Neural differentiation of enbryonic stem cells: clonal properties and sensitivity to cytomegalovirus infection

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Thesis

Reference

Neural differentiation of enbryonic stem cells: clonal properties and sensitivity to cytomegalovirus infection

MARTINEZ, Yannick

Abstract

In this thesis we focused on stem cell differentiation into neural lineage and an application of this differentiation. We have shown that all selected clonal ESC express markers of the early ICM, have the capacity to be differentiated towards more than one cell type and can generate embryoid bodies including cells of the three germ layers. We also studied cell tropism, infectious dynamics of CMV infection on proliferation, regeneration and differentiation of neural cells. We used ESC differentiated into neural lineage to investigate neural permissiveness to CMV infections. We showed that undifferentiated ESC are well protected against CMV infection but their differentiation into NPC makes them permissive to the virus.

MARTINEZ, Yannick. Neural differentiation of enbryonic stem cells: clonal properties and sensitivity to cytomegalovirus infection. Thèse de doctorat : Univ. Genève, 2011, no.

Sc. 4389

URN : urn:nbn:ch:unige-180636

DOI : 10.13097/archive-ouverte/unige:18063

Available at:

http://archive-ouverte.unige.ch/unige:18063

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

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UNIVERSITE DE GENEVE

Département de biologie cellulaire FACULTE DES SCIENCES Professeur J.-C. Martinou Département de pathologie et immunologie FACULTE DE MEDECINE

Professeur K.-H. Krause

Département de médecine interne FACULTE DE MEDECINE

Professeur L. Kaiser _____________________________________________________________________

Neural differentiation of embryonic stem cells: clonal properties and sensitivity to cytomegalovirus infection

THESE

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

par

Yannick MARTINEZ de

Annecy (France)

Thèse n° 4389 Genève

2011

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ACKNOWLEDGMENTS

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Remerciements

Je remercie en premier lieu mes directeurs de thèse pour m’avoir accueilli dans leurs laboratoires et m’avoir donné la possibilité de mener à bien ces travaux: le Professeur Karl-Heinz Krause, son enthousiasme pour la recherche, ses connaissances et ses critiques constructives. Le Professeur Laurent Kaiser pour ses compétences, sa compréhensive patience et sa gestion d’équipe exemplaire. Tous deux sont des exemples à suivre pour tout jeune chercheur.

Je remercie le Professeur Andras Dinnyes pour avoir accepté de venir d’aussi loin juger ce travail et malgré un emploi du temps déjà bien remplis. Son vaste intérêt pour de nombreux domaines de la biologie et ses idées m’ont ouvert de nouvelles portes de recherche.

Je remercie mon répondant pour la Section de biologie, le Professeur Jean-Claude Martinou ainsi que tous les membres du jury pour avoir accepté de juger ce travail.

Cette thèse n’aurait pas été possible sans l’aide de Miche Dubois-Dauphin et Olivier Preynat-Seauve. Je tiens à leur exprimer ma gratitude pour leur compétence, le temps qu’ils m’ont consacré, leurs suggestions et les innombrables lectures et corrections de textes scrupuleuses. Leurs commentaires détaillés m’ont été très utiles.

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6 Je remercie Christophe Delgado et Diderik Tirefort pour leur immense aide technique, leur investissement et leur enthousiasme. Je suis également reconnaissant à mes anciens collègues : David Suter et Jérome Bonnefont pour les discussions enrichissantes et m’avoir montré l’envers du décor.

Mes collègues présents et passé du laboratoire du Professeur Karl-Heinz Krause : Maxime Feyeux, Li Bin, Silvia Sorce, Christine Deffert, Cécile Guichard, Vincent Jacquet, Michela Tempia-Caliera, Yuan Huiping, Vannary Tieng-Caulet, Mitra Nayernia, Karen Bedard, Olivier Plastre, Stefania Schiavone, Stéphanie Julien, Olivier Basset et Hélène Gfeller-Tillmann pour leur aide au quotidien.

Tous les membres du laboratoire du Professeur Laurent Kaiser et très spécialement : Delphine Garcia, Noëlle Roguet, Pascal Cherpillod, Caroline Tapparel, Samuel Cordey, Manuel Schibler et Sanda Van Belle pour leur aide et discussions scientifiques intéressantes tout au long de ces années.

Tous les membres de la plateforme génomique de la faculté de Médecine de Genève, et en particulier Celine Vivier, Didier Chollet et Isabelle Durussel-Gerber pour leur aide dans la réalisation et analyses des expériences de microarrays.

Dominique Garcin et Mario-Luca Suva pour leurs conseils, informations, échantillons, bref les milles petites choses qui rendent la vie d’un thésard plus facile.

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7 Tous les membres de la plateforme de bioimagerie, et en particulier Serguei Startchik pour son aide pour l’analyse automatisée d’image et en particulier l’utilisation du logiciel métamorph.

Je remercie toutes les personnes qui ont montré de l’intérêt scientifique ou qui ont participé à ce travail. Au-delà de la biologie, la découverte des gens a été une expérience inoubliable au cours de laquelle de nombreux souvenirs se sont créés.

Je remercie mes amis qui m’ont supporté pendant ces année et permis de garder contact avec le monde réel ou pas… : Alex, Capu, Carmen, Dan, Dr No, Greg, Will et les autres.

Je remercie enfin mes parents pour leur soutien au cours de toutes ces années, pour leur confiance et leur amour. Et « last but not least » mon sünike pour sa compréhension lors de cette dernière année et son soutien quotidien indéfectibles.

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ABSTRACT

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Abstract

In this thesis we focused on stem cell differentiation into neural lineage and an application of this differentiation. We tried to obtain a homogenous cell population of neuroprecursors (NPC) or a mature population of a defined neural type from embryonic stem cells (ESC). We observed that neural colonies derived from ESC often show a curious heterogeneity. From this observation, we tested the differentiation potential of individual cells in established ESC lines and found a various differentiation capacity. These variations are not due to precursors of extraembryonic tissues, but represent cells expressing the typical marker profile for the early inner cell mass (ICM). The sublines within established ESC showed stable cellular individualities. The individualities of the sublines are reflected in a distinct gene expression pattern in undifferentiated ESC, and a distinct cell fate potential upon differentiation. We have shown that all selected clonal ESC express markers of the early ICM, have the capacity to be differentiated towards more than one cell type and can generate embryoid bodies including cells of the three germ layers. Their differentiation potential toward neural or cardiac lineage differs.

One of the interests in ESC differentiation toward neural lineage is to provide models for brain development and diseases, for toxicity and drug screening and to explore the mechanism of action of different molecules. Using the expertise obtained in ESC differentiation toward neural lineage we created a model to study cytomegalovirus (CMV) tropism in fetal brain.

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10 We studied cell tropism, infectious dynamics of CMV infection on proliferation, regeneration and differentiation of neural cells. We used ESC differentiated into neural lineage to investigate neural permissiveness to CMV infections. We evaluated the susceptibility to CMV infection in the different stages of ESC differentiation. We showed that undifferentiated ESC are well protected against CMV infection but their differentiation into NPC makes them permissive to the virus. This permissiveness decrease when NPC become mature neural cells. After infection we saw an inhibition of the G1/S phase transition. In vitro we found a loss of neural cells and a decreased proliferation of NPC. We used a method developed in our laboratory to generate three-dimensional neural culture using an air-liquid interface system. These cultures of engineered neural tissues (ENTs) have phenotypic and structural similarities to the early human fetal brain. The comparison of our results with samples of fetal brain showed similarities between ENTs and fetal brain infection patterns.. We showed that an abnormal cell population consequent to infection appears and that cells are mostly infected around neural tubes. CMV disrupts the normal progression of neuronal cell differentiation in an engineered neural tissue.

The developmental brain abnormalities seen during congenital CMV infection could be due in part to viral infection of these susceptible NPC and the subsequent phenotypic modifications. As present antivirals used against HCMV suffer from complications associated with prolonged treatments ENT model may provide advanced in vitro models for secondary antiviral testing in combination with non- destructive techniques. Cell proliferation, migration, differentiation and synaptogenesis could be followed in ENTs and gave critical information about the effects of HCMV on proliferation and toxicity.

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RESUME

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Résumé

Les cellules souches embryonnaires : un outil au service de la médecine

Dans ce travail nous nous sommes penchés sur la différentiation des cellules souches embryonnaires (ESC) en cellules de type neural ainsi que sur une application biomédicale pour ces cellules. Nous avons tentés d’obtenir une population cellulaire homogène de neuroprécurseurs (NPC) ainsi qu’une population de cellules plus matures de neurones. Nous avons alors observé que les colonies neurales dérivées des ESC montrent une curieuse hétérogénéité. A partir de cette observation, nous avons testé le potentiel de différentiation individuel des cellules dans les lignées ESC établies. Nous avons trouvé une capacité de différentiation différente pour les cellules. Ces variations ne sont pas dues à la présence de précurseurs des tissus extraembryonaires en raison de l’expression des marqueurs typiques de la masse cellulaire interne. Les sous-lignées cellulaires établies à partir des lignées classiques d’ESC montrent des individualités cellulaires stables. Celles- ci se reflètent dans un profile d’expression des gènes spécifique lorsqu’elles sont indifférenciées. Ces sous-lignées montrent également un potentiel de différentiation distinct. Nous avons montrés que toutes les sous lignées sélectionnés expriment les marqueurs de la masse cellulaire interne. Ils ont la capacité de se différencier en plus d’un type cellulaire différent dans chacune des 3 lignées germinales, peuvent générer des corps embryoides ce qui prouve leur pluripotence. Leur potentiel de différentiation vers les lignées de cellules cardiaques diffère également.

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13 L’un des intérêts de la différentiation des ESC vers des lignées neurales est de fournir des modèles pour le développement du cerveau ainsi que pour l’étude de maladies. Nous avons utilisés les connaissances acquises dans cette différentiation pour générer un modèle d’étude du tropisme du cytomegalovirus (CMV) dans le cerveau fœtal.

Nous avons étudié le tropisme cellulaire, la dynamique d’infection, la prolifération et l’impact du CMV dans notre modèle. Nous avons utilisé les ESC différentiées vers des lignées neurales pour investiguer la capacité d’infection du CMV. Différentes étapes de la différentiation des ESC ont été testées. Nous montrons que les ESC indifférenciées sont bien protégées contre l’infection au CMV. En revanche leur différentiation les rend permissives au virus. Cette permissivité diminue au fur et à mesure de la différentiation vers des cellules neurales plus matures. Après infection nous avons pu voir une inhibition de la phase de transition G1/S. De plus in vitro nous observons une perte des cellules neurales et une diminution de la prolifération des NPC. Nous avons utilisé une méthode développée dans notre laboratoire pour générer une culture neurale tridimensionnelle (ENT) à partir d’une interface air/liquide. Cette culture d’ENT a des similarités phénotypiques et structurelles avec le cerveau fœtal dans les premières phases de développement. La comparaison de nos résultats d’infection avec des observations réalisées sur des échantillons de cerveau fœtal humain nous montre de fortes similarités. Notre model ENT pourrait être utilisé pour des études de screening secondaires d’antiviraux en combinaison avec des techniques d’imagerie récentes.

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A. ABBREVIATIONS & ACRONYMES

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15 ALS amyotrophic lateral sclerosis

ABC avidin biotin complex BBB blood brain barrier

BMP2 bone morphogenetic protein 2 BSA bovine serum albumin

BDNF brain-derived neurotrophic factor bFGF basic fibroblasts growth factor β-FGF fibroblasts β growth factor CMV cytomegalovirus

CNS central nervous system

DMEM Dulbecco's modified Eagle medium DMSO dimethyl sulphoxide

EBs embryoid bodies

EGF epidermal growth factor ENT engineered neural tissue ESC embryonic stem cells

FACS fluorescence-activated cell sorting FCS fetal calf serum

GFAP glial fibrillary acidic protein HBSS Hank’s buffered salt solution hESC human embryonic stem cells hCMV human cytomegalovirus HSPG heparin sulfate proteoglycans HSV herpes simplex virus

HEK human embryonic kidney IPS induced pluripotent stem cell IE1 immediate early protein 1

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16 IE2 immediate early protein 2

ir Immuno reactive

IRF3 interferon regulatory factor 3 IRF7 interferon regulatory factor 7 MEF mouse embryonic fibroblast mESC mouse embryonic stem cells MIEP major immediate early promoter MNC mature neural cells

MS5 murine bone marrow-derived stromal cells

NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells NSC neural stem cells

NPC neural precursor cells PGC primordial germ cells PS penicillin / streptomycin PSC pluripotent stem cells RT room temperature

SR KnockOut Serum Replacement SSEA stage specific embryonic antigen TGF transforming growth factor

TLR2 Toll like receptor 2

Vh human foreskin fibroblasts

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B. INTRODUCTION

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18 Introduction:

The idea of stem cells, and their usefulness, goes back to at least 1883 (Rey 2003). Modern stem cell vision implies two qualities:

- A physiological self-renewing ability, which means that after each mitosis at least one cell with the same characteristics as the mother cell is generated. The cycle can be associated with a quiescent phase.

- The capacity to generate, after a differentiation process, a cell phenotypically differentiated.

This notion is central to developmental biology that generates a pluricellular organism organized from a single cell.

Embryonic stem cells are a transitory cell type. From the zygote, holoblastic cleavage creates a morula after 3 to 4 days in mice and after 4 to 5 days in human.

At this point, morula cells called blastomeres have the same size and an equivalent differentiation capacity. The structure has a blackberry shape and undergoes a compaction (Matte, Doggenweiler et al. 1987; Chazaud, Yamanaka et al. 2006). During this compaction, external and internal blastomeres derived from 32-cell embryos are determined and lose their totipotent capacity. After compaction, a cavitation process occurs. The blastocoel, a fluid-filled cavity, is formed by the secretion of a fluid by the outermost layer of cells (Fleming and Pickering 1985; Cheng and Costantini 1993). This reorganization forms the blastocyst (Schema 1) andthe implantation into the endometrium happens during this stage. Blastocoel and external blastomeres forming the trophoblast will form extra-embryonic tissues (Garbutt, Chisholm et al. 1987; Dardik and Schultz 1991). Internal blastomeres make the inner cell mass (ICM), which will form all somatic tissues.

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19 Schema 1: First steps of embryogenesis in mouse and human.

After sampling, ICM cells can grow under controlled conditions between 4 and 6 days post- fecundation for mouse and between 5.5 and 7.5 days post-fecundation for human.

The process of cell isolation from the ICM to obtain a stable embryonic stem cells ESC line is called derivation. This process was described for the first time for mice in 1981 by two independent teams. One team was led by Sir Martin John Evans (Nobel Prize in Medicine and Physiology in 2007) from Great Britain and Matthew H. Kaufman. The other team was led by Gail R. Martin from the University of California, San Francisco (Evans and Kaufman 1981; Martin 1981). In 1998, the James A. Thomson team from the Genome Center of Wisconsin (Madison, United States), published the derivation of five human ESC lines from

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20 men and women. Two were derived from female (XX) and three from male (XY), from fourteen ICM isolated from human blastocysts (Thomson, Itskovitz-Eldor et al. 1998).

In our present work, we used CGR8 and D3 cell lines for mESC and H1 and H9 cell lines for hESC (Fig. 1).

Figure 1: mESC cell line (CGR8) et hESC cell line (H1). Phase contrast.

The obtained cell lines could be propagated in culture in an undifferentiated state and could be differentiated towards cells of the three germ layers (Itskovitz-Eldor, Schuldiner et al.

2000). ESC are characterized by three distinguishing features:

1. They are capable of differentiating into all cell types derived from the three germ layers; in other words, they arepluripotet. ESC are not totipotent, as it is sometimes written (Bernstein and Breitman 1989; Robbins, Doetschman et al. 1992). This term is usually reserved for blastomeres during the cleavage stage of the embryo and for the fertilized egg, meaning that cells have complete developmental potency. This

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21 description was used because ESC can make germ cells (Pease and Williams 1990).

Although ESC can produce all somatic cells, they cannot produce cells of the trophectoderm. Because of that, they cannot give rise to a complete blastocyst and cannot generate an embryo.

2. They havevirtually limitless proliferation. These cells synthesize telomerase so they do not have the replication limit common to normal primary cell lines.

3. They are self-renewing and can maintain an undifferentiated state (Nichols and Smith 2011). ESC is of major interest for the study of early developmental steps. Indeed, they allow mimicking in cell culture numerous events which occur during embryogenesis (Trounson 2005).

In the first part of this thesis, we focus on mouse embryonic stem cells (mESC), their differentiation toward neural lineage and their intrinsic heterogeneity. The second part of this thesis focuses on the cytomegalovirus infection potential on differentiated hESC. mESC and hESC have obvious similarities, but their important differences made one ideal for each study.

mESC: Origins, properties and propagation.

The field of ESC research has been expanding and progress has been made in the generation of ESC-derived mature tissue cells, including neurons. The first mouse ESC (mESC) were derived from mouse blastocysts in 1981. Such derivation was first reported by two groups (Evans and Kaufman 1981; Martin 1981). Before their finding, it was only

possible to establish growing cultures of pluripotent cells in vitro after teratocarcinoma

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22 formation in vivo. They reported the establishment of pluripotent cell lines isolated directly from in vitro cultures of mouse blastocysts. Their protocol of derivation was simple. It

consisted of the plating onto a feeder layer of the ICM after its immunosurgical isolation from embryos in the blastocyst stage. The culture was maintained for several days in medium supplemented with 20% fetal calf serum (FCS) and 0.1 mM 2-mercaptoethanol. The culture of ICM was disaggregated mechanistically, or with enzymes such as trypsin, and then plated again onto feeder cells. Cells are subsequently organized into colonies with a characteristic undifferentiated morphology (fig. 1).

After several passages, if these morphological cell particularities remain stable and if they can be expanded, an ESC line is established. mESC in culture proliferate quickly. Moreover, from single cells, clonal populations can be initiated (Amit and Itskovitz-Eldor 2009).

Expansion of mESC gives an undifferentiated population, expressing a range of

characteristic markers such as Nanog, Oct4, Sox2, SSEA1, Tra1-60, STELLAR and KLF4 (Berrill, Tan et al. 2004; Nunomura, Nagano et al. 2005). The cell morphology is relatively homogenous, consisting of small, round and stringent cells. ESC self-renewal is

symmetrical. It is confirmed when we obtain chimeric mouse from ESC injection in the ICM.

They have an infinite expansion and important populations can be quickly generated.

Contrary to other primary cultures, ESC do not show senescence and thus appear to be immortal. Finally, mESC lines are defined based on retrospective function. A chimera must be generated from ESC injection into the blastocyst. Usually, 10 to 15 cells are injected but only one or two will provide most of the somatic tissues (Wang and Jaenisch 2004).

The maintenance of these cells requires some critical factors in addition to the necessary nutrients and metabolites for cell survival. The first protocols considered feeder cells as essential. The Smith team showed that feeders cells can be substituted by special

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23 components that will stimulate cell trophism and keep the cell undifferentiated (Smith and Hooper 1987). Two publications one year later showed that a cytokine, the leukemia inhibitory factor LIF, was enough to maintain ESC self-renewal even in the absence of feeder cells (Smith, Heath et al. 1988; Williams, Hilton et al. 1988). This cytokine, originally produced by feeder cells in presence of ESC, is now directly added to the medium (Rathjen, Nichols et al. 1990). In an ESC culture of feeder cells, if they have genetic deficiency of the Lif gene, the ESC cannot grow effectively. ESC still proliferate but a differentiation can be observed fastly (Stewart, Kaspar et al. 1992). After few days, the cells become differentiated and do not express pluripotency markers.

The receptor gp130 seems to be involved in the LIF effect (Yoshida, Chambers et al. 1994).

Differentiation is blocked due to the activation of gp130 by LIF. The signaling processes involve the recruitment of a JAKkinase followed by the activation of transcription factor STAT3 (Burdon, Chambers et al. 1999; Burdon, Stracey et al. 1999). The activation of STAT3 is necessary to keep mESC undifferentiated, but the activation of other pathway’s members is not essential (Matsuda, Nakamura et al. 1999). STAT3 activation or inhibition can also promote differentiation in various cell types such as myeloid cells, astrocyte

precursors and hepatocytes (Kishimoto 1994). The signal transduction pathway is therefore nonspecific to mESC. In mouse early embryo, gp130 and LIF are expressed but not

necessary in pre-gastrulation events (Stewart, Kaspar et al. 1992; Ware, Horowitz et al.

1995; Nichols, Davidson et al. 1996; Yoshida, Anand-Apte et al. 1996).

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24 Comparison with hESC.

After derivation of mouse (Evans and Kaufman 1981; Martin 1981) and monkey (Thomson, Kalishman et al. 1995) ESC lines, hESC were finally derived in 1998 from in vitro fertilization spare embryos by the group of J.A. Thomson (Thomson, Itskovitz-Eldor et al. 1998).

Though mESC differentiate readily when deprived of LIF, this is not the case of hESC. hESC need a feeder support or a special matrix for growing and maintaining their undifferentiated state. We still do not know which factors are involved in maintaining the undifferentiated status and the proliferation in hESC. In the absence of feeder cells or a matrix, various differentiated morphologies emerge (Reubinoff, Pera et al. 2000). Markers of endoderm and mesoderm appear. The major cell types that appear have not yet been fully characterized (Mandal, Tipnis et al. 2006; Chen, Kuo et al. 2007).

As for mESC, hESC were isolated from the inner cell mass of blastocyst-stage embryos. The Thomson team used mouse embryonic fibroblasts (MEF) as feeder cells to support hESC and the original medium contained serum. Cell propagation is now well controlled and hESC can be propagated for extended periods (Aguilar-Gallardo, Poo et al. 2010; Wu, Xu et al.

2010). Experiments were performed to improve the medium for more defined constituents (Hisamatsu-Sakamoto, Sakamoto et al. 2008; Tecirlioglu, Nguyen et al. 2010). Indeed, serum is problematic because of the unknown protein composition. Some variability in culture is observed and this limits the robustness of the culture. Subsequently, animal serum was replaced by KnockOut Serum Replacement (SR). SRs have a more defined formula and contain transferrin, insulin and bovine albumin (Inzunza, Gertow et al. 2005). Unlike MEF, hESC can be cultivated on laminin-coated plates, on fibronectin matrices or on matrigel- coated plates (Xu, Inokuma et al. 2001; Amit, Shariki et al. 2004; Pakzad, Totonchi et al.

2010). The exact composition of Matrigel is not divulged by manufacturer, but it is known to

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25 contain collagen IV entactin, laminin and heparin sulfate proteoglycans (HSPGs). Medium can be supplemented with basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), nicotinamide, activin A and transforming growth factor (TGF) (Beattie, Lopez et al.

2005).

Most of hESC properties are the same as those seen in mESC. They have 1) an unlimited self-renewal capacity, 2) a stable diploid karyotype, 3) the potential to generate all somatic cell types of fetus and adult, 4) no checkpoint of G1 cell cycle, 5) an absence of X

chromosomal inactivation, 6) a potential for derivation without immortalization or transformation (Burdon, Smith et al. 2002). However, contrary to mESC, hESC do not require gp13 stimulation to remain undifferentiated (Rose-John 2002). Differentiation for hESC is longer than for mESC, which can be explained by the formation processes of gastrulation and organogenesis that needs approximately 4 days in vivo for mouse (Shemer, Kafri et al. 1991) but in humans needs between two and five weeks post-fecundation. We still ignore many of the precise events occurring during human primary tissue specification. It is thus hard to efficient;y differentiate specific cell types from hESC.

mESC and hESC differentiation.

mESC and hESC provide an exceptional framework to understand the cellular mechanisms involved in developmental and pathological processes (Wdziekonski, Villageois et al. 2007;

Shiraki, Umeda et al. 2008; Borowiak, Maehr et al. 2009). They allow the dissection of interactions between the cell and its cellular environment. They theoretically give access to all cell phenotypes that may play a role in these processes. This understanding is made possible thanks to the modern techniques of molecular and cell biology. Now the ability to amplify a more specific cell population from ESC, or to purify a cell population of interest,

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26 allows for genomic, transcriptomic and proteomic studies (Doss, Chen et al. 2007; Doss, Wagh et al. 2010; Tsai, Chou et al. 2011). The costs of these approaches are decreasing, so liked with ESC culture they provide a powerful tool in many biological fields. Among these areas, the modeling of diseases such as those caused by neurotropic viruses is one important application in basic science (Markus, Grigoryan et al. 2011). Some viral infections involve tissues hard to obtain and study. For example it is hard to take a tissue sample from the brain to study a neurotropic virus like herpes simplex 1 or from the lung to study influenza virus. In such cases, primary cell lines are rare and studies with non-immortalized cell lines are complex. mESC or, more recently, hESC can provide such cells and make possible these studies.

mESC differentiated into embryoid bodies.

The three distinguishing features are maintained in part in mESC by LIF. This cytokine is routinely added to the culture medium of mESCs and the removal of LIF results in a rapid differentiation of mESCs. mESC differentiation is usually triggered by putting them in suspension culture without LIF. This protocol allows the formation of multi-differentiated structures. The resulting cell aggregates are called embryoid bodies (EBs). The generation of various cell types is due to the reactivation of the ICM, the epiblastic developmental program in ESC. The protocol using cell culture suspension was originally developed by the Martin team with embryonic carcinoma (Martin and Evans 1975; Martin, Wiley et al. 1977).

In EBs, due to the absence of axial organization and body plan elaboration, the differentiation process is disorganized (Doetschman, Eistetter et al. 1985). Despite this, the timing of differentiation is similar to the early developing embryo and numerous cell types are created. Some cell types can be promoted with the addition of specific factors, medium components or a subsequent attachment (Kurosawa, Imamura et al. 2003). For example, the

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27 addition of retinoic acid causes ESC differentiation into neurons (Rohwedel, Guan et al.

1999). Even if factors are added, EBs cultures always contain a heterogenous cell population. Using EBs formation, cells expressing gut endoderm marker gene, pancreatic markers or hepatocyte markers can be obtained. Such cells have been reported in several publications since 1996 (Abe, Niwa et al. 1996; Levinson-Dushnik and Benvenisty 1997;

Hamazaki, Iiboshi et al. 2001; Lumelsky, Blondel et al. 2001).

Cells with the neural crest phenotype can also be found in varying proportions. (Motohashi, Aoki et al. 2007; Pomp, Brokhman et al. 2008). EBs staining will invariably show markers from the three germ layers (Table 1). The percentage of cells from each category will change depending on the EB protocol used (Imamura, Cui et al. 2004; Niimi, Kim et al. 2005;

Weitzer 2006).

The induction of a particular lineage needs a specific condition. For most of the lineages of interest, such conditions are not yet well defined. Factors, environment, and cell interactions are important for such differentiation. The protocol determination for EBs is purely empirical.

Results depend on the addition of retinoic acid, phenazopyridine or fibroblast growth factor (FGF) to the medium (Suter, Preynat-Seauve et al. 2009).

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Germ layer Organogenesis Markers

Endoderm Epithelial lining of digestive tube.

Lungs, Liver, Pancreas, Thyroid…

chymotrypsin, collagen iv, glucagon, insulin, nestin, neuro d, pancreatic,

islet cell, PAX-6 , PDX-1…

Mesoderm Heart, skeletal muscle, kidney, spleen…

cartilage proteoglycan, cardiotin, cd34, gata-4 [gata binding factor],

myosin, prominin-1, renal cell, carcinoma, troponin i…

Ectoderm Central nervous system, nerves, mammary glands…

GFAP, nestin, PAX6, SOX1, gbx, ren, prominin1, O1, LIM1,

vimentin…

Table 1 : Germ layers, organogenesis and some typical markers.

The Nishikawa team researching EB differentiation plated the cells on a collagen substratum (Selezneva, Savintseva et al. 2006). They showed that differentiation is not systematically linked with complex cell interactions occurring in EBs. Mesodermal subsets were also generated with the same protocol, producing endothelial and hematopoietic progenitors isolated by FACS (Nishikawa, Nishikawa et al. 1998). Though EBs are interesting to obtain in a mixed cell population, the major interest in mESC culture is to obtain a particular cell type. For this purpose, selective culture conditions are needed.

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29 mESC differentiated into cardiac cells.

mESC culture protocols have dramatically evolved over the last two decades. Laboratories developed their own techniques to obtain cells with interesting phenotype for them. Major advances have been made in the understanding of the pathways involved in differentiation and the epigenetic mechanisms that are linked. The main protocols use EBs formation to initiate cell differentiation. Cardiac subtypes that can be obtained are phenotypically similar to atrial, nodal and ventricular cells (Fuegemann, Samraj et al. 2010).

A battery of markers can be used to determine cardiac cell type, including Annexin VI, Caveolin-2, PECAM1 or CD66 (Table 2). Cardiac cells are particularly interesting for biomedical applications such as cell replacement therapies, drug screening and basic research (Lee, Jeong et al. 2001; Fujiki, Johnson et al. 2009). Obtaining cardiac cell subtypes allows the investigation of differentiation and development of cardiomyocytes. Most classical differentiation methods involve a hanging-drop or mass culture method (Fuegemann, Samraj et al. 2010; Chen, Lin et al. 2011). Specific factors, such as the bone morphogenetic protein 2 (BMP2), can improve cardiac differentiation, when added early in the EB medium (Zhang and Bradley 1996; Sachinidis, Fleischmann et al. 2003; Kawai, Takahashi et al. 2004). Most recent protocols use hanging-drop method in medium supplemented with BMP2 (Chiriac, Nelson et al. 2010). Cardiomyocytes are isolated from EBs with trypsin. Another way to generate cardiomyocytes is to proceed in a mESC co- culture with neonatal cardiac mesenchymal cells with specific growth factors (Condorelli, Borello et al. 2001).

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30 mESC differentiated into neural cell lines.

ESCs theoretically give access to any cell type in the adult organism after directed differentiation (Burdon, Smith et al. 2002). To achieve this goal, we must master the steps leading undifferentiated ESC to the cell population of interest. Developmental biology over the last century provides the basics to understand the cellular and molecular mechanisms of embryonic development (Zimmerman, Parr et al. 1994; Vittet, Prandini et al. 1996). During development, epiblastic cells are directed to the three germ layers during gastrulation (Rasweiler and Badwaik 1996). Once cells have this identity, the next developmental stages generate more than 200 different cell types. The international research community is trying to recreate the sequence of signals discovered by embryology studies that have a function in cell differentiation (Schnerch, Cerdan et al. 2010).

At present, it is largely possible to reproduce the first stages of embryonic neurogenesis with ES cells in vitro. Even if it is possible now to obtain high numbers of NPC and neurons in cell culture, we are still unable to obtain a homogenous cell population with a specific phenotype (Ko, Park et al. 2007; Erceg, Ronaghi et al. 2009; Nasonkin, Mahairaki et al. 2009;

Mahairaki, Lim et al. 2011). Moreover, when we use the protocols described above, some non-neural groups of cells frequently persist during differentiation.

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31

Cell line Main markers

Undifferenciated ESC Genesis (FOXD3), KLF4, Nanog, Oct4, SOX2, SSEA3 and 4, SSEA1 (only for mESC), Telomerase, TRA-1.

Cardiac cells GATA6, cardiac troponin, annexin VI, CD66 Neuroprecursor cells Musashi, nestin, vimentin, PAX6, SOX1

Neurons DAT, GABA, MAP2, peripherin, synapsin 1, TAU, βIII- tubulin, neurofilament.

Astrocytes GFAP, S100-protein, vimentin

Oligodendrocytes A2B5, CNPase, O1, O4

Table 2: Cell lines of interest obtained after ESC differentiation in this work and main markers used.

hESC differentiated into neural cell lines.

Neural induction of hESC is based on 1) the EBs formation, 2) the ESC culture on a feeder layer of stromal cells, 3) the spontaneous differentiation of ESC (Guillaume, Johnson et al.

2006; Ko, Park et al. 2007). These protocols were developed primarily in mice, and lead to the formation of neural precursor cells (NPC). In culture, NPC take the form of neurospheres in suspension or adherent neural rosettes (Fig. 2).

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32 Figure 2: Neurosphere (left) and adherent neural rosette (right), phase contrast.

These precursors can be differentiated into functional neurons or into glial cells (Tian, Bai et al. 2005; Di Giorgio, Boulting et al. 2008). The term “neurosphere” refers to multicellular aggregates derived from adult or fetal neural stem cells cultured in vitro in the presence of mitogenic factors EGF and FGF2 (Sanalkumar, Vidyanand et al. 2010; Zeng, Fabb et al.

2011). Neural rosettes are formed by neuroepithelial cells or NPC that are polarized. They are organized in circular structures and are characterized by the expression of neuroectodermal markers such as Nestin, Sox1, Pax6 and NCAM (Table 1). This organization is similar to neural tube organization. Neural induction of hESC has been explored for more than 10 years (Zeng, Fabb et al. 2011).

Undifferentiated hESC maintained for several weeks in culture on feeder cells (MEF), without passaging or medium changes, lead to the formation of areas with the characteristic morphology of rosettes. These areas are characterized by the expression of neuroectodermal markers Nestin and Pax6 then by the marker NCAM (Jelitai, Anderova et al. 2004; Parekkadan, Berdichevsky et al. 2008). These cells can be isolated mechanically and cultured in suspension as neurospheres with growth factors EGF and FGF2 in the medium (Kalyani, Mujtaba et al. 1999). This technic was described first for fetal or adult

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33 NSC. However, these conditions generate mixed cell populations containing various ectodermal, endodermal and mesodermal cells. Based on the work with the mESC, various strategies were then considered. In the early 2000s, a new protocol, based on the co-culture of ESC with bone marrow stromal cells (PA6, MS5 and S17), was described for mESC (Wang, Zhao et al. 2005; Shintani, Nakao et al. 2008). This technique, also known as Stromal Cell-Derived Inducing Activity (SDIA) allows, for mESC, a fast and efficient neural induction without expression of mesodermal markers (Kawasaki, Mizuseki et al. 2000;

Kawasaki, Mizuseki et al. 2002). The idea of using feeder cells derived from mesoderm is based on the observation that mesodermal cells produce, in vivo, important signals for ectodermal induction. For example, bone morphogenetic proteins (BMPs) antagonists like noggin or chordin are essential during morphogenesis for ectodermal induction (Kuroda, Wessely et al. 2004). The transposition of this protocol to hESC, after culture on MS5 stromal cells, allows production of many NPC with neural rosette organization. After approximately 4 weeks of co-culture in serum-free medium, over 90% of the NPC obtained express the neuroectodermal markers Nestin, Sox1 and Pax6. These cells also do not maintain differentiation markers Oct-4 and Nanog. Moreover, there is no expression of meso-or endodermal markers (Rieske, Krynska et al. 2005). However, the use of stromal cell lines induces variation in the induction of cell function (Yang, Tsang et al. 2009). It is then possible to overcome the stromal cells. For example, a laminin support can promote neural induction (Kuang, Xu et al. 1998; Miyazaki, Futaki et al. 2008; Vuoristo, Virtanen et al. 2009).

Very few growth factors are currently used to improve neural induction. Inhibition of neuroectoderm formation by BMPs signals in vivo has led some teams to test in vitro the action of BMPs antagonists such as Noggin. Their work demonstrated that neural induction of hESC cells, based on the formation of EBS or on co-culture with feeder cells, was enhanced in the presence of Noggin (Munoz-Sanjuan and Brivanlou 2002; Cazillis, Rasika et al. 2006). Culture medium also plays an essential role in neural induction. Indeed, the use of

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34 medium supplemented with serum, as described in the first experiments in mice, is not good for neural induction. One explanation is that serum may contain BMPs, which have an inhibitory role on neural pathways. Then, the protocols for neural induction of undifferentiated hESC cells rapidly converged on the use of minimal serum-free medium supplemented with N2 (Peng and Chen 2005). Initially developed for primary cultures of neurons and subsequently used for the neuronal differentiation of mESC, this medium combines five molecules: transferrin, insulin, putrescine, progesterone and selenium.

Transferrin and insulin are essential for the proliferation of neuronal precursors. Transferrin, putrescine, and progesterone have neuroprotective activity. Selenium protects against excitotoxicity and promotes neuronal survival (Suter and Krause 2008). Finally, putrescine plays also a role in axonal growth. Some protocols use another supplement called B27 (Svendsen, Fawcett et al. 1995), which is more complex than N2 and has more than twenty compounds including, vitamins, hormones, fatty acids, antioxidants and growth factors. The exact composition of this supplement is not revealed by the manufacturer (Suter and Krause 2008). With these supplements it was possible to define conditions for neural induction from undifferentiated hESCs to be as effective and fast as on MS5 cells, but without the need for feeder cells.

hESC lines have been derived using many different culture conditions. The first hESC lines were derived in the presence of irradiated MEFs, in a medium containing 80% DMEM high glucose, 20% FBS, L-Glutamine, β-mercaptoethanol and non- exxential amino-acids (Thomson, Itskovitz-Eldor et al. 1998). Shortly after, the group of J.A. Thomson described a more suitable medium containing 4 ng/ml basic fibroblast growth factor (bFGF) and, instead of FBS, 20% KnockOut Serum Replacement (SR), a defined formulation from Invitrogen (Itskovitz-Eldor, Schuldiner et al. 2000). In this work for ESC derivation we used the following protocol in three

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35 steps: In Vitro Differentiation of Neurons from hESC require a first step with the aggregation of ES cells and therefore the formation of embryonic bodies. The second stem leads to neuroepithelial differentiation (formation of neural tube-like rosettes). The third step is the isolation of neuroepithelial cells in the Neural Tube- Like Rosettes and Differentiation of neurons (Zhang, Wernig et al. 2001; Dhara and Stice 2008).

Heterogeneity of mESC.

mESC lines are defined based on retrospective function. A chimera must be generated from ESC injection into the blastocyst. Usually, 10 to 15 cells are injected. Only one or two will provide most of the somatic tissues (Wang and Jaenisch 2004). mESC are considered as a homogenous cell population in most of the studies. However, mESC subpopulations have been found in established mESC lines. These subpopulations are phenotypically and functionally heterogeneous. They are partially characterized and support the hypothesis that mESC contain distinct subpopulations with various differentiation potential (Hayashi, Lopes et al. 2008), in a dynamic equilibrium. Despite their differences, their detection is complicated partly because they are transient. mESC are similar to three cell groups of mouse embryo. All of them express pluripotency marker genes such as Oct4 (Pesce, Gross et al. 1998; Chambers and Smith 2004). They exist in primordial germ cells (PGC), cells from the epiblast and cells from the ICM (Zwaka and Thomson 2005; Surani, Hayashi et al.

2007).

These various cells have similar properties to mESC. They are pluripotent and have indefinite self-renewal capacity. In established mESC lines, heterogeneous expression of

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36 markers such as SSEA1, Pecam1 or Nanog is proved (Cui, Johkura et al. 2004; Furusawa, Ikeda et al. 2006; Payer, Chuva de Sousa Lopes et al. 2006; Chambers, Silva et al. 2007;

Toyooka, Shimosato et al. 2008). One marker, Stella, is used to differentiate epiblastic cells with cells from the ICM. This marker is repressed in the epiblast and expressed in PGC only after their specification. The gene Stella is regulated by chromatin-based modifications and repressed by DNA methylation (Sato, Kimura et al. 2002; Payer, Chuva de Sousa Lopes et al. 2006), but a staining for the Stella marker in mESC shows intrinsic cell heterogeneity.

Several studies have attempted to elucidate how this heterogeneity is compatible with self- renewal and pluripotency. The team of Katsuhiko Hayashi showed that a relatively constant percentage of Stella imunoreactive cells and Stella negative cells is maintained in a mESC population (Hayashi, Lopes et al. 2008). Two hypotheses remain about the origin of these two populations: 1) an epigenetic origin and 2) an environmental origin. Stella is not the only marker found as heterogenous in mESC. Nanog and Stella have also various expression levels (Singh, Hamazaki et al. 2007; Toyooka, Shimosato et al. 2008).

The functionality of these subpopulations was then investigated (Kalmar, Lim et al. 2009).

Studies showed that Nanog-negative cells cannot generate germinal cells but can contribute to all other somatic linages (Chambers, Silva et al. 2007).

ESC, culture targets.

Over 30 years ago, embryonic stem cells were found to spontaneously differentiate toward many cell types in vitro, including to neurons (Martin 1981). Unfortunately it is not yet possible to obtain a pure population of a particular cell type. At present one of the major

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37 challenges in the use of hESC pluripotency is to direct their cell fate to a specific phenotype (Yabut and Bernstein 2011). It is important to obtain a pure cell population because of the risk of tumor formation upon in vivo implantation (Dressel, Schindehutte et al. 2008). In fact, even after differentiation, there is a persistence of poorly differentiated cells.

Initial works on neural induction of hESC, described above, have demonstrated the ability of neural precursors to differentiate into functional neurons and into glial cells. The nervous system development is controlled by interactions between neural precursors and morphogens in a precise spatial and temporal anteroposterior sequence. From embryologic data, several studies tries to determine the essential molecules needed to guide the NPC specification to a specific neuronal or glial subtype. A protocol published in 2003 by Barberi demonstrated the ability to control the fate of neural precursors, obtained by culturing cells on a layer of stromal cells such as PA6, MS5 and S17 (Barberi, Klivenyi et al. 2003). These NPC grow according to a specific temporal sequence of growth factors put in the medium.

The aim is to mimic some in vivo neural development signals. During such process, NPC can differentiate into various subtypes of neurons and glial cells.

Despite years of research and the generation of a multitude of different protocols, ESC differentiation into neurons still does not provide a homogeneous population (Francis and Wei 2010). A significant heterogeneity in cell phenotype appears in the early stages of differentiation (Martinez, Bena et al. 2011).

Based on this observation, we designed a protocol to understand the reasons for this heterogeneity. Several studies already demonstrate the fact that ESC lines used are already a heterogeneous cell population. No studies could explain the difficulties in obtaining a

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38 specific neural cell type consisting of 100% cells expressing the same phenotype at the same time (Martinez, Bena et al. 2011). Previous studies showed that the ICM is already heterogeneous 3.5 days after fertilization. Even if all the cells express transcription factors such as Oct4 or Nanog that are characteristics markers of undifferentiated ESC, some factors like GATA-6 do not appear in all inner cell mass cells (Singh, Hamazaki et al. 2007).

Still, none of these studies showed the presence of stable subpopulations in ESC cultures.

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Neural differentiation of enbryonic stem cells: clonal properties and sensitivity to cytomegalovirus infection

Thesis

Reference