nature of their harvest [28].

In humans, adult MSCs have been detected in various tissues such as the dermis, blood, muscle and the trabecular bone [35]. The cell population from some of these sources may have a more limited capacity for differentiation, containing monopotent or bipotent cells that have differentiation potentials developmentally adapted and restricted to the tissues in which they were found. This could lead to issues involving ease of isolation, cell yield, and donor site complications, suggesting that certain sources may be more suitable than others [3].

In equine medicine, which centers generally on musculoskeletal repair, the bone marrow is the most common source for isolation of multipotent MSCs and adipose tissues as well, due to the low morbidity associated with their harvest and their renewable nature. Herein we compare the advances made in both domains.

2.1 Adult adipose tissue-derived stem cells

Adult adipose tissue (AT) originates from the embryonic mesenchyme and consists mainly of adipocytes and a supportive stroma, composed of

fibroblast-like precursor cells known as preadipocytes [5]. The latter were initially believed only be able to differentiate into cells of its tissue of origin, but recent studies have shown that these stromal cells are actually capable of differentiation into multiple other cell-lines [41]. This supportive stroma represents an important source of adult MSCs since its cells can be easily isolated in large quantities. AT-MSC can be isolated from its tissue when digested in collagenase type I [7], expanded in vitro and then inoculated into the damaged tissue.

There are many advantages to using adipose tissue as a source of MSCs in equine regenerative medicine. First, the presence of adipose tissue in horses is quite substantial and its harvest is much simpler and less prone to complications than bone marrow extraction [10]. Most horses have enough fat around their tail head to obtain the required amount for stem cell injection into their damaged tissue. The adipose tissue is collected either under sedation and local anesthesia or under a quick general anesthesia. In humans it has been reported that AT-MSCs can be quickly isolated from adipose tissue and the resulting stromal vascular cell fraction (SVF) contains a greater proportion of stromal/stem cells per unit volume in comparison to bone marrow [14]. In horses, it has been shown that for the same quantity of tissue sample, the total quantity of AT-MSCs attained after 21 days in culture is significantly larger that for bone marrow MSCs [36]. This represents an important advantage since tissue lesions in horses will usually require a large quantity of cells to ensure successful and long lasting therapeutic repair. Adipose tissue seems to be an accessible and abundant source of adult derived stem cells.

Some disadvantages of AT-MSCs are the slightly lower osteogenic capability than that of BM-MSCs, the non-sterile conditions and risk of pathogen agents at the collection site and the difficulty to obtain fat from highly fit athletes [3]. Also, although the use of autologous AT-MSCs allows a lower risk of immunosuppression, it also requires a longer wait before treatment, due to time period required for tissue collection, stem cell isolation, culture and chara-cterization. Although there is a higher possibility of rejection and risk of disease transmission, allogenic AT-MSCs present certain advantages as well for

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Chapitre 1. Recension de la littérature (suite)

 

1.2 Technique de reprogrammation par induction à la pluripotence (iPS)

Plusieurs facteurs sont importants lors de la dérivation de cellules somatiques en cellules iPS. En premier lieu, le type de cellule reprogrammé peut avoir un impact considérable sur le succès de la reprogrammation. Il existe plusieurs niveaux de différentiation chez les cellules somatiques, certaines se trouvant à un état plus avancé que d’autres. Ensuite, il est important de bien choisir les facteurs de pluripotence à introduire dans les cellules à reprogrammer, ainsi que la méthode de livraison et d’activation de ces transgènes. Finalement, les conditions de culture de la reprogrammation induite doivent être bien établies.

1.2.1 Sélection du type de cellule à reprogrammer

Plusieurs facteurs entrent en jeux lors de la sélection du type de cellule optimal pour la reprogrammation tels l’accessibilité, le niveau de réplication, l’efficacité d’intégration des vecteurs et le niveau de différenciation. La plupart des iPS ont été dérivées à partir de cellules fibroblastes de la peau, car elles sont facilement accessibles, ne causent pas de douleur majeure au donneur sans oublier qu’elles sont faciles à cultiver. Elles ont également un bon taux de réplication en culture in vitro, ce qui à été démontré comme étant un facteur essentiel pour la reprogrammation (Ruiz et al, 2011). Par contre, les cellules fibroblastes de la peau d’un donneur adulte sont à un niveau de différenciation avancé, ce qui signifie que les gènes endogènes de pluripotence sont inactivés et demandent une reprogrammation plus complexe (Serrano et al., 2013). Plusieurs études ont remédié à cette limitation par l’utilisation de cellules fibroblastes provenant de la peau d’un fœtus (Cao et al., 2012; Spinelli et al., 2013). Au stade fœtal, les cellules fibroblastes semblent être plus malléables, en raison du niveau moins différencié de ces

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cellules. Par contre, d’un point de vue clinique, il est toujours intéressant de développer des méthodes pour reprogrammer des cellules adultes dans le but de pouvoir obtenir des cellules souches autologues pour des approches thérapeutiques personnalisées aux patients.

1.2.2 Sélection des facteurs de reprogrammation

Il existe plusieurs combinaisons de facteurs de reprogrammation qui peuvent être introduits dans les cellules somatiques à reprogrammer. Les quatre facteurs qui ont mené à la dérivation des premières cellules iPS chez la souris sont Oct4, Sox2, c-Myc et Klf4 (Takahashi et al., 2006). Cette combinaison « conventionnelle » a permis par la suite de produire des cellules iPS chez plusieurs autres espèces. Oct4 et Sox2 sont des facteurs de transcription essentiels pour maintenir la pluripotence (Boyer et al., 2005; Loh et al., 2006). c-Myc est un facteur qui s’associe au complexe d’histone acetyltransferase (HAT) et induit l’acétylation (souvent associée à l’activation) de plusieurs gènes incluant les gènes de pluripotence (Adhikary et Eilers, 2005). Le facteur Klf4 inhibe le facteur p53, permettant l’activation de Nanog et d’autres gènes de pluripotence (Rowland et al., 2005).

L’addition ou le remplacement par certains facteurs tels que Nanog, Lin28, SV40LT et/ou hTERT ont permis d’améliorer l’efficacité de reprogrammation ainsi que la production de cellules iPS à partir de cellules difficiles à reprogrammer comme des cellules vasculaires musculaires lisses (Park et al., 2008; Lee et al., 2010). Tout comme Oct4, Nanog et Lin28 sont des facteurs de transcription qui permettent de maintenir la pluripotence. SV40LT et hTERT protègent les terminaisons des chromosomes, ce qui empêche la sénescence des cellules et augmente leur niveau de prolifération.

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1.2.3 Sélection de la méthode de livraison

Initialement, la plupart des cellules iPS sont dérivées par l’insertion des facteurs de reprogrammation par l’entremise de rétrovirus ou lentivirus. Ces méthodes de livraison sont très efficaces, avec de très hauts taux de transduction des cellules, et les vecteurs sont développés afin que les transgènes s’inactivent lorsque la cellule devient pluripotente. Par contre, l’insertion des ectogènes dans le génome par la méthode virale est aléatoire et irréversible, et l’inactivation n’est pas assurée, ce qui augmente les risques de mutagenèse d’insertion (Varas et al., 2009). Ces méthodes de livraisons sont donc déconseillées pour les approches thérapeutiques. Une autre méthode de livraison utilise des vecteurs dérivés de transposons, qui intègrent le génome, mais peuvent être excisés par la suite. Un exemple est le transposon piggyBac (PB) qui, à l’aide d’une transposase qui catalyse l’insertion et l’excision du transposon, permet l’activation et l’inactivation facile et sans traces des ectogènes.

1.2.4. Sélection des facteurs à ajouter au milieu de culture

Les composantes du milieu de culture pour les cellules iPS sont similaires à celles des cellules ES. La présence de facteurs de croissance et de certains inhibiteurs de différenciation est essentielle pour maintenir l’état non différencié des cellules pluripotentes. Bien sûr, les conditions varient d’une espèce à l’autre, mais ils nécessitent tous la présence de certains de ces facteurs. Bien qu’il ne soit pas nécessaire chez les cellules ES ou iPS de primates ou humaines, LIF est habituellement essentiel chez les autres espèces. LIF promeut fortement la pluripotence à travers l’activation de la voie Jak-Stat3 (Burdon et al., 2002). Une fois cette voie activée, le transducteur de signal et activateur de transcription 3 (Stat3) se déplace vers le noyau pour activer

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les gènes de pluripotence. Le bFGF induit la prolifération des cellules en activant la voie MEK/ERK et permettant la transcription de plusieurs facteurs de croissance (Thisse et al., 2005). L’addition de certaines molécules inhibitrices augmente aussi l’efficacité de reprogrammation des cellules. L’utilisation d’inhibiteurs de la voie de signalisation MEKK et GSK3 permettent d’augmenter le nombre de cellules reprogrammées. L’inhibition de la voie MEK et ALK aide à la transformation des cellules fibroblastes en cellules pluripotentes et empêche la différenciation (Li et al., 2009).

Finalement, la présence d’une couche de cellules nourricières inactivées, habituellement des cellules fibroblastes embryonnaires de souris (MEF), est aussi essentielle pour maintenir la morphologie des colonies iPS et maintenir leur capacité de prolifération (Takahashi et al., 2009). Chez la souris, il a été démontré que la présence de bFGF et d’activin A peut permettre la reprogrammation des cellules différenciées en l’absence de MEFs (Chen et al., 2009).

1.3 Phases de reprogrammation

Certaines théories conceptualisent la reprogrammation en deux phases (Samavarchi-Tehrani et al., 2010 ; Golipour et al., 2012). Tout d’abord, la phase limitante d’initiation, où les gènes de pluripotence sont activés et les cellules tentent d’entrer en mode reprogrammation. Les voies que peuvent entreprendre les cellules dans cette étape sont nombreuses, comme l’apoptose, la sénescence, la transformation, la transdifférentiation et parfois la reprogrammation. Dans cette étape initiale, les cellules en mode reprogrammation augmentent leur taux de prolifération, subissent des modifications au niveau des histones, initient la transition mésenchymale-à-épiteliale et activent la réparation d’ADN et le traitement de l’ARN. Par la suite, les cellules en mode reprogrammation entrent dans la deuxième phase, soit celle de maturation et de

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stabilisation. Cette phase est initiée par l’activation de Sox2 qui cascade en une série d’évènements qui produisent la cellule iPS stable (Buganim et al., 2013).

1.4 Reprogrammation secondaire

Lors de la reprogrammation, la population de cellules iPS dérivées est hétérogène, avec peu de cellules dans la phase initiale de reprogrammation aboutissant en une cellule iPS. Plusieurs groupes de recherche ont tenté de remédier à cette basse efficacité et cette variété dans les colonies iPS en développant un système de reprogrammation secondaire (2eRS). Des études chez la souris et l’humain utilisent des cellules iPS qui sont différenciées en fibroblaste ou en kératinocyte pour ensuite faire une deuxième étape de reprogrammation (Hockemeyer et al., 2008; Maherali et al., 2008). Puisque les transgènes introduits dans les cellules lors de la première étape de reprogrammation sont inductibles par une drogue telle que la doxycycline, l’addition de la drogue dans le milieu de culture contenant les iPS différenciées induit automatiquement la deuxième reprogrammation, sans le besoin de retransfection. Cette technique de 2eRS permet une augmentation significative de l’efficacité de reprogrammation ainsi qu’une population de colonies iPS plus homogène. À partir de ces résultats, plusieurs autres groupes ont développé leur propre version de 2eRS et ont non seulement été convaincus de l’efficacité de cette technique, mais ont pu produire des cellules iPS en quantité suffisante pour pousser l’analyse moléculaire et génomique. Par exemple, ce sont ces études qui ont démontré que la transition mésenchymale-à-épitheliale (MET) est réellement cruciale pour la production d’une cellule iPS stable (Samavarchi-Tehrani et al., 2010).

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1.5 Caractérisation des cellules pluripotentes

Pour s’assurer d’avoir dérivé et maintenu d’authentiques cellules pluripotentes, plusieurs méthodes de caractérisation de pluripotence ont été développées. Écartant la capacité de se propager sans différentiation à travers plusieurs passages, les cellules pluripotentes possèdent des marqueurs distinctifs reflétant leur état indifférencié. L’un des premiers marqueurs identifiés comme marqueur de la pluripotence est l’activité de la phosphatase alcaline (AP) (Evans and Kaufman, 1981; Thomson et al., 1998). Cette enzyme est responsable de la déphosphorylation de plusieurs molécules, mais son rôle chez les cellules pluripotentes n’a toujours pas été élucidé. Cependant, la perte de l’activité de l’AP est l’un des premiers indicatifs de la différenciation (Palmqvist et al. 2005). Bien que l’AP soit active dans plusieurs tissus, elle demeure tout de même un des marqueurs les plus utilisés dans la caractérisation de cellules souches pluripotentes. La détection de certains marqueurs de surface comme les antigènes embryonnaires de stade spécifique (SSEA) et les antigènes de rejet tumoral (TRA) est une autre méthode d’identification (Umlauf et al., 2010). Les cellules ES de la souris et de l’humain expriment TRA-1-60 et TRA-1-81 (Evans et Kaufman, 1981). Quant à SSEA-1, il est fortement exprimé chez les ES de souris, et SSEA-3 et 4 sont seulement exprimés chez les cellules différenciées (Thomson et al., 1998). Au contraire, SSEA 3-4 sont fortement exprimés chez les ES humaines et SSEA-1 est exprimé chez les cellules différenciées (Solter, 2006). Chez l’équin, il a été démontré que les cellules iPS expriment SSEA 1, SSEA-3 et SSEA-4, TRA-1-60 et TRA-1-81 ainsi que REX-1 et LIN28 (Donadeu, 2012).

Une autre méthode de caractérisation est l’expression génique de certains gènes de pluripotence. Les gènes Oct4, Nanog et Sox2 sont essentiels à l’embryogenèse chez les

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mammifères et, comme mentionné plus haut, exercent des rôles cruciaux dans l’auto-renouvellement et le maintien de la pluripotence (Boiani and Scholer, 2005).

Finalement, l’évaluation de la capacité de différenciation des cellules souches pluripotentes est l’étape finale de caractérisation. D’abord, la culture in vitro des cellules souches pluripotentes en suspension doit permettre l’agglomération et la différenciation des cellules en corps embryonnaire (EB). Ces EB sont morphologiquement similaires à des embryons, mais sans organisation axiale (Doetschman et al., 1985). À l’aide de facteurs de croissance précis, ces EB remis en culture doivent être capables de différenciation en cellules des trois lignées germinales. Ensuite, la technique de différenciation in vivo la plus simple est l’injection des cellules pluripotentes dans des souris immunodéficientes pour observer la formation de tératomes. Ces tératomes sont des tumeurs qui contiennent plusieurs types de cellules représentant les trois feuillets germinatifs embryonnaires (Solter, 2006; Damjanov et Andrews, 2007). Enfin, la production d’un embryon chimérique et la complémentation tétraploïde sont les deux méthodes les plus robustes démontrant l’état de pluripotence des cellules (Tam et Rossant, 2003; Nagy et al., 1993).

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Chapitre 2. Hypothèse et Objectifs

2.1. Hypothèse

Puisque le clonage par transfert nucléaire offre une étape additionnelle de reprogrammation des cellules différenciées, la dérivation d’une ligné stable de cellules iPS autologues sera plus rapide et efficace à obtenir des cellules embryonnaires et fœtale clonées que directement des cellules somatiques de chevaux adultes.

2.2 Objectifs

2.2.1 Objectif général

Développer un protocole efficace de dérivation de cellules iPS équines autologues à partir de fibroblastes de la peau équine adulte.

2.2.2 Objectifs spécifiques

1) Notre premier objectif est de vérifier si les cellules fibroblaste fœtales clonées (ntFF) et cellules ES clonées (ntES) peuvent être reprogrammée en cellules iPS.

2) Notre deuxième objectif est de comparer le niveau de pluripotence entre les cellules équines ES non-transfectées aux cellules équines ntES dérivées en iPS.

2.3 Conception expérimentale

Ce projet compare deux types de production de cellules pluripotentes induites autologues équines, la reprogrammation de cellules fibroblaste fœtales ainsi que la reprogrammation de cellules embryonaire. Les cellules sont obtenues à partir de fibroblastes de la peau de chevaux adultes, passant par une étape de reprogrammation initiale de clonage par transfert nucléaire.

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Figure 1 : Dérivation de cellules souches pluripotentes induites (iPS) autologues de cheval.

Obtention et culture de cellules fibroblastes de la peau équine, suivi du transfert et la fusion d’une cellule dans un ovocyte énucléé pour permettre l’activation embryonnaire et la formation d’un blastocyst ou d’un fœtus cloné. 1) Reprogrammation de cellules fibroblastes fœtales d’origine SCNT (ntFF) en cellules iPS (ntFF-iPS); 2) Reprogrammation de cellules souches embryonnaires d’origine SCNT (ntES) en cellules iPS (ntES-iPS). Toutes les cellules ont été reprogrammées par l’électroporation des vecteurs piggyBac MKOS (éclair jaune).

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Chapitre 3. Résultats

Article 2 : Secondary reprogramming of cloned fetuses and embryos to produce autologous

pluripotent stem cells in horses

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Secondary reprogramming of cloned fetuses and embryos to produce autologous pluripotent stem cells in horses

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ABSTRACT

Autologous pluripotent stem cell lines can be derived from adult cells either by direct reprogramming through induced expression of pluripotency genes (iPS) or indirectly by reprogramming through somatic cell nuclear transfer (SCNT) followed by the derivation of embryonic stem cells (ESC). However, outcome efficiency of SCNT and iPS protocols are invariably low, indicating that, alone neither of these reprogramming routes is sufficient for deriving genetically and epigenetically stable pluripotent stem cells. We hereby report on the production of autologous equine iPS cells by combining SCNT and iPS reprogramming protocols. Adult skin fibroblasts were used for SCNT, and the resulting cloned embryos were either used to obtain fetal fibroblasts (ntFF), or used for ESC culture (ntES). Cells were then transfected using the piggyBac transposon-based doxycycline-inducible iPS derivation system. The ntFF-iPS colonies appeared at day 20-30 post-transfection with an estimated 0.03%

reprogramming efficiency. In contrast, ntES-iPS colonies appeared at day 7 post-transfection, with 0.17% reprogramming efficiency, and showed improved pluripotency marker expression when compared with ntES cells. These results indicate that stable equine iPS cell lines may be readily obtained from secondary reprogramming of cloned ntFF and ntES cells, opening novel avenues for developing autologous pluripotent stem cell therapies.

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INTRODUCTION

The high proliferation properties and capacity for multilineage differentiation of stem cells have instigated a wave of new technology developed in the field of human and veterinary regenerative medicine. Advancements in this field have played an important role in disease modeling (Tan and Scotting, 2013) and animal studies have shown that stem cell therapy can induce improvement in conditions such as neurodegenerative disease (Lescaudron et al., 2012), cardiac disease

(Kanashiro-Takeuchi et al., 2011) and tissue repair (Oldershaw 2012). Multipotent somatic stem cells, such as bone marrow-derived mesenchymal stem cells or neural progenitor stem cells, can be easily collected and allow autologous transplantation (Jahagirdar, 2005). However, the collection of these cells results in heterogeneous populations with low percentages of true multipotent stem cells (Vidal et al., 2006) and once isolated, these cells have more limited differentiation capacities.

Pluripotent stem cells, on the other hand, have unlimited proliferation and are capable of

differentiation into any cell type of all three germ layers (Lanza and Rosenthal, 2004). Such cells are isolated from early embryos, such as embryonic stem cells (ESC) and epiblast stem cells (EpiSC), or from the genital ridges of developing fetuses (embryonic germ cells). Because of their fragility and due to ethical concerns, autologous pluripotent stem cells are difficult to obtain from humans. Some approaches have successfully been developed to circumvent these ethical and technical issues. Co-culture of differentiated cells with oocyte cell-free extracts has led to partial reprogramming of cells to an embryonic stem (ES)-like state (Miyamoto et al., 2009).

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Recently, somatic cell nuclear transfer (SCNT) was successfully performed in humans and stable ESC lines were isolated (Tachibana et al, 2013).

Although these techniques have been widely used in domestic animals, attempts to obtain stable ESC lines have been largely unsuccessful (Talbot et al., 2008). Because of their similarities in physiology with humans, domestic species are considered excellent models for long-term experiments in biomedical research and more specifically, regenerative medicine. Derivation of pluripotent stem cell lines from large animals such as horses and pigs will thus benefit both human and veterinary clinical applications.

An important advance in recent years was the establishment of pluripotent stem cells by the Yamanaka group, who showed that the ectopic expression of four transcription factors, cMyc, Klf4, Oct4 and Sox2 (MKOS), induced the reprogramming of mouse and human differentiated somatic cells, known as induced pluripotent stem (iPS) cells (Takahashi et al., 2006; Takahashi et al., 2007). The iPSC have been derived from various other mammalian species, including

domestic ungulates including pigs (Ezashi et al., 2009), sheep (Li et al., 2011), cattle (Sumer et al., 2011) and horses (Nagy et al., 2011; Breton et al., 2013). However, iPS cell generation remains inefficient, with very few transformed cells achieving complete reprogramming.

Moreover, whereas many studies show that iPS cells and ESC share key pluripotency properties, others indicate that important differences remain between these two cell types, especially in genome integrity (Laurent et al., 2011; Mayshar et al., 2010).

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generations (Wakayama et al, 2013) and, in pigs, serial re-cloning has even been suggested to restore epigenetic errors arising during a first generation nuclear transfer (Cho et al, 2007). By inducing the expression of pluripotency transgenes in differentiated tissues derived from iPS cells, secondary reprogramming systems have also been shown to accelerate reprogramming and to produce more homogeneous iPS population, suggesting that a second wave of reprogramming significantly increases the reprogramming factor expression and the amount of successfully reprogrammed iPS cells (Nagy, 2013).

In the present study, we hypothesized that autologous equine iPS cells may be obtained from adult fibroblasts through the sequential approach in which fibroblasts are reprogrammed initially by SCNT to obtain cells at earlier stages of development, and subsequently reprogrammed using genetically induced iPS-derivation systems. We report the rapid and efficient generation of equine iPS (eiPS) cell lines from SCNT-derived cloned fetal fibroblasts and ES-like cells using a piggyBac (PB) transposon delivery system used previously to derive eiPS from fetal fibroblasts (Nagy et al., 2011).

MATERIAL AND METHODS

Preparation of Equine Adult Fibroblast (eAF) and Fetal Fibroblast (ntFF) Cells

All procedures using live animals were performed in compliance with the institution’s guidelines for the care and use of laboratory animals, approved by the local animal care committee as sanctioned by the Canadian Council on Animal Care. Following asepsis of an area in the neck,

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local anesthesia was administered and a 6 mm diameter full-thickness skin biopsy was collected from two male and two female adult horses. Skin samples were maintained in saline on ice and transported immediately to the laboratory for further processing.

Autologous fetal fibroblast cells were obtained from cloned fetuses produced by somatic cell nuclear transfer (SCNT) using skin fibroblasts from the four adult horses above. Pregnant

recipient mares at 40 days of gestation were tranquilized with xylazine (0.2 mg/kg; Bayer Health Care) and acepromazine malate (0.02 mg/kg, Vétoquinol) and a catheter was introduced in the uterus to flush out the fetus in PBS (Invitrogen), which was transported on ice to the laboratory for recovery of skin samples.

Adult and fetal-derived skin was dissected and exposed to a 5 mg/ml collagenase I (Sigma) solution in DMEM (Invitrogen) for 3 h at 37°C. After dissociation, cells were washed twice in fresh DMEM with 10% FBS (Invitrogen), plated, cultured, and passaged once to obtain first passage equine adult fibrobalsts (eAF) and fetal (ntFF) fibroblasts. Stocks were frozen in DMEM with 10% FBS and 10% DMSO (Sigma).

Somatic Cell Nuclear Transfer (SCNT)

The procedure of SCNT was performed as described previously (Galli et al., 2003). Briefly, slaughterhouse-derived ovaries were dissected and follicles were aspirated using a 16 G needle to obtain cumulus oocyte complexes (COC) with multilayered compacted cumulus. COCs were cultured in 50 µl droplets of DMEM-F12 (Sigma), 10% FBS (Invitrogen), 2 mg/mL FSH (follicle

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stimulating hormone; Bioniche), 5 mg/mL LH (luteinizing hormone; Bioniche), 40 µg/mL IGF-1 (insulin-like growth factor 1; Sigma) and 40 µg/mL EGF (epidermal growth factor; Sigma) at 38oC in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2. After 24 h, secondary oocytes with expanded cumulus were denuded and microsurgically enucleated in the presence of 0.8 M cytochalasin B (Sigma) by removal of the metaphase spindle and first polar body. After

microsurgery, fibroblast cells were injected into the perivitelline space of enucleated oocytes and the couplets were electrofused using a 1.2 KV/cm DC current. Fused couplets were activated by 4 minute exposure to 5 mM ionomycin (Sigma) followed by 3 h exposure to 100 mM

6-(Dimethylamino)purine (6-DMAP; Sigma). After activation, reconstructed oocytes were washed and cultured in DMEM-F12 in vitro for 8-9 days to develop to the blastocyst stage.

Preparation of Equine Embryonic Stem-Like (ntES) cells

The ntES cells were obtained from somatic cell nuclear transfer (SCNT) cloned embryos. Viable day 8-9 blastocysts were cultured on mitomycin inactivated feeder layers consisting of mouse embryonic fibroblasts isolated from day 14.5 embryos with standard procedure (MEFs) (Jozefczuk, J. et al., 2012) using eiPS medium (see below). Once embryos attached to the well and began proliferating, mechanical separation was used to propagate the embryonic stem-like (ntES) cells to a first passage. The cells were then separated by TrypLE (Life Technologies) enzymatic separation for the second passage and once propagation was sufficient, cells were collected for transfection. Stocks were frozen in 90% FBS and 10% DMSO.

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Equine fetal fibroblasts were maintained in DMEM High Glucose (Invitrogen) supplemented with 2 mM GlutaMaxTM (Invitrogen), 0.1 mM Non-essential amino acids (Invitrogen), 0.1 mM Betamercaptoethanol (Sigma), 1 mM Sodium Pyruvate (Invitrogen), 50 U/ml

Penicillin/Streptomycin (Invitrogen) and 15% fetal bovine serum (HyClone). Culture media for eiPS and ntES cells was the same as above, supplemented with 1000 U/ml leukemia inhibitory factor (LIF; ESGRO, Millipore), 10 ng/ml bFGF (Peprotech), 1.5 µg/ml Doxycycline (Sigma) 3 µM GSK (Glycogen Synthase Kinase) inhibitor (StemGent), 0.5 M MEK (mitogen-activated protein kinase) inhibitor (StemGent), 2.5 µM TGF-β (transforming growth factor) inhibitor (StemGent) and Thiazovivin (StemGent). From day 8 of the reprogramming process until day 15, the media was also supplemented with 25 µM ALK (activin receptor-like kinase) receptor

inhibitor (StemGent) for the adult and fetal cells.

Gene transfection protocol

Plasmids for the piggyBac procedure (PB-TET-MKOS, PB-GFP, PB-CAG-rtTA and pCyL43 PBase) were constructed as previously described (Nagy et al., 2011). All cells (ntFF and ntES) were transfected with the Neon electroporation device (Invitrogen) according to the

manufacturer’s instruction, using preset program 14. For each electroporation in 10 µl tips, 2.5×105 cells were used with a total of 1 µg mixed DNA. The DNA mixture consisted of equal weight ratios of all four plasmids. Cells were seeded in wells of a 9.6 cm2 surface area for culture.

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Establishment of eiPS Cell Lines

As soon as well-defined colonies appeared post-transfection and prior to passaging of the cells, colonies were picked mechanically. The first passage of the isolated colonies used mechanical dissociation. From the second passage onward, the lines were dissociated enzymatically with TrypLE Select (Invitrogen) and cultured on MEFs in eiPS media. The cells were passaged every 3-4 days at a 1:5 ratio. At the appropriate level of expansion, the cells were cryopreserved in 90% FBS and 10% DMSO.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)

RNA was collected from cells grown in 60 mm tissue culture plates by brief enzymatic

dissociation, resuspension with culture media, centrifugation, and snap freezing of the pelleted cells. RNA was extracted by isopropanol and purified with RNeasy Mini kit (Qiagen #74104) following manufacturer’s protocol. Reverse transcription was performed using QuantiTect Reverse Transcription kit (Qiagen #205313) with the RT primers and Mix provided by the kit. The reaction was performed at 42° C for 30 min. For all samples, a negative RT was used as a control, consisting of an RT reaction omitting the reverse transcriptase. The following RT-PCR primers were used to detect and quantify the expression of equine specific KLF4 (forward: GTGCCCCAAGATCAAGCAG, reverse: TGCTGAGAGGGGGTCCAGT, amplify 89 bp, Roche probe #94), NANOG (forward: CGGGGCTCTATTCCTACCACC, reverse:

GGTTGCTCCAAGACTGGCTGT, amplify 129 bp, Roche probe #3), OCT4 (forward:

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Roche probe #3) and SOX2 (forward: CTTGGCTCCATGGGTTCG, reverse:

TGGTAGTGCTGGGACATGTGA, amplify 188 bp, Roche probe #70). The relative

concentration of mRNA was quantified using RotorGene comparative quantification analysis system (Qiagen). These measurements were normalized with the equine specific housekeeping genes GAPDH (forward: GAGATCCCGCCAACATCAAA, reverse:

AAGTGAGCCCCAGCCTTCTC, amplify 97 bp, Roche probe #159), SDHA (forward: GCACCTACTTCAGCTGCACG, reverse: AACTCCAAGTCCTGGCAGGG, amplify 94 bp, Roche probe #37) and RPL32 (forward: GAAGCACATGCTGCCCAGT, reverse:

CTTTGCGGTTCTTGGAGGAG, amplify 89 bp, Roche probe #132). We have established that none of these primer pairs amplify mouse transcripts (data not shown).

Bisulfite Sequencing

Approximately 400–500 ng of total genomic DNA was used for a bisulfite treatment reaction using the EpiTect Bisulfite kit (Qiagen). Primers specific for bisulfite-converted DNA were designed within the equine OCT4 region to amplify a 513-bp fragment, spanning from 124 to 658 bp downstream of exon 1 (GenBank accession no. NW_001867389). Each PCR reaction was performed in triplicate using the OCT4 primers (forward: TTTAGTGGGTTAGGAATTGGGT, reverse: CCAACTTCCAACTCCCCCAAA). The PCR reaction was carried out in a final volume of 50 µl, containing 1–2 µl of bisulfite-treated DNA, 0.2 µM each primer, 0.3 mM mixed dNTP, 13 PCR buffer, 1.5 mM MgCl2, and 2.5 units of Platinum Taq DNA polymerase (Invitrogen). The reactions were performed using an initial 2-min step at 94°C followed by 50 cycles of 30 sec at 94°C, 30 sec at 53°C, 1 min at 72°C, and a final 3-min step at 72°C. The PCR products were

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resolved in 1% agarose gels, followed by purification using the QIAquick Gel Extraction kit (Qiagen). Purified fragments were subcloned in pGEM-T Easy Vector (Promega), and cell transformation was performed using competent Escherichia coli DH5a cells (Invitrogen). To ensure that reliable data were collected, a total of 20–23 clones for each sample were picked and sequenced.

Immunocytochemistry

Cells grown in 4-well tissue culture plates (Corning) on coverslips were washed twice with DPBS (Invitrogen) and fixed with 4% paraformaldehyde solution for 15 min. After washing with DPBS twice, cells were permeabilized with chilled 0.1% triton for 10 min, washed twice with DPBS and blocked 60 min with 5% normal goat serum (Jackson ImmunoResearch) in DPBS. Cells were then incubated overnight at 4°C with the following primary anti-mouse, anti-rat or anti-human antibodies; (i) anti-Nanog (Reprocell), (ii) anti-SSEA1 (Stemgent), (iii) anti-SSEA3 (Stemgent) (iv) anti-SSEA4 (Stemgent), (v) anti-TRA-1-60 (Stemgent), (vi) anti-Rex-1

(Millipore). After two washes in DPBS, cells were incubated for 60 min at room temperature with Cy3 or Cy5 conjugated secondary antibodies (Stemgent). For control experiments, the primary antibody was omitted.

Statistical Analysis

All values are expressed as mean ± SD. To determine significance between two groups, comparisons were made using Student’s t-test. Analysis of multiple groups was performed by

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one-way ANOVA using Graphpad Prism V5.0 (Graphpad Software. San Diego, CA, USA). P < 0.05 was considered significant.

RESULTS

Reprogramming of Fibroblast from SCNT-Derived Fetuses

Equine adult fibroblasts from four horses were used for somatic cell nuclear transfer (SCNT) to obtain blastocysts that were transferred to surrogate mares for implantation. Gestations were terminated to recover 40 days fetuses that were then used to obtain fetal fibroblast (ntFF)

cultures. (Figure 1.A) Using the previously established PB transposon-based system (Wang et al., 2008), the four reprogramming factors c-Myc, Klf4, Oct4 and Sox2 (MKOS) were transfected in SCNT-derived ntFFs to induce reprogramming. The cell survival rate after transfection was estimated to be 75% and, from the ratio of GFP positive cells 24h after plating, transfection efficiency was estimated at 46% (data not shown).

GFP positive ES-like colonies began to appear 21-25 days after transfection of SCNT-derived ntFF. These colonies were morphologically similar to fetal eiPS cells previously obtained (Nagy et al., 2011) with well-defined edges, monolayer organization (Figure 1.B) elevated nucleus to cytoplasm ratio and positive for alkaline phosphatase staining (data not shown). A total of four independent SCNT-derived ntFF primary cell cultures were transfected producing an average of 25 (± 2) colonies per cell culture. Of those colonies, 4 ntFF-iPS lines were successfully

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cells surviving electroporation divided by the number of colonies found on each plate) was estimated to 0.029% (Table 1).

Using reverse-transcriptase PCR and comparative quantification assays, endogenous pluripotent gene expression was measured in both ntFF and ntFF-iPS cells. ntFF-iPS cells showed significant increase in OCT4 (18 fold), NANOG (18 fold) and SOX2 (30 fold) expression (Figure 2),

indicating a consistent transcriptional upregulation of the endogenous pluripotency network. However, KLF4 expression was significantly higher in ntFF, which may be indicative of their dermal (skin) origin. Together, these results indicate that SCNT-derived ntFF can be efficiently reprogrammed into pluripotent eiPS lines thereby circumventing the limitations of producing iPS directly from adult horses by allowing the production of autologous iPS cell lines from cloned fetuses.

Reprogramming of SCNT-Derived Embryonic Stem-like Cells

Equine adult fibroblasts from the same four horses were used for somatic cell nuclear transfer (SCNT) to derive day 8 blastocysts that were then allowed to attach and cultured on gelatin and inactivated MEF covered dishes to derive ES-like lines (ntES). After 7 days in culture, these ntES cells were transfected with reprogramming factors and cultured to produce iPS lines (ntES-iPS). As for fetal fibroblasts, survival rate after transfection of ntES was estimated to be 75%, and 46% of the surviving cells expressed GFP 24 h after plating (data not shown). Various small but distinct GFP positive colonies appeared 2-3 days after transfection and at day 5-7 the colonies were large enough for manual picking and transfer as individual colonies to fresh culture dishes

(51)

 

(Figure 3.A-B). These ntES-iPS colonies were GFP positive (Figure 3.C) and morphologically similar to the control early passage ntES cultures, with well-defined edges and monolayer organization. Since the colonies were already present at the time of transfection, the ALK receptor inhibitor SB431542 was not added to the iPS initial culture media.

Four independent ntES lines were transfected, and an average of 144 (± 30) ntES-iPS colonies were produced per cell line. The reprogramming efficiency was estimated to 0.167% (Table 1). Of those colonies, four (one from each horse) were selected and expanded into established ntES-iPS lines that were maintained up to at least 15 passages and all showed alkaline-phophatase staining (Figure 3.D).

The non-transfected ntES lines used as controls in this study were cultured in parallel with the ntES-iPS lines for comparative analysis. At early passage, ntES cells showed the typical morphology of rounded and monolayered colonies with well defined edges. However, from passages 6 to 8, ntES cells showed slower proliferation, as indicated by fewer colonies and delayed confluence compared with ntES-iPS lines (Figure 3.E-F). Of the four ntES lines used as controls in this study, two reached senescence at 10 passages (line 1 and 2), one at passage 15 (line 3) and the other at passage 17 (line 4). Moreover, most ntES cells cryopreserved at passage 13 and further did not survive after thawing (data not shown). This sluggish growth pattern and freezing sensitivity contrasted to ntES-iPS lines that continued growing vigorously without slowing for over 20 passages even after cryopreservation (data not shown). Together, these results indicate that transfection of ntES cells with the four exogenous reprogramming factors used in this study leads to the efficient and rapid derivation of morphologically and kinetically

(52)

 

   40  

stable ntES-iPS cell lines that reliably withstand cryopreservation. In support to the phenotypic characteristics of the ntES-iPS lines, immunofluorescence analyses revealed the expression of key pluripotency markers NANOG, TRA1-60, REX-1, SSEA1, SSEA-3 and SSEA4 in the ntES-iPS cell lines further supporting their pluripotent status (Figure 4).

Pluripotency Expression in ntES and ntES-iPS Cell lines

Our next step was to determine whether the expression levels of pluripotency genes were

upregulated in ntES-iPS cell lines in comparison to their respective ntES counterpart. Using qRT-PCR with equine-specific primers for the endogenous pluripotency markers, we observed a wide variation in the expression patterns of these genes among the four ntES-iPS cell lines. OCT4 and KLF4 expression increased significantly in both lines 3 and 4. In contrast, the SOX2 expression increase was significant in lines 1 and 3 and NANOG expression increased significantly only in line 1. In contrast to the other lines, line 2 showed no significant increase in gene expression for any of the pluripotency genes analyzed (Figure 5). These results indicate that the pluripotency genes are reactivated in ntES cells by induced expression of the MKOS genes, but secondary reprogramming efficiency varies between the lines obtained.

To further understand the control mechanisms involved in the increased expression of endogenous OCT4, we performed bisulfite sequence analysis to determine the methylation pattern of the equine OCT4 region between bp 124 and bp 658 at passage 7 in ntES and ntES-iPS cells from line 4 (Figure 6). Analysis of the nine CpG dinucleotides positioned 5’ of intron 1 (nt405 to nt650) revealed that the endogenous OCT4 gene was significantly hypomethylated in

Figure

Figure 1. Overview of the pluripotent stem cells used in equine regenerative medicine
Figure 1 : Dérivation de cellules souches pluripotentes induites (iPS) autologues de cheval
Table  I.  Number  of  colonies  obtained  and  reprogramming  efficiency  of  ntFF  and  ntES  cells
Figure 2 : Estimation du temps pour l’obtention de cellules souches pluripotentes induites  (iPS)  autologues  chez  le  cheval

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