Fontes alternativas de células β têm sido estudadas para o tratamento de Diabetes mellitus tipo 1, dentre as quais a mais promissora consiste das células-tronco diferenciadas em células produtoras de insulina (IPCs). Alguns trabalhos demonstram a capacidade de células-tronco embrionárias murinas (mESCs) de formarem estruturas semelhantes a ilhotas pancreáticas, porém, os níveis de produção de insulina são insuficientes para a reversão do diabetes em camundongos diabetizados. Este trabalho visa desenvolver um protocolo adequado para geração de IPCs e contribuir para a identificação e caracterização funcional de novos genes associados à organogênese pancreática. Logo no início da diferenciação das mESCs em IPCs, foi possível verificar o surgimento de células progenitoras, evidenciado pela expressão de marcadores importantes da diferenciação beta-pancreática. Ao final do processo de diferenciação in vitro, ocorreu a formação de agrupamentos (clusters) semelhantes a ilhotas, corando positivamente por ditizona, que é específica para células β-pancreáticas. Para avaliar seu potencial in vivo, estes clusters foram microencapsulados em Biodritina® e transplantados em camundongos diabetizados. Apesar dos níveis de insulina produzidos não serem suficientes para estabelecer a normoglicemia, os animais tratados com IPCs apresentaram melhores condições, quando comparados ao grupo controle, tendo melhor controle glicêmico, ganho de massa corpórea e melhor aparência da pelagem, na ausência de apatia. Além disso, análise dos clusters transplantados nestes animais indicou aumento da expressão de genes relacionados à maturação das células β. Porém, quando estes clusters foram microencapsuladas em Bioprotect® e submetidos à maturação in vivo em animais normais, ocorreu um aumento drástico na expressão de todos os genes analisados, indicando sua maturação completa em células beta. O transplante destas células completamente maturadas em animais diabetizados, tornou-os normoglicêmicos e capazes de responder ao teste de tolerância à glicose (OGTT) de forma semelhante aos animais normais. A segunda parte do trabalho visou analisar genes diferencialmente expressos identificados em estudo anterior do nosso grupo, comparando, através de DNA microarray, mESCs indiferenciadas e diferenciadas em IPCs. Um dos genes diferencialmente expressos é aquele que codifica para a Purkinge cell protein 4 (Pcp4), sendo 3.700 vezes mais expresso em IPCs. Para investigar o possível papel do gene Pcp4 em células β e no processo de diferenciação β-pancreática, adotou-se o enfoque de genômica funcional, superexpressando e inibindo sua expressão em células MIN-6 e mESCs. Apesar da alteração na expressão de Pcp4 em células MIN-6 não ter interferido de forma expressiva na expressão dos genes analisados, quando inibido, modificou o perfil da curva de crescimento celular, aumentando seu tempo de dobramento de forma significativa e diminuindo da viabilidade celular em ensaios de indução de apoptose. Já na diferenciação de mESCs em IPCs, a superexpressão de Pcp4 interferiu de forma positiva apresentando uma tendência a aumentar a expressão dos genes relacionado à diferenciaçãoβ-pancreática. Concluindo, desenvolvemos um novo protocolo de diferenciação de mESCs em IPCs as quais foram capazes de reverter o diabetes em camundongos diabetizados e descrevemos, pela primeira vez, o gene Pcp4 como sendo expresso em células β-pancreáticas, podendo estar relacionado à manutenção da viabilidade celular e maturação destas células. / New cellular sources for type 1 Diabetes mellitus treatment have been previously investigated, the most promising of which seems to be the insulin producing cells (IPCs), obtained by stem cells differentiation. Some reports show that murine embryonic stem cells (mESCs) are able to form islet-like structures, however, their insulin production is insufficient to render diabetic mice normoglycemic. This work aims at developing an adequate protocol for generation of IPCs and searching for new genes which could be involved in the pancreatic organogenesis process. Early on during mESCs differentiation into IPCs, we observed the presence of progenitor cells, which were able to express pancreatic β-cell markers. At the end of the differentiation process, the islet-like clusters positively stained for the insulin-specific dithizone. These clusters were microencapsulated in Biodritin® microcapsules, and then transplanted into diabetized mice. Although the levels of insulin production were insufficient for the animals to achieve normoglycemia, those which received IPCs displayed improved conditions, when compared to the control group, as judged by a better glycemic control, body weight gain and healthy fur appearance, in the absence of apathy. In addition, when these transplantated clusters were retrieved, high levels of expression of the genes related to β-cell maturation were detected. IPCs were also microencapsulated in Bioprotect® and subjected to in vivo maturation in normal animals. A dramatic increase of the analyzed genes expression was observed, indicating complete maturation of the differentiated cells. When these cells were transplanted into diabetized mice, these animals achieved normoglycemia and were able to display glucose tolerance test (OGTT) response very similar to that of normal mice. In the second part of this work, we analyzed upregulated genes described in previous work from our group, comparing undifferentiated mESCs to IPCs using a microarray platform. One of these genes is that coding for the Purkinje cell protein 4 (Pcp4), which is 3,700 more expressed than in undifferentiated mESC cells. We adopted a functional genomics approach to investigate the role played by the Pcp4 gene in β-cells and in β-cell differentiation, by inducing overexpression and knocking down this gene in MIN-6 and mESC cells. Although the differential expression of Pcp4 in MIN-6 was not able to interfere with the expression of the genes analyzed, we observed different cell growth rates, with increased doubling time and decreased cell viability when its expression was knocked down. In addition, overexpression of Pcp4 in mESCs subjected to differentiation into IPCs apparently increases the expression of genes related to β-cell differentiation. In conclusion, we developed a new protocol for ESCs differentiation into IPCs, which is able to revert diabetes in diabetized mice, and we also describe here, for the first time, the Pcp4 gene as being expressed in pancreatic β-cells and possibly being related to maintenance of cell viability and β-cell maturation.

GNAS est un locus complexe soumis à l'empreinte parentale. Il code pour cinq transcrits alternatifs dont l’expression est régulée de manière parentale, tissulaire et développementale : la sous-Unité alpha stimulatrice de la protéine G hétérotrimérique (Gαs), XLαs, NESP55, et deux ARNnc, A/B et GNAS-AS1. Gαs est une protéine clé dans la transduction hormonale partageant avec XLαs la capacité de produire l'AMPc intracellulaire après stimulation des récepteurs couplés à Gαs.Dans la première partie de ma thèse, je me suis concentrée sur l'étude du rôle des transcrits de GNAS, en particulier XLαs, dans la croissance fœtale et post-Natale. J’ai profité du modèle unique des pseudohypoparathyroïdies (PHPs), pathologies humaines rare de l’empreinte, causées par des anomalies génétiques ou épigénétiques du locus GNAS altérant le dosage génique des transcrits de GNAS. La croissance anormale est une caractéristique majeure des PHPs.Dans la deuxième partie de ma thèse, j’ai étudié le profil épigénétique du locus GNAS (méthylation de l'ADN et expression des transcrits) dans les cellules souches humaines embryonnaires -HESCs-, dans les cellules pluripotentes induites dérivées à partir de fibroblastes de sujets sains -IPSCs- et dans les cellules redifférenciées en cellules souches neurales et mésenchymateuses. La caractérisation précise du locus humain GNAS en physiologie (cellules souches) et pathologie (PHP) est essentielle pour une meilleure compréhension des processus développementaux importants comme la croissance. L'exploration du phénotype "croissance" de différents types de PHPs a permis de mieux comprendre le rôle des transcrits du locus GNAS dans la physiologie et la physiopathologie. L'analyse de cellules des PHPs a permis de mieux caractériser l’impact des anomalies moléculaires du locus GNAS en pathologie humaine. Les hiPSCs peuvent être un outil utile pour étudier les modifications épigénétiques au niveau du locus GNAS. / GNAS is a complex locus subjected to parental imprinting encoding five parental-, tissue- and developmental-Manner regulated transcripts : the alpha stimulatory subunit of the G protein (Gαs), XLαs, NESP55, and two ncRNAs, A/B and the antisens GNAS-AS1. Gαs is a key protein in hormonal signaling sharing with XLαs the ability to produce intracellular cAMP upon stimulation of Gαs-Coupled receptors. In the first part of my thesis, I focused on studying the role of the GNAS transcripts, particularly XLαs, in fetal and postnatal growth. I took advantage of the unique model of pseudohypoparathyroidism (PHP), a rare human disease, caused by genetic or epigenetic abnormalities at the GNAS locus leading to various combinations of GNAS transcripts alterations. Abnormal growth appears to be a major feature of PHP. In the second part of my thesis, I studied the epigenetic pattern of GNAS (DNA methylation and transcripts expression) in human embryonic stem cells -HESCs-, in induced pluripotent stem cells -IPSCs- derived from fibroblasts from healthy individuals, and in cells re-Differentiated from these stem cells in neuronal and mesenchymal cells. The precise characterization of the human GNAS locus in physiology (stem cells) and pathology (PHP) is critical for a better understanding of major processes like growth. Through exploration of the "growth" phenotype of different groups of PHPs we have participated to the better understanding of the role of the GNAS transcripts in the physiology and pathophysiology. Human iPSCs may be an useful tool to study epigenetic modifications at the GNAS locus.

This dissertation is the compilation of three separate works of research revolving around the theme of regulation of biomedical technologies that are either emerging or that have undergone significant developments over the past decade or so. Each of these three research works examines a legal response to a technological development in the areas of biotechnology and/or medicine and addresses one or more challenges - ethical, constitutional, legal or one that is related to public policy - created by that response. The first work of research, which was published in the Administrative Law Review in March 2008, examines the legality of the restrictions imposed by the administration of President George W. Bush on the funding of research involving human embryonic stem cells. Reaching the conclusion that the Bush Administration's actions were outright illegal in more than one way, the research highlights existing tensions in the division of decision-making power between the President and executive agencies and between Congress and the President. The second work of research, which was published in the Columbia Science and Technology Law Review in August 2010, reviews the regulation of genetic screening and testing of donated reproductive tissue in the United States. Analyzing the regulation in the federal, state and industry level, the research highlights significant shortcomings of the regulation of this area and, drawing on the experience of other countries, advocates the regulation of this area by the FDA. The third and last work of research of which this dissertation consists is dedicated to the examination of the newly created regime of statutory exclusivities afforded to biological pharmaceuticals under the Biologics Price Competition and Innovation Act (BPCIA) as it compares to the protection afforded to such products under patent law. The research concludes that allowing biological pharmaceuticals to benefits from parallel protection under both patent law and the statutory exclusivities regime established under BPCIA does not contribute to incentivizing innovation and might have undesirable ramifications from a public policy perspective. Hence, the research proposes limiting the protection afforded to biological pharmaceutical products, namely to the protection under either patent law or BPCIA, by suspending the ability to enforce patents covering biological pharmaceuticals against generic applicants under BPCIA. In addition, the research examines the proposition that under some circumstances it would be possible to substitute patent protection for statutory exclusivities.

Die vorliegende Arbeit beschäftigt sich mit der Bildgebung magnetisch markierter Stammzellen im ZNS mittels Magnetresonanztomographie. Dazu wurden Stammzellen mit Eisenoxidnanopartikeln (VSOP, very small superparamagnetic iron-oxide particles) in vitro effizient und ohne zusätzliche Lipofektionsagenzien magnetisch markiert. Es zeigte sich keine wesentliche Beeinflussung der Vitalität, Proliferation und Differenzierungsfähigkeit sämtlicher untersuchter Zellpopulationen. Zur Evaluierung der Grenzen der zellulären MR-Bildgebung wurde das Detektionslimit magnetisch markierter embryonaler Stammzellen in vivo nach intrastriataler Injektion im Gehirn der Ratte untersucht. Es ließen sich bei einer Feldstärke von 17,6 T weniger als 100 magnetisch markierte Zellen sicher vom Hirnparenchym abgrenzen. Die histologische Korrelation bestätigte den zellulären Ursprung der beobachteten T2*-Hypointensitäten. In einem Rattenmodel des Morbus Parkinson konnte eine spezifische Detektion der intrastriatal injizierten magnetisch markierten embryonalen Stammzellen über einen Zeitraum von 6 Monaten erreicht werden. Es konnte keine signifikante Migration der Zellen festgestellt werden, jedoch fanden sich große interindividuelle Unterschiede in ihrer räumlichen Verteilung. In der histologische Analyse stellten sich auch sechs Monate nach der Transplantation im Bereich des Stichkanals eisenoxidmarkierte Stammzellen dar. In einem Mausmodell der cerebralen Ischämie wurde erstmals die Anreicherung systemisch injizierter magnetisch markierter mononukleärer Zellen kernspintomographisch erfasst. 24 - 48 h nach der Injektion magnetisch markierter Zellen stellten sich T2*-gewichtete Signalhypointensitäten im Randbereich der Ischämie dar. Insgesamt zeigte sich in dieser Studie die zelluläre Magnetresonanztomographie zu einem nicht-invasiven Nachweis einer geringen Anzahl magnetisch markierter Zellen über einen langen Zeitraum mit hoher Sensitivität in der Lage. / This thesis is dealing with the imaging of magnetically labeled stem cells in the CNS using magnetic resonance imaging (MRI). Stem cells were efficiently magnetically labeled with very small superparamagnetic iron-oxide particles (VSOP), without any lipofection agents. No significant impact on vitality, proliferation and ability to differentiate could be observed after the magnetic labeling of all cell populations investigated. Magnetically labeled embryonic stem cells were injected into the striatum of rats to evaluate their detection limit by MRI. At field strengths of 17.6 T, less than 100 cells could be discriminated from the brain parenchyma as T2*-weighted hypointensities. Histology proved the cellular origin of MRI-signal changes. In a rat model of Parkinsons’s Disease, magnetically labeled embryonic stem cells could be detected by MRI after intrastriatal injection for a time period of more than 6 months. No significant migration of transplanted cells could be observed, however significant inter-individual differences concerning the spatial distribution of cells could be found. Histologically, transplanted iron-oxide-labeled cells could still be detected in the vicinity of the injection tract six months after transplantation. In a mouse model of cerebral ischemia, the enrichment of systemically injected magnetically labeled mononuclear cells was detected non-invasively by MRI. 24 to 48 hours after injection of magnetically labeled cells, T2*-weighted hypointense signal changes could be observed in the border zone of the ischemia. Over all, this study showed that cellular MRI is capable of the sensitive non-invasive detection of small numbers of magnetically labeled cells over a long period of time.

Daten des humanen Genomprojektes zeigen, dass ca. 8% des gesamten humanen Genoms aus retroviralen Sequenzen besteht. Der überwiegende Teil dieser Proviren ist aufgrund verschiedener Mutationen defekt. Im Gegensatz zu allen anderen HERV Proviren scheinen einige HERV-K Proviren intakt zu sein und besitzen offene Leserahmen für alle viralen Proteine. Die Familie des humanen endogenen Retrovirus K HML2 umfasst ca. 30 eng verwandte Proviren. Zusätzlich zu den Strukturproteinen Gag und Env und der Reversen Transkriptase, exprimiert HERV-K zwei regulatorische Proteine, Rec und Np9. Beide sind im Nukleus lokalisiert und tumorigene Eigenschaften bzw. eine Expression in Assoziation mit Tumorgeweben wurde nachgewiesen. Neben Zelllinien, wie die Teratokarzinomzelllinie GH und einigen Brustkrebszelllinien, für die die Expression von HERV-K mRNA und die Produktion von Viruspartikeln bekannt ist, konnte die Expression von HERV-K Proteinen und Partikeln für Melanomzellen gezeigt werden. Volllängen mRNA von HERV-K war in allen untersuchten humanen Proben nachweisbar. Gespleißtes env und rec war in 39% der Gewebe und in 38% der Melanomzelllinien exprimiert. Zusätzlich werden HERV-H, -R und -W exprimiert. Von den auf spezifische Antikörper gegen HERV-K Proteine untersuchten Seren der Melanompatienten waren 16% positiv für das transmembrane Hüllprotein, jedoch reagierte kein Serum mit Re oder Np9. Da im Zuge der Entstehung von Tumoren immer auch eine Dedifferenzierung der entarteten Zellen diskutiert wird, wurde die Expression von HERVs in undifferenzierten, embryonalen Stammzellen bestimmt. In den untersuchten embryonalen Stammzellen lässt sich Volllängen mRNA, sowie gespleißte env, rec und np9 mRNA nachweisen. Während der Differenzierung zu neuronalen Vorläuferzellen sinkt die Expression jedoch wieder auf ein mit normalen Zellen vergleichbares Niveau. Obwohl gespleißte RNA und virale Proteine von HERV-K vor allem in Tumoren und Tumorzelllinien exprimiert werden, ist deren Funktion während der Tumorentstehung noch immer ungeklärt. Auch die Bedeutung der HERV-K Expression in humanen Stammzellen ist noch unklar, insbesondere in Hinblick auf eine mögliche Tumorigenität. / In contrast to all other human endogenous retroviruses, proviruses of the human endogenous retrovirus family HERV-K have maintained open reading frames for all viral proteins. Although most proviruses are defective, structural proteins Gag and Env, the reverse transcriptase and two regulatory proteins, Rec and Np9, have been described. Rec resembles the Rev protein of HIV and tumourigenic potential was confirmed. Np9 as well is located in the nucleus and expression in association with tumour tissues was observed. Additionally to cell lines known to produce HERV-K virus particles, such as the teratocarcinoma cell line GH and breast cancer cell lines, recently melanoma cells were described to express HERV-K proteins and particles. In order to study the expression of HERV-K, -H, -R and -W, in melanoma cell lines and biopsies primer sets were used. Antisera specific for HERV-K proteins were used for immunohistochemistry and sera from melanoma patients were investigated for HERV-K specific antibodies. Full length mRNAs of all HERVs were found in all human cells. Spliced env and rec of HERV-K were detected in 39% of the melanoma biopsies and in 38% of the melanoma cell lines. Expression of HERV-K in situ was shown by immunohistochemistry. In addition, 16% of the patients sera tested showed antibodies against the HERV-K transmembrane envelope protein, but no antibodies against Np9 or Rec could be detected. A certain dedifferentiation of cells as a consequence of tumour development is discussed. Therefore the expression of HERV-K in undifferentiated embryonic stem cells was investigated. The investigated stem cells showed expression of HERV-K full length, env, rec and np9 mRNA. Although the expression decreased with differentiation to neuronal precursor cells. Even though HERV-K mRNA and proteins were expressed in a high percentage of melanomas their function in tumour development is still unclear. As well as the meaning of the HERV-K expression in embryonic stem cells, particularly for a tumourigenic potential.

L’osteopetrosi autosomale recessiva (ARO) è un gruppo di malattie dovute a un difettoso funzionamento degli osteoclasti che preclude un rimodellamento osseo corretto. Nel 50% dei casi umani il difetto è dovuto ad una delezione nel gene Tcirg1. Il modello murino mutante oc/oc porta lo stesso difetto genetico e fenotipico umano. Nel lavoro di tesi si è dimostrato che gli epatociti fetali di 12.5 giorni di gestazione trapiantati in utero in feti mutati di 13.5 giorni di gestazione sono in grado di curare il fenotipo malato. Si è inoltre derivata una sottolinea di cellule staminali embrionali murine transgeniche per il costrutto plasmidico GOF18eGFP. Si vuole utilizzare la GFP sotto il controllo del promotore del gene Oct-4 come marcatore del livello di staminalità cellulare per microiniettare le ESC in blastocisti murine mutate oc/oc. / Autosomal recessive osteopetrosis (ARO) is a group of genetic disorders due to defects that preclude normal function of osteoclasts. In half the cases, human ARO is due to mutations in the Tcirg1 gene. The oc/oc mutant mouse closely recapitulates human Tcirg1-dependent ARO. In ths work we demonstrate that in utero injection of allogenic fetal liver cells on 12.5 days into oc/oc fetuses at 13.5 day post coitum completely rescue the osteopetrotic phenotype. Moreover, an embryonic stem cells line transgenic for GOF18eGFP was produced. The goal is to use the GFP under the transcriptional control of the Oct-4 promoter as a marker of pluripotency of the ESC that are to microinject into oc/oc blastocysts.

Embryonic stem cells (ESCs) are characterized by their unlimited capacity for self-renewal and the ability to contribute to every lineage of the developing embryo. The promoters of developmentally regulated loci within these cells are marked by coincident epigenetic modifications of gene activation and repression, termed bivalent domains. Trithorax group (TrxG) and Polycomb Group (PcG) proteins respectively place these epigenetic marks on chromatin and extensively colocalize with Oct4 in ESCs. Although it appears that these cells are poised and ready for differentiation, the switch that permits this transition is critically held in check. The derepression of bivalent domains upon knockdown of Oct4 or PcG underscores their respective roles in maintaining the pluripotent state through epigenetic regulation of chromatin structure. The mechanisms that facilitate the recruitment and retention of Oct4, TrxG, and PcG proteins at developmentally regulated loci to maintain the pluripotent state, however, remain unknown. Oct4 may function as either a transcriptional activator or repressor. Prevailing thought holds that both of these activities are required to maintain the pluripotent state through activation of genes implicated in pluripotency and cell-cycle control with concomitant repression of genes required for differentiation and lineage-specific differentiation. More recent evidence however, suggests that the activator function of Oct4 may play a more critical role in maintaining the pluripotent state (Hammachi et al., 2012). The purpose of the studies described in this dissertation was to clarify the underlying mechanisms by which Oct4 functions in transcriptional activation and repression. By so doing, we wished to contextualize its role in pluripotent cells, and to provide insight into how changes in Oct4 function might account for its ability to facilitate cell fate transitions. As a result of our studies we find that Oct4 function is dependent upon post-translational modifications (PTMs). We find through a combination of experimental approaches, including genome-wide microarray analysis, bioinformatics, chromatin immunoprecipitation, functional molecular, and biochemical analyses, that in the pluripotent state Oct4, Akt, and Hmgb2 participate in a regulatory feedback loop. Akt-mediated phosphorylation of Oct4 facilitates interaction with PcG recruiter Hmgb2. Consequently, Hmgb2 functions as a context dependent modulator of Akt and Oct4 function, promoting transcriptional poise at Oct4 bound loci. Sumoylation of Oct4 is then required to maintain Hmgb2 enrichment at repressed loci and to transmit the H3K27me3 mark in daughter progeny. The expression of Oct4 phosphorylation mutants however, leads to Akt inactivation and initiates the DNA Damage Checkpoint response. Our results suggest that this may subsequently facilitate chromatin reorganization and cell fate transitions. In summary, our results suggest that controlled modulation of Oct4, Akt, and Hmgb2 function is required to maintain pluripotency and for the faithful induction of transcriptional programs required for lineage specific differentiation.

Pluripotent stem cells (PSCs) are specialized cells, which have remarkable ability to maintain in an undifferentiated state and are capable of undergoing differentiation to three germ-layer lineage cell types, under differentiation-enabling conditions. PSCs include embryonic stem (ES)-cells, embryonal carcinoma (EC)-cells and embryonic germ (EG)-cells. ES-cells are derived from the inner cell mass (ICM) of day 3.5 blastocysts (mouse). On the other hand, EC- and EG-cells have different source of origin and exhibit some differences in terms of their differentiation abilities and culture requirements. These PSCs act as an ideal in-vitro model system to study early mammalian development and cell differentiation and, they could potentially be used for experimental cell-based therapy for a number of diseases. However, one of the problems encountered is the immune rejection of transplanted cells. For this, immune-matched induced pluripotent stem (iPS)-cells have been derived from somatic cells, by forced expression of a few stemness genes. Although, human PSCs lines are being experimented, their cell-therapeutic potential is still far from being thoroughly tested due to lack of our understanding regarding lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked PSCs and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse: GU-3 line (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. Recently, we have derived a ‘GS-2’ ES-cell line from the GU-3 mouse line (Singh et al., 2012). Additionally, we envisaged the need for developing an iPS-cell line from the GU-3 mouse and then use them for studying cell differentiation. Thus, aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked iPS-cell line from a genetically non-permissive FVB/N mouse strain, characterize the established iPS-cell line and achieve differentiation of various cell types from EGFP-expressing iPS-cell line; (2) to study differentiation phenomenon, in particular to cardiac lineage, using select-cardiogenesis modulators and (3) to assess the gene-expression profiles and signaling system associated with cardiomyocyte differentiation of PSCs. This thesis is divided into four chapters with the 1st chapter being a review of literature followed by three data chapters. In the chapter I of the thesis, a comprehensive up-to¬date review of literature is provided pertaining to PSCs, their classification, derivation strategies especially for reprogramming of somatic cells for iPSC generation, their differentiation potential and characterization, particularly to cardiac lineage. Various molecular regulators involved in cardiac differentiation of PSCs with emphasis on epigenetic regulation involving DNA methylation and signaling pathways involved are described in detail. Subsequently, various approaches used for enhanced cardiac differentiation of PSCs and the therapeutic potential of PSC-derived differentiated cell types to treat disease(s) are discussed. Chapter-II describes the successful establishment of a permanent iPS-cell line (named ‘N9’ iPS-cell line) from the non-permissive FVB/N EGFP-transgenic GU-3 ‘green’ mouse. This chapter provides results pertaining to detailed derivation strategy and characterization of the ‘N9’ iPS-cell line which includes colony morphology, expansion (proliferation) efficiency, alkaline phosphatase staining, pluripotent markers’ expression analysis by qPCR and immunostaining approaches and karyotyping analysis. Further, in order to thoroughly assess the differentiation competence of the ‘N9’ iPS¬cell line, assessment of in-vitro and in-vivo differentiation potential of the ‘N9’ iPS-cell line by embryoid body (EB) formation and teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives were performed, followed by the generation of chimeric blastocysts by aggregation method. This established N9 iPS-cell line could potentially offer a suitable model system to study cardiac differentiation along with other established PSC lines such as the GS-2 and D3 ES-cell lines and the P19 EC-cell line. Following the establishment of the system to study cardiac differentiation of PSC lines, efforts were made to understand the biology of cardiac differentiation of PSCs (wild¬type and EGFP-transgenic PSC lines and P19 EC-cell line) using small molecules as modulators. Data pertaining to this is described in Chapter-III. The possible involvement of epigenetic regulation of cardiogenesis for example, DNA methylation changes in cardiogenesis-associated genes is studied using 5-aza cytidine as one of the chromatin modifiers. In order to understand the cardiac differentiation phenomenon, as a consequence of using 5-aza cytidine in cell culture, it was important to investigate its ability to induce/mediate cardiac differentiation. This involved an assessment by quantitating the cardiac beating phenotype and correlating this with enhanced cardiac-gene expression profiles. Further, DNA methylation regulation of cardiogenesis¬associated genes is described using various DNA methylation analysis techniques. Moreover, the possible involvement of other signaling members in mediating the cardiac differentiation is also studied using the P19 EC-cells. Results pertaining to the above findings are described in detail in the Chapter-III. Chapter-IV is focused on various efforts made towards investigating the ability of ascorbic acid to enhance cardiac differentiation of mouse ES-cells (GS-2 and D3 lines). Ascorbic acid has been implicated to be influencing cardiogenesis and it is reported to enhance differentiation of various cell types under certain culture conditions. Results pertaining to enhancement of cardiac differentiation of PSCs using ascorbic acid are presented in this chapter. This included assessment by quantitating cardiac beating phenotype and its correlation with enhanced cardiogenesis-associated gene expression profiles. Besides, estimation on the sorted cardiomyocyte population, derived from PSCs was also made using mature-cardiac marker. The possible underlying signaling mechanism involved was also studied in detail, using specific inhibitors for pERK (U0126), integrin signaling (pFAK; PP2) and collagen synthesis (DHP), in order to ascertain their involvement in ascorbic acid-mediated cardiac differentiation of mouse ES-cells. Subsequent to the three data chapters (II-IV), separate sections are provided for ‘Summary and Conclusion’ and for ‘Bibliography’, cited in the thesis. The overall scope of the study has been to understand the basic biology of cardiac differentiation from PSCs (EC-cells, iPS-cells and transgenic and wild-type ES-cells) and to assess, by using certain small molecules, whether PSCs could be coaxed to enhance the differentiation to a particular cell type (cardiac). The data contained in this thesis addresses the above theme.

La découverte des cellules souches embryonnaires et de leur immense potentiel thérapeutique a fait naître de grands espoirs. De nouvelles thérapies révolutionnaires pour traiter certaines des maladies les plus graves dont souffre l'humanité sont désormais envisageables. Le traitement de la vie à son stade le plus précoce est mis en cause. Le statut juridique reconnu au foetus et à l'embryon humain a des répercussions directes sur le domaine de la recherche et sur leur utilisation à des fins thérapeutiques. Nous avons examiné l'état du droit canadien quant au statut juridique du foetus et de l'embryon. De cette étude, nous avons constaté l'incertitude qui prévaut au Canada quant à leur statut. Par la suite, nous avons étudié les différentes normes canadiennes établies pour encadrer l'utilisation des cellules souches embryonnaires à des fins thérapeutiques et nous les avons analysées et comparées pour faire ressortir leurs similitudes et leurs différences. II est ressorti de notre analyse que les textes canadiens se rejoignent généralement sur l'essentiel et qu'il y a eu peu de changements de 1993 à aujourd'hui, en regard des activités de recherche interdites au Canada. Puis, nous avons examiné les systèmes normatifs applicables à ces recherches à l'étranger, soit aux États-Unis et en GrandeBretagne. Nous avons effectué une analyse comparative des trois systèmes normatifs étudiés, en évaluant différents paramètres communs à ces systèmes. Il est ressorti de cette analyse, que la Grande-Bretagne est le pays le plus libéral relativement à ces domaines de recherche, que les États-Unis sont les plus conservateurs sur ces questions et que le Canada se situe entre les deux. / The embryonic stem cells discovery and the immense therapeutic potential glven to them has created big hopes in the world of today. The appearance of new revolutionary therapies to treat sorne of the most serious known diseases are now conceivable. However, the treatment of life to its earliest stage is questionned. The legal status recognized to the foetus and the embryo has, in fact, a direct effect to the research area and industry as weil as to its therapeutic use. Therefore, we have examined and studied the CUITent canadian law with respect to the legal status of the foetus and embryo. Following this study, we have noticed the uncertainty that prevails in Canada concerning the said legal status. Afierwards, we have examined ail the different canadian norms and regulations already established regarding the use of embryonic stem cells for therapeutic ends. We also did the comparaison between those norms and regulations so as to see their differences and similarities. It appears from our analysis that ail the canadian litterature generally treat the subject in the same way and that there have been few changes from 1993 up until now with respect to the forbidden researchs activities in Canada. We also have analysed the foreign law standards and regulations in United States and Great Britain concerning those forbidden researchs activities. We did the exercise of comparing the state of the law in these three countries with different parameters. It emerges from that that Great Britain is the most liberal country, United States being the most conservative and Canada being in between them. / "Mémoire présenté à la Faculté des études supérieures en vue de l'obtention du grade de Maîtrise en droit (L.L.M.) Option recherche"

Les cellules souches embryonnaires (ES) sont porteuses de grands espoirs en recherche biomédicale dans le but d’apporter un traitement définitif à l’ostéoarthrose. Parce que certaines articulations des chevaux sont similaires à celles des humains, cet animal représente un modèle important dans l’évaluation de stratégies de régénération du cartilage. Cependant, pour expérimenter un traitement par les cellules ES chez le cheval, des cellules ES équines (eES) n’ont toujours pas pu être dérivées. Dans ce contexte, l’objectif principal de cette étude est de dériver des lignées de cellules eES. Le premier objectif de notre étude consiste à optimiser la technique de dérivation des cellules eES. Nous démontrons que la lignée de cellules nourricières et le stade de développement des embryons influencent l’efficacité de la technique de dérivation tandis que l’inhibition de voies de signalisation menant à la différenciation des cellules ES ne l’influence pas sous nos conditions. Le deuxième objectif de notre étude est de caractériser de façon plus approfondie les lignées de cellules eES obtenues. Nous démontrons que les cellules eES dérivées expriment autant des marqueurs associés aux cellules pluripotentes qu’aux cellules différenciées et que l’inhibition de voies de signalisation menant à la différenciation n’influence pas l’expression de ces marqueurs. Pour conclure, nous confirmons avoir dérivé des lignées de cellules semblables au cellules eES (eES-like) ne correspondant pas complètement aux critères des cellules ES. / Embryonic stem (ES) cells carry high hopes for biomedical research in order to provide definitive treatment for osteoarthritis. The horse is considered to be an important animal model for examining osteoarthritis treatments. However, despite almost thirty years of research, authentic equine ES (eES) cells have not yet been derived. In this context, the main objective of this study was to derive eES cell lines. The first objective of our study was to optimize the technique for deriving eES cells. We show that different feeder cell lines and embryo development stages influence the effectiveness of this technique while the use of cell signalling inhibitors does not influence eES cell derivation. The second objective was to characterize markers of pluripotency and differentiation in eES cell lines by RT-PCR. We demonstrate that the eES cells express both markers associated with pluripotent cells and differentiated cells and that the presence of cell signalling inhibitors in the culture medium does not influence the expression of these markers. In conclusion, we confirm having derived eES-like cells but these do not meet all the molecular criteria of authentic ES cells.