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

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.

Identification de déterminants impliqués dans la différenciation des cellules souches embryonnaires

Les cellules souches ont attiré l’attention du public ces dernières années, grâce non-seulement à leur utilisation comme thérapies visant à s’attaquer à certains types de cancers, mais aussi en relation avec leur potentiel dans le domaine de la médecine regénérative. Il est établi que le destin cellulaire des cellules souches embryonnaires (ESC) est régulé de façon intensive par un groupe de facteur clés agissant sur leur pluripotence. Il est néanmoins envisageable que certains déterminants influençant l’auto-renouvellement et la différenciation de ces cellules soient toujours inconnus. Afin de tester cette hypothèse, nous avons généré, en utilisant une méthode par infections virales, une collection de ESC contenant des délétions chromosomales chevauchantes que nous avons baptisée DelES (Deletion in ES cells). Cette librairie contient plus de 1000 clones indépendants dont les régions délétées couvrent environ 25% du génome murin. À l’aide de cette ressource, nous avons conduit un criblage de formation de corps embryoïdes (EB), démontrant que plusieurs clones délétés avaient un phénotype de différenciation anormal. Nos études de complémentation sur un groupe de clones ont par la suite permis l’identification de Rps14 - un gène codant pour une protéine ribosomale (RP) comme étant haploinsuffisant pour la formation de EB. Dans un deuxième temps, l’analyse approfondie des résultats de notre crible a permis d’identifier un groupe de gènes codants pour des RP qui semblent essentiels pour la différenciation des ESC, mais dispensables pour leur auto-renouvellement. De manière intéressante, les phénotypes anormaux de formation en EB les plus marqués sont associés à des délétions de RP qui se retrouvent au site de sortie des ARN messagers (ARNm) du ribosome, soit Rps5, Rps14 et Rps28. Étonnament, alors qu’un débalancement des RP conduit généralement à une réponse de type p53, l’haploinsuffisance de ces trois gènes ne peut être renversée par une simple réduction des niveaux d’expression de ce gène suppresseur de tumeurs. Finalement, nos études de profilage polysomal et de séquençage à haut-débit montrent une signature spécifique de gènes liés au mésoderme chez un clone hétérozygote pour Rps5, suggérant ainsi une explication au phénotype de différenciation p53-indépendant identifié chez ces ESC. Nos travaux rapportent donc la création d’une ressource intéressante de génomique fonctionnelle qui a permis de mettre à jour le rôle essentiel que jouent les RP dans le processus de formation de EB. Nos résultats permettent aussi de documenter une réponse p53-indépendante suite à un débalancement de RP dans un contexte opposant l’auto-renouvellement et la différenciation des ESC.

O impacto das novas biotecnologias no pensamento político

A dissertação de mestrado “O Impacto das Novas Biotecnologias no Pensamento Político – A problemática das células estaminais embrionárias” partiu do pressuposto basilar de que a Humanidade se depara com uma ruptura de modelo de pensamento sem paralelo na História. O Homem detém hoje um conhecimento científico sem precedentes e vê-se perante o potencial das novas biotecnologias que, pela primeira, vez podem alterar a forma de olhar sobre si próprio, não apenas enquanto ser social mas sobretudo como entidade biológica. Todo o enquadramento da dissertação tem em consideração os diferentes momentos da História em que certos homens levados pela inevitabilidade do progresso intelectual e científico contribuíram decisivamente para alterar profundamente os modelos de pensamento. Modelos que, surgidos em determinado contextos históricos, foram considerados de ruptura e revolucionários. Em sentido contrário, numa espécie de reacção conservadora, foram surgindo forças de autoridade e de poder, rejeitando novos modelos e paradigmas que, de uma maneira ou de outra, pudessem pôr em causa o sistema de sociedade instituído. As grandes rupturas na História da Humanidade resultaram desse confronto de ideias, entre um modelo de pensamento vigente e um novo paradigma proposto. Ao longo da dissertação apresentada são analisados vários períodos de ruptura, com particular enfoque para o advento da genética no século XIX e posterior revolução biotecnológica nos Estados Unidos que, num futuro próximo, poderá vir a curar doenças congénitas e degenerativas, funcionando como uma espécie de “kit de reparação do corpo humano, e, num horizonte mais alargado, poderá potenciar a possibilidade da criação de um “outro eu”, produto do Homem e não do livre arbítrio. Pela primeira vez, o Homem tem conhecimento e técnica para criar um mundo pós-humano, onde cada um é resultado da vontade individual dos seus progenitores, dando-se, assim, início a uma nova História. Mas, tudo isto levanta uma série de questões morais, éticas e políticas. Dilemas quanto aos processos de investigação e quanto às consequências que a sua aplicação poderá trazer para a própria Humanidade. Como trabalho de Ciência Política não cabe no propósito deste tecer cenários filosóficos quanto ao futuro do Homem face aos avanços da investigação genética, mas sim tentar analisar e procurar encontrar um padrão de comportamento na forma como os legisladores e governantes, mediante a sua base doutrinária, têm abordado uma matéria cujas implicações terão eventualmente impacto na concepção da própria Humanidade.

Repressor element 1 silencing transcription factor couples loss of pluripotency with neural induction and neural differentiation

Neural differentiation of embryonic stem cells (ESCs) requires coordinated repression of the pluripotency regulatory program and reciprocal activation of the neurogenic regulatory program. Upon neural induction, ESCs rapidly repress expression of pluripotency genes followed by staged activation of neural progenitor and differentiated neuronal and glial genes. The transcriptional factors that underlie maintenance of pluripotency are partially characterized whereas those underlying neural induction are much less explored, and the factors that coordinate these two developmental programs are completely unknown. One transcription factor, REST (repressor element 1 silencing transcription factor), has been linked with terminal differentiation of neural progenitors and more recently, and controversially, with control of pluripotency. Here, we show that in the absence of REST, coordination of pluripotency and neural induction is lost and there is a resultant delay in repression of pluripotency genes and a precocious activation of both neural progenitor and differentiated neuronal and glial genes. Furthermore, we show that REST is not required for production of radial glia-like progenitors but is required for their subsequent maintenance and differentiation into neurons, oligodendrocytes, and astrocytes. We propose that REST acts as a regulatory hub that coordinates timely repression of pluripotency with neural induction and neural differentiation.

REST: transcriptional and epigenetic regulator

Acquisition and maintenance of cell fate and potential are dependent on the complex interplay of extracellular signaling, gene regulatory networks and epigenetic states. During embryonic development, embryonic stem cells become progressively more restricted along specific lineages, ultimately giving rise to the diversity of cell types in the adult mammalian organism. Recent years have seen major advances in our understanding of the mechanisms that regulate the underlying transcriptional programmes during development. In particular, there has been a significant increase in our knowledge of how epigenetic marks on chromatin can regulate transcription by generating more or less permissive chromatin conformations. This article focuses on how a single transcription factor, repressor element-1 silencing transcription factor, can function as both a transcriptional and epigenetic regulator, controlling diverse aspects of development. We will discuss how the elucidation of repressor element-1 silencing transcription factor function in both normal and disease conditions has provided valuable insights into how the epigenome and transcriptional regulators might cooperatively orchestrate correct development.

Serum protein layers on parylene-C and silicon oxide: effect on cell adhesion

Among the range of materials used in bioengineering, parylene-C has been used in combination with silicon oxide and in presence of the serum proteins, in cell patterning. However, the structural properties of adsorbed serum proteins on these substrates still remain elusive. In this study, we use an optical biosensing technique to decipher the properties of fibronectin (Fn) and serum albumin adsorbed on parylene-C and silicon oxide substrates. Our results show the formation of layers with distinct structural and adhesive properties. Thin, dense layers are formed on parylene-C, whereas thicker, more diffuse layers are formed on silicon oxide. These results suggest that Fn acquires a compact structure on parylene-C and a more extended structure on silicon oxide. Nonetheless, parylene-C and silicon oxide substrates coated with Fn host cell populations that exhibit focal adhesion complexes and good cell attachment. Albumin adopts a deformed structure on parylene-C and a globular structure on silicon oxide, and does not support significant cell attachment on either surface. Interestingly, the co-incubation of Fn and albumin at the ratio found in serum, results in the preferential adsorption of albumin on parylene-C and Fn on silicon oxide. This finding is supported by the exclusive formation of focal adhesion complexes in differentiated mouse embryonic stem cells (CGR8), cultured on Fn/albumin coated silicon oxide, but not on parylene-C. The detailed information provided in this study on the distinct properties of layers of serum proteins on substrates such as parylene-C and silicon oxide is highly significant in developing methods for cell patterning.

För Sverige i framtiden : Bio-objektifiering av ny medicinsk teknologi

Taking Sweden into the future – Bio-objectification of new medical technology In this article, we analyze how contemporary discursive silences around new biotechnologies such as cybrids and induced pluripotent stem cells (iPSC), have been enabled by earlier policy processes in the area, e.g. boundary work around what is human and non-human, living and non-living, subject and object. The analysis of policy processes around xenotransplantations and the use of human embryonic stem cells, shows that the stem cells’ and xenografts’ “bio-identities” become stabilized through high expectations for the future, a lack of therapeutic possibilities and struggles over definitions of life. The policy processes around human embryonic stem cells and organs from other animals, are characterized by a normalization of certain understandings of ”life”, trust in scientific progress and it’s national financial potentials and a categorization of criticism as irrational. Through these “bio-objectification processes”, debate and decision making has been moved from a political and public context into ethical committees and research funding bodies. The article concludes by discussing consequences of this political non-handling of biomedical technologies and how these bioobjects could be re-politicized.

Algumas polêmicas envolvendo a utilização de células-tronco embrionárias no Brasil: um desafio à inovação jurisdicional

A finalidade da presente dissertação será o exame de algumas das muitas polêmicas éticas e jurídicas que envolvem a utilização das células-tronco embrionárias humanas para fins de pesquisa e terapia. A utilização de tais células-tronco foi aprovada pela Lei n.º 11.105 de 2005, conhecida como a nova Lei de Biossegurança, cujo artigo 5º permitiu, apenas para fins de pesquisa e terapia, a utilização das citadas células obtidas de embriões humanos provenientes do processo de fertilização in vitro, não utilizados no respectivo procedimento, atendidas certas condições. Assim que entrou em vigor, o citado dispositivo sofreu por parte do Procurador Geral da República a Ação Direta de Inconstitucionalidade (ADI) nº 3510, que gerou amplos debates de âmbito multidisciplinar. Apesar da declaração de constitucionalidade do referido artigo, ainda são muitas as polêmicas de ordem jurídica e ética. Questiona-se, principalmente, as divergências existentes acerca da natureza jurídica do embrião, produzido in vitro e excedente nos processos de fertilização, bem como a adequação do princípio constitucional da Dignidade Humana neste contexto. São demonstradas, ainda, questões pertinentes ao patenteamento de material genético e, consequentemente, das células-tronco embrionárias.

Efeitos da tricostatina A sobre a acetilação de histonas, proliferação celular e diferenciação de células tronco embrionárias murinas

Background: Embryonic stem cells are cells derived from early-stage embryos that are characterized by pluripotency and self-renewal capacity. The in vitro cultured murine embryonic stem cells can indefinitely propagate in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). However, when stimulated, these cells can differentiate into cell lines derived from all three embryonic germ layers. The trichostatin A (TSA) is an epigenetic modifier agent and several studies have used the TSA to stimulate cellular differentiation. However, most of these studies only assessed one TSA concentration. Therefore, this study aimed to evaluate the effects of different TSA concentrations on histone hyperacetylation during in vitro cell differentiation of murine pluripotent embryonic stem cells, cultured with or without LIF, in the quest of to standardize their application on early cultures of embryonic stem cells.Materials, Methods & Results: Undifferentiated murine embryonic stem cells were plated in the presence of different TSA concentrations (0 nM, 15 nm, 50 nM and 100 nM) in the presence or absence of LIF. Thus, the treatments were evaluated in undifferentiated embryonic stem cells cultured in the presence of LIF (Control group: 0 nM LIF(+); Group 15 nM LIF+; Group 50 nM LIF+ and Group 100 nM LIF+), and in embryonic stem cells cultured in the absence of LIF (Control group: 0 nM LIF; Group 15 nM LIF(-); Group 50 nM LIF(-) and Group 100 nM LIF-). Treatment with TSA was performed for 24 h. After that the medium was replaced with fresh medium without TSA. Samples were collected at 0, 12, 24, 36 and 48 h after the beginning of the experiment. Three replicates were performed in each experimental group. The relative amount of Histone H3 lysine 9 acetylation was analyzed in all groups, as well as the cell proliferation in the embryonic stem cells cultured in the presence of LIF. In the control group (0 nM), the absence of LIF resulted in higher levels (P < 0.05) of H3lys9ac compared to the cultures supplemented with LIF. In the embryonic stem cells cultured in the presence of LIF, the 50 nM and 100 nM treatments resulted in higher levels (P < 0.05) of H3lys9ac when compared with 0 nM and 15 nM treatments. Evaluating the Hoechst area in the 0 nM group, it was observed that the number of cells increased (P < 0.05) according to the time of culture. Treatment with 15 nM also reflected a similar distribution, but the Hoechst area in 15 nM group was lower (P < 0.05) at 24 and 48h when compared to the observed in the control group. In the 100 nM treatment, was observed that the area of Hoechst was lower (P < 0.05) to that obtained in the control group at 12, 24 and 48h. In addition, it was observed that treatment with TSA induces greater cellular differentiation when compared to control groups in stem cells cultured in the presence of LIF as well as in the absence of LIF.Discussion: In the present study it was observed that TSA treatment increased the levels of histone acetylation in murine embryonic stem cells at a 50 nM concentration, making it possible to reduce the concentration recommended in the literature (100 nM). In addtion, it was concluded that the lower TSA concentrations utilized (15 nm and 50 nM) was less harmful to cellular proliferation than the 100 nM TSA concentration.

A necessidade atual em se continuar a pesquisar com células-tronco embrionárias

Embryonic stem cells are pluripotent and able to generate all cell types of the body, being the most promising cells to the study for regenerative medicine. Following the differentiation path, there are adult stem cells, which are committed with a specific cell lineage, creating limitations on their application. On the extreme opposite of embryonic stem cells there are the induced pluripotent stem cells, originated from a somatic cell after genetic reprogramming. Induced pluripotent stem cells are a recent science discovery and may substitute the use of embryonic stem cells in future research. But with nowadays knowledge, the use of adult stem cells and induced pluripotent stem cells are limited due to high expenses and long time process demand. Moreover, the development achieved on all kinds of stem cells study are, in some part, due to the study of embryonic stem cells, what makes the study of these cell type still mandatory. © Todos os direitos reservados a.

Cultivo e caracterização de células-tronco embrionárias de bovinos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção de embriões por fecundação in vitro e ativação partenogenética visando o isolamento de células tronco embrionárias felinas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Obtenção, manutenção e caracterização de células-tronco embrionárias equinas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cultivo e caracterização de celulas-tronco obtidas de blastocistos equinos frescos e refrigerados

Pós-graduação em Medicina Veterinária - FMVZ