The governance of cell fate decisions during development is a fundamental biological problem. An important aspect of this is how cells exit a multipotent state and choose their fates in a correct manner and proportion. To tackle an aspect of this problem, I have focused on 2 multipotent models: one infinite self-renewal pluripotency in an artificial environment, and the other, bipotent progenitors in the context of the mouse embryo. The first model aimed to explore the effects of chromatin-associated factors on the ability of pluripotent mouse Embryonic Stem Cells (ESCs) to self-renew, via monitoring gene expression heterogeneity of key genes. The second model focused on Neural Mesodermal Progenitors (NMPs), a bipotent cell population found in the Caudal Lateral Epiblast (CLE) of mammalian embryos, which contributes to the spinal cord and paraxial mesoderm. The aim here was to derive NMPs in vitro which exhibit similar gene expression patterns and function like their mouse embryo counterpart and study their renewal and differentiation in detail. The first multipotent model explores the effects of chromatin remodelling on cell fate decisions, specifically investigating the consequences of inhibiting the histone acetyltransferase Kat2a on the ESCs fate. I found first, that the effect of Kat2a inhibition depends on the pluripotent state of the cells; cells in a ground state exhibit a resistance to Kat2a inhibition and maintain their pluripotency, whereas cells in a naïve state experience destabilization of their pluripotency gene regulatory network and shift towards differentiation. Second, that Kat2a inhibition in the naïve state results in a decline in the gene expression noise strength contributed by the promoter activation operation, which suggests that when ESCs become lineage-primed their transcriptional noise is constrained. In the bipotent model, the NMPs are identified as cells coexpressing Sox2 and T/Brachyury, a criterion used to derive NMP-like cells from ESCs in vitro. Comparison between the different NMPs protocols stresses that Epiblast Stem Cells (EpiSCs) are an effective source for deriving a multipotent population resembling the embryo Caudal Epiblast (CE), that generates NMPs. Furthermore, self-organization of this CE-like population, resulted in axially organized aggregates. Exploiting the mouse embryo CLE as a reference shows that EpiSCs derived NMPs, monolayers and aggregates, consist of a high proportion of cells with the embryo's NMP signature. Importantly, studying this system in vitro sheds light on the sequence of events which lead to NMP emergence in vivo. On this basis, I conclude that understanding the initial state of cells at a crossroads is important to reveal the limitations it imposes on the cells fate exploration, hence makes it possible to mimic more precisely the fate decision process in vitro.

As células-tronco embrionárias (CTEs), extraídas da massa celular interna do blastocisto, tem como características principais a capacidade de auto-renovação e a pluripotência. Durante o desenvolvimento, as células perdem seu potencial de diferenciação e adquirem um perfil de expressão gênica mais restrito, modulado por mecanismos epigenéticos, assim como por microRNAs. Membros da família miR-29 têm como transcritos alvos enzimas responsáveis pela metilação da citosina em 5mC (DNMT3a e 3b) e também da desmetilação (TET1, 2 e 3) do DNA, pela hidroxilação de 5mC em 5hmC. Recentes trabalhos sugerem que a modulação do miR-29 sobre estes alvos teria um papel no início da diferenciação em CTEs de camundongos e no aumento de eficiência da geração de iPS em células humanas. No presente trabalho, buscou-se compreender o papel regulatório do miR-29a em seus alvos epigenéticos no contexto da pluripotência e no início da diferenciação com atRA. Para tanto, duas linhagens celulares pluripotentes humanas (H1 e NTera- 2) foram submetidas a indução de diferenciação com atRA e ao ganho de função do miR-29a durante quatro dias de cultivo para análises de expressão gênica. Ademais, em NT2, realizamos ensaios funcionais por microscopia de imunofluorescência quantitativa para avaliar os efeitos do ganho e perda de função do miR-29a, DNMT3b e TET1, sobre a expressão nuclear de OCT4 e os perfis globais de 5mC e 5hmC após 96 horas de transfecção. Neste ensaio, também avaliamos o papel específico da regulação pós-transcricional de DNMT3b e TET1 pelo miR-29a, utilizando moléculas bloqueadoras dos sítios alvo (TSB) do miR-29a nestes transcritos. Observamos que sob a indução do atRA, os níveis de expressão do miR- 29a e de seus genes alvos (com exceção de DNMT3b), assim como dos marcadores de endoderme e ectoderme, aumentaram, seguido da diminuição dos marcadores de pluripotência em ambas as linhagens. A transfecção de moléculas mímicas do miR-29a, reduziu os níveis de seus transcritos alvos após dois e quatro dias em NT2 e H1, além de reduzir os níveis nucleares de DNMT3b em NT2. Ainda, ocorreu um aumento na expressão de genes da endoderme, mesoderme e ectoderme em H1 e a queda da expressão gênica e nuclear de OCT4 em NT2. Com o uso de siRNA específicos, demonstramos que o knockdown dos níveis nucleares de DNMT3b foi acompanhado de uma queda nos níveis globais de 5mC e um aumento de OCT4 e de 5hmC. Já o knockdown de TET1, elevou os níveis de 5mC, mas também os níveis de 5hmC e OCT4 nuclear. As avaliações com o uso de TSB contra os sítios de ligação do miR-29a em seus transcritos alvo, TET1 e DNMT3b, demonstraram que em células NT2, o bloqueio da ligação do miR endógeno aos seus alvos resultam no aumento dos níveis globais de 5hmC, indicando que a regulação póstranscricional destes alvos pelo miR-29 teria um importante papel na regulação epigenética de células pluripotentes. / Embryonic stem cells (CTEs), extracted from the internal cell mass of the blastocyst, are main characterized by the capacity for self-renewal and pluripotency. During development, the cells lose their differentiation potential and acquire a restricter gene expression profile, modulated by epigenetic mechanisms, as microRNAs. Members of the miR-29 family have as target transcripts enzymes for cytosine methylation in 5mC (DNMT3a and 3b) and for DNA demethylation (TET1, 2 and 3), by hydroxylation of 5mC in 5hmC. Recent studies suggest that the modulation of miR-29 on these targets plays a role in early differentiation of mouse CTEs and in increasing human iPS cell generation efficiency. In the present study, we sought to understand the regulatory role of miR-29a in its epigenetic targets in the context of pluripotency and in early differentiation with atRA. For this, two human pluripotent cell lines (H1 and NTera-2) were submitted to differentiation induction with atRA and function gain of miR-29a during four days of culture for gene expression analysis. Furthermore, in NT2, we performed functional assays by quantitative immunofluorescence microscopy to evaluate the gain- and loss-of-function of miR-29a, DNMT3b and TET1 in the OCT4 nuclear expression and global profiles of 5mC and 5hC, 96 hours posttransfection. In this assay, we also evaluated the specific role of post-transcriptional regulation of DNMT3b and TET1 by miR-29a, using target site blocking molecules (TSB) of miR-29a. We observed that under the induction of atRA, the miR-29a expression levels and its target genes (except of DNMT3b), further the markers of endoderm and ectoderm, increased, followed by decreased pluripotency markers in both cell lines. Transfection of mimic molecules of miR-29a reduced the levels of their target transcripts after two and four days in NT2 and H1, and reduced nuclear levels of DNMT3b in NT2. In addition, the expression of endoderm, mesoderm and ectoderm genes increased in H1 and gene and nuclear expression of OCT4 decreased in NT2. With the use of specific siRNA, we demonstrated that the knockdown of nuclear levels of DNMT3b was accompanied by a drop in global 5mC levels and an increase of OCT4 and 5hmC. While, the knockdown of TET1 increased the levels of 5mC, 5hmC and nuclear OCT4. Evaluations using TSB against the miR- 29a binding sites in their target transcripts, TET1 and DNMT3b, show that in NT2 cells blocking the binding of endogenous miR to their targets results in an increase in global 5hmC levels, indicating that the post-transcriptional regulation of these targets by miR-29 would play an important role in the epigenetic regulation of pluripotent cells.

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by over expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Using serial live cell immunofluorescence imaging of human fibroblasts undergoing reprogramming, we traced the emergence of nascent iPS cell colonies among heterogeneous cell populations and defined the kinetics of marker expression. We identified distinct colony types that morphologically resemble embryonic stem (ES) cells yet differ in molecular phenotype and differentiation potential. By analyzing expression of pluripotency markers, methylation at the OCT4 and NANOG promoters, and differentiation into teratomas, we determined that only one colony type represented bona fide iPS cells, whereas the others represented reprogramming intermediates. Proviral silencing and expression of TRA-1-60, DNMT3B, and REX1 distinguished the fully reprogrammed state, whereas Alkaline Phosphatase, SSEA-4, GDF3, hTERT and NANOG proved insufficient as markers. Reprogramming in chemically defined medium favored formation of bona fide iPS cell colonies relative to partially reprogrammed colonies. These data highlight the need for rigorous characterization and standardization of putative iPS cells.

No description available.

Regeneration is a process that renews damaged or lost cells, tissues, or even of entire body structures, and is a phenomenon which is widespread in the animal kingdom. Urodeles such as newts and salamanders have a remarkable regeneration ability. They can regenerate organs such as gills, lower jaws, retina, appendages like fore- and hind limbs, and also the tail including the spinal cord. The regeneration process requires the use of resident stem cells or somatic cells, which have to be reprogrammed. In both cases the reprogrammed cells are less differentiated, meaning the cell would have the ability to form any kind of fetal or adult cell which rose from the three different germ layers, the ectoderm, mesoderm and endoderm. Artificial reprogramming of differentiated mammalian somatic cell had been reported previously. It was shown that four pluripotency factors, OCT4 (also called POU5f1), SOX2, c-MYC and KLF4 are sufficient to generate an induced pluripotent stem (iPS) cell. It has been shown that some of these factors are also involved in regenerating processes. In newt limb and lens tissue, Sox2, c-Myc and Klf4 mRNA levels were upregulated in the beginning of blastema formation when compared to non-amputated tissue. Oct4 mRNA however, was not detected. During xenopus tail regeneration, Sox2 and c-Myc were expressed, while the xenopus Pou homologs Pou25, Pou60, Pou79, Pou91 were not detected. In regenerating zebrafish fin tissue, Sox2, Pou2, c-Myc and Klf4 mRNA were not upregulated. The mammalian transcription factor OCT4, a class V POU protein, is responsible in maintaining pluripotency in gastrula stage embryos. It was reported that mouse OCT4 is also expressed in the caudal node of embryos having 16 somites. It is further known that progenitors exist in mouse tailbud, which give rise to neural and mesodermal cell lineage. This suggests that the OCT4 expressing cells in caudal node might be a stem cell reservoir. Oct4 was detected in axolotl during embryonic development, and prior to my work we found Oct4 when screening the axolotl blastema cDNA library. In addition, we also identified Pou2, another class V POU gene. Phylogenetic analysis showed a clear distinction of both genes in the axolotl. We determined the mRNA pattern of Pou2 during embryogenesis and compared it to Oct4 mRNA and protein. Both genes are expressed in the primordial germ cells and the pluripotent animal cap region of the embryo. Apart from this similarity, both genes have a different expression pattern in the embryo. We are interested in the involvement of OCT4, POU2, as well as the transcription factor SOX2 in regenerating axolotl spinal cord. We asked whether the cellular pluripotent character conferred by POU factors is limited to mammals or if it is an ancient characteristic of lower vertebrates. To answer the question we performed in vitro and in vivo studies. Hence this thesis is separated into two chapter. By in vitro studies we investigated the pluripotent PouV orthologs from different species. Therefore, we performed reprogramming experiments using mouse or human fibroblasts and transduced them with axolotl Oct4 or Pou2, in combination with human or axolotl Sox2, c-Myc and/or Klf4. The generated iPS cells with the different sets of factors had similar endogenous pluripotency gene expression profiles to embryonic stem cells. Further, iPS cells expressed the pluripotency markers like OCT4, NANOG, SSEA4, TRA1-60 and TRA1-81. Another evaluation of the iPS cells was the formation of embryoid bodies. Immunouorescence staining showed that tissue from all three germ layers was formed after induction. We observed a positive staining for the endoderm marker !-FEROPROTEIN, the mesoderm marker !-SMOOTH MUSCLE ACTIN and the ectoderm marker \"III TUBULIN in the generated cells. This indicated that the iPS cells generated using axolotl Oct4 and Sox2 in combination with mammalian Klf4 and with or without c-Myc, as well as iPS cell generated with axolotl Pou2 and mammalian Sox2 and Klf4 and with or without c-Myc have a pluripotent potential. In addition, the axolotl factors are able to form heterodimers with the mammalian proteins. Furthermore, we compared the reprogramming ability with POU factors from mouse, human, zebrash, medaka and xenopus. We showed that xenopus Pou91, as the only non-mammalian example, is nearly as efficient as mouse and human Oct4 cDNAs in inducing GFP expressing cells. Also axolotl Pou2, axolotl Oct4 and medaka Pou2 showed reprogramming character however at a much lower efficiency. In contrast, zebrash Pou2 is not able to establish iPS cells. This indicates that a reprogramming ability to a pluripotent cell state is an ancient trait of Pou2 and Oct4 homologs. By in vivo studies we investigated the role of Oct4, Pou2 and Sox2 gene expression in regenerating spinal cord tissue. Performed in situ hybridizations and antibody staining studies in the regenerating spinal cord showed that Oct4, Pou2 and Sox2 were expressed during spinal cord regeneration. Knockdown experiments in regenerating spinal cord using morpholino showed that Pou2-morpholino does not have an effect. In contrast, SOX2 was required for spinal cord regeneration but to a lesser extent, than OCT4, which decreased the regenerated length signicantly compared to control. Even though, with Sox2-morpholino we did not observe the phenotype as a significantly shorter regenerated spinal cord, about 45% of SOX2 knocked down cells were not cycling and proliferating anymore. This indicates that axolotl SOX2 has an effect in regeneration. Therefore we wanted to know whether spinal cord cells would also have a pluripotent character in vivo and form other tissue types. Regenerating cells of the spinal cord are only able to form the same cell type and thus they keep their cell memory. However, when we performed transplantations of OCT4/SOX2 expressing spinal cord cells into somite stage embryos, we could show the formation of muscle cells. This shows that the spinal cord cells have the potential to change their fate in an embryonic context, where the normal environment of spinal cord has changed. However, our data do not indicate whether muscle is formed directly from the spinal cord or whether spinal cord cells fuse to developmental myoblasts, a cell type of embryonic progenitors, which give rise to muscle cells. To clearly state whether regenerating OCT4/SOX2 expressing spinal cord cells are pluripotent we have to perform OCT4 knock down in spinal cord and transplant these less proliferating cells into embryos, observing their cell fate.

Le microenvironnement apporte une multitude d'informations aux cellules régulant ainsi leurs fonctions et leur organisation. L'objectif de cette thèse a été de contrôler différents paramètres du microenvironnement cellulaire in vitro afin de moduler l'autorenouvellement et la destinée des cellules souches embryonnaires (CSE).Cette thèse comporte 3 parties. Premièrement, des films de polyélectrolytes multicouches à base de poly(L-lysine) et de hyaluronane ont été utilisés comme substrats modulables. Les propriétés mécaniques ainsi que la chimie des films régulent la proportion de sous-populations de CSE qui reflètent soit le stade masse cellulaire interne, soit le stade épiblaste.Dans un deuxième temps, un équilibre entre l'expression des marqueurs embryonnaires de l'axe proximodistal a été mis en évidence dans la culture de CSE. L'emploi de micro-patrons adhésifs permettant de contrôler la géométrie des colonies a révélé l'importance des contraintes topologiques sur la distribution des cellules exprimant le marqueur proximal Brachyury. Enfin, l'action combinée de BMP2 et de wnt3a mimant l'environnement biochimique du stade tardif de la ligne primitive a permis d'isoler une population pure et très précoce de progéniteurs cardiaques SSEA1+ multipotents. / The microenvironment provides stem cells with numerous pieces of information. Biochemical and mechanical cues synergize to regulate cell function and organization. The aim of this PhD thesis was to control specific microenvironmental parameters to modulate embryonic stem cell (ESC) self-renewal and fate.First, poly(L-lysine) and hyaluronan based polyelectrolyte multilayer films were used as tunable substrates. Both mechanical and chemical properties of the films influenced the balance between ESC subpopulations reflecting different embryonic stages (inner cell mass versus epiblast)Second, a dynamic equilibrium was found between the expression of embryonic proximal and distal markers within ESC culture. The uses of micropatterned substrates to control colony shape uncovered a key role for geometrical constraints in the distribution of Brachyury expression.Last, BMP2 was used together with secreted wnt3a to mimic the late streak stage of the embryo and to trigger the differentiation of pluripotent cells towards the cardiogenic mesoderm. Responsive cells could be sorted out based on SSEA1 expression. This purified population represents the earliest ESC derived multipotent cardiac progenitor population identified to date.

O estabelecimento de cultivo de células-tronco embrionárias (ESC) ainda não foi realizado com sucesso. Verificação de marcadores de pluripotência e diferenciação nos três folhetos germinativos são necessárias para validação de uma linhagem celular pluripotente. O objetivo deste estudo foi estabelecer e caracterizar o cultivo de ESC suínas usando Matrigel e comparar a expressão dos marcadores de pluripotência Oct-4, CD9 e α6-integrina em embriões. Blastocistos in vitro ou in vivo foram submetidos à imunocirurgia para cultura da massa celular interna, fixados para imunocitoquímica ou extração de RNA total para RT-PCR. Nenhuma colônia de ESC foi obtida usando co-cultivo em fibroblastos embrionários murinos (MEF) ou em Matrigel. Expressão de Oct-4, CD9 e α6-integrina foi detectada por PCR. Os produtos de PCR de CD9 e α6-integrina tiveram suas sequências nucleotídicas determinadas e comparadas com bases de dados públicas. O produto de CD9 foi idêntico à seqüência do CD9 suíno e o produto de α66integrina foi similar à humana e eqüina. Reação de Imunocitoquímica revelou a presença de Oct-4 no citoplasma de células da massa celular interna e do trofoblasto. CD9 e α6-integrina foram observados preferencialmente em células do trofoblasto. Não foi possível comparar a expressão dos marcadores de pluripotência entre ESC e embriões em suínos. Porém, este estudo descreve pela primeira vez a expressão de CD9 e α6-integrina em blastocistos suínos, os quais podem não estar relacionados com células pluripotentes embrionárias suínas. / Establishment of embryonic stem cell (ESC) culture in pigs has not been achieved. Verification of pluripotency markers and differentiation in the three embryonic layers are necessary for validation of a pluripotent cell line. The objective of this study was to establish and characterize porcine ESC culture using Matrigel and compare the expression of pluripotency markers Oct-4, CD9 and α6-integrin with embryos. In vitro or in vivo porcine blastocysts were submitted to immunosurgery for culture of inner cell mass, fixation for immunocytochemistry or total RNA extraction for RT-PCR. No ESC colonies were obtained using co-culture on mouse embryonic fibroblasts (MEF) or on Matrigel. Expression of Oct-4, CD9 and α6-integrin was detected by PCR. CD9 and α6-integrin PCR products had their nucleotide sequence assessed and compared with public nucleotide database. CD9 product was identical to CD9 porcine sequences and α6-integrin product was similar to human and equine α6-integrin. Immunocytochemistry revealed Oct-4 expression in cytoplasm of the inner cell mass and trophoblast cells. CD9 and α6-integrin were observed preferentially on trophoblast cells. It was not possible to compare expression of pluripotency markers between porcine ESC and embryos. However, this study describes for the first time expression of CD9 and α6-integrin in porcine blastocysts, which may not be related to pluripotent porcine embryonic cells.

A família Bromeliaceae apresenta importância ecológica e econômica, desta forma, o desenvolvimento de protocolos para a micropropagação de espécies dessa família, faz-se necessário, a fim de suprir sua demanda comercial e mesmo ecológica. A escolha do meio de cultura e do explante utilizado durante a micropropagação são fundamentais para um protocolo eficaz. Nesse contexto, o objetivo deste estudo foi avaliar as diferenças quantitativas e qualitativas no desenvolvimento de explantes de Neoregelia sp em meios de cultura e monitorar as vias morfogênicas dos propágulos obtidos em explantes foliares. Para tanto, brotos de microcepas e explantes foliares procedentes de um micro jardim clonal, foram transferidos para os meios de cultura de multiplicação MS, ½ MS e WPM, todos suplementados com 0,050 mg.L-1 ANA e 0,50 mg.L-1 de BAP, onde foram mantidos por 120 dias e submetidos a diversas análises morfofisiológicas. Paralelamente, explantes foliares foram mantidos em meio de cultura MS de multiplicação para o monitoramento das vias morfogênicas durante os processos regenerativos. Para os experimentos com brotos de microcepas verificou-se que o meio de cultura MS proporcionou a melhor taxa de multiplicação, maior crescimento dos brotos, obtendo os valores mais elevados de peso de matéria fresca e seca, além disso, apresentaram maior acúmulo de nitrogênio total e proteico. No entanto, os meios de cultura ½ MS e WPM promoveram uma taxa de multiplicação semelhante a do MS, mas com brotos menores e menos vigorosos, porém, mais homogêneos, com isso, na dependência do objetivo do cultivo in vitro, não deve ser desconsiderada a possibilidade de utilização dos meios de cultura ½ MS e WPM. Os explantes foliares não se desenvolveram bem no meio de cultura WPM, não havendo diferença entre os meios MS e ½ MS, visto que ambos apresentaram resultados satisfatórios. As análises histológicas e histoquímicas identificaram células parenquimáticas, que atuam como células-tronco, manifestando capacidade morfogênica para toti ou pluripotência, dando origem respectivamente a embriões somáticos e gemas adventícias, em resposta aos estímulos in vitro. / The Bromeliaceae family has an ecological and economic importance, therefore, the protocols development for micropropagation of species of this family becomes necessary in order to meet its business and even its ecological demand. The choice of culture medium and the explant used during micropropagation are essential for an effective protocol. Thus, the aim of this study was to evaluate the quantitative and qualitative differences in the explants development of Neoregelia sp in the culture media and monitor the morphogenetic pathways of obtained propagules from leaf explants. Consequently, shoots and leaf explants coming from microcloning garden were transferred to the MS, ½ MS and WPM multiplication culture media, all supplemented with 0.050 mg.L-1 NAA and 0.50 mg.L-1 BAP, where they were held for 120 days and submitted to morphological and physiological analysis. Therefore, leaf explants were kept on MS-medium multiplication for monitoring morphogenetic pathways during the regenerative processes. Furthermore, MS medium showed the best multiplication rate for the sprouts of the microstumps, increased growth of shoots, obtaining the highest values of fresh and dry matter weight, and also showed higher accumulation of total nitrogen and protein. However, the ½ MS and WPM culture media promoted a similar multiplication rate to the MS medium, with the development of the smaller and less vigorous shoots, but with greater homogeneity. This way, depending on the purpose of in vitro culture, their use in the micropropagation for this species should not be disregarded. The leaf explants are not well developed in WPM medium, and don\'t had significant difference between the MS and ½ MS culture medium, as both showed satisfactory results. The histological and histochemical analysis identified the presence of the parenchymatic cells, which act as stem cells, expressing morphogenic ability for toti or pluripotency, leading respectively to somatic embryogenesis or adventitious organogenesis in response to in vitro stimuli.

As células-tronco embrionárias (CTEs), extraídas da massa celular interna do blastocisto, tem como características principais a capacidade de auto-renovação e a pluripotência. Durante o desenvolvimento, as células perdem seu potencial de diferenciação e adquirem um perfil de expressão gênica mais restrito, modulado por mecanismos epigenéticos, assim como por microRNAs. Membros da família miR-29 têm como transcritos alvos enzimas responsáveis pela metilação da citosina em 5mC (DNMT3a e 3b) e também da desmetilação (TET1, 2 e 3) do DNA, pela hidroxilação de 5mC em 5hmC. Recentes trabalhos sugerem que a modulação do miR-29 sobre estes alvos teria um papel no início da diferenciação em CTEs de camundongos e no aumento de eficiência da geração de iPS em células humanas. No presente trabalho, buscou-se compreender o papel regulatório do miR-29a em seus alvos epigenéticos no contexto da pluripotência e no início da diferenciação com atRA. Para tanto, duas linhagens celulares pluripotentes humanas (H1 e NTera- 2) foram submetidas a indução de diferenciação com atRA e ao ganho de função do miR-29a durante quatro dias de cultivo para análises de expressão gênica. Ademais, em NT2, realizamos ensaios funcionais por microscopia de imunofluorescência quantitativa para avaliar os efeitos do ganho e perda de função do miR-29a, DNMT3b e TET1, sobre a expressão nuclear de OCT4 e os perfis globais de 5mC e 5hmC após 96 horas de transfecção. Neste ensaio, também avaliamos o papel específico da regulação pós-transcricional de DNMT3b e TET1 pelo miR-29a, utilizando moléculas bloqueadoras dos sítios alvo (TSB) do miR-29a nestes transcritos. Observamos que sob a indução do atRA, os níveis de expressão do miR- 29a e de seus genes alvos (com exceção de DNMT3b), assim como dos marcadores de endoderme e ectoderme, aumentaram, seguido da diminuição dos marcadores de pluripotência em ambas as linhagens. A transfecção de moléculas mímicas do miR-29a, reduziu os níveis de seus transcritos alvos após dois e quatro dias em NT2 e H1, além de reduzir os níveis nucleares de DNMT3b em NT2. Ainda, ocorreu um aumento na expressão de genes da endoderme, mesoderme e ectoderme em H1 e a queda da expressão gênica e nuclear de OCT4 em NT2. Com o uso de siRNA específicos, demonstramos que o knockdown dos níveis nucleares de DNMT3b foi acompanhado de uma queda nos níveis globais de 5mC e um aumento de OCT4 e de 5hmC. Já o knockdown de TET1, elevou os níveis de 5mC, mas também os níveis de 5hmC e OCT4 nuclear. As avaliações com o uso de TSB contra os sítios de ligação do miR-29a em seus transcritos alvo, TET1 e DNMT3b, demonstraram que em células NT2, o bloqueio da ligação do miR endógeno aos seus alvos resultam no aumento dos níveis globais de 5hmC, indicando que a regulação póstranscricional destes alvos pelo miR-29 teria um importante papel na regulação epigenética de células pluripotentes. / Embryonic stem cells (CTEs), extracted from the internal cell mass of the blastocyst, are main characterized by the capacity for self-renewal and pluripotency. During development, the cells lose their differentiation potential and acquire a restricter gene expression profile, modulated by epigenetic mechanisms, as microRNAs. Members of the miR-29 family have as target transcripts enzymes for cytosine methylation in 5mC (DNMT3a and 3b) and for DNA demethylation (TET1, 2 and 3), by hydroxylation of 5mC in 5hmC. Recent studies suggest that the modulation of miR-29 on these targets plays a role in early differentiation of mouse CTEs and in increasing human iPS cell generation efficiency. In the present study, we sought to understand the regulatory role of miR-29a in its epigenetic targets in the context of pluripotency and in early differentiation with atRA. For this, two human pluripotent cell lines (H1 and NTera-2) were submitted to differentiation induction with atRA and function gain of miR-29a during four days of culture for gene expression analysis. Furthermore, in NT2, we performed functional assays by quantitative immunofluorescence microscopy to evaluate the gain- and loss-of-function of miR-29a, DNMT3b and TET1 in the OCT4 nuclear expression and global profiles of 5mC and 5hC, 96 hours posttransfection. In this assay, we also evaluated the specific role of post-transcriptional regulation of DNMT3b and TET1 by miR-29a, using target site blocking molecules (TSB) of miR-29a. We observed that under the induction of atRA, the miR-29a expression levels and its target genes (except of DNMT3b), further the markers of endoderm and ectoderm, increased, followed by decreased pluripotency markers in both cell lines. Transfection of mimic molecules of miR-29a reduced the levels of their target transcripts after two and four days in NT2 and H1, and reduced nuclear levels of DNMT3b in NT2. In addition, the expression of endoderm, mesoderm and ectoderm genes increased in H1 and gene and nuclear expression of OCT4 decreased in NT2. With the use of specific siRNA, we demonstrated that the knockdown of nuclear levels of DNMT3b was accompanied by a drop in global 5mC levels and an increase of OCT4 and 5hmC. While, the knockdown of TET1 increased the levels of 5mC, 5hmC and nuclear OCT4. Evaluations using TSB against the miR- 29a binding sites in their target transcripts, TET1 and DNMT3b, show that in NT2 cells blocking the binding of endogenous miR to their targets results in an increase in global 5hmC levels, indicating that the post-transcriptional regulation of these targets by miR-29 would play an important role in the epigenetic regulation of pluripotent cells.

As células pluripotentes (CPs), em teoria, são capazes de dar origem a todos os mais de 200 tipos de células do organismo. Na natureza, há três tipos de células pluripotentes: células-tronco embrionárias, células germinais embrionárias e células de carcinoma embrionário. As características das CPs têm permitido um importante avanço para a pesquisa básica e apontam uma grande aplicabilidade na medicina regenerativa. No núcleo das CPs existem fatores atuantes responsáveis pela manutenção da identidade pluripotente; dentre eles destacam-se OCT4, NANOG, SOX2, KLF4 e MYC. Muito já se sabe sobre os mecanismos que estes fatores atuam para promover a manutenção da pluripotência celular. Baseados nestes estudos foi possível gerar células de pluripotência induzida (iPSCs). Porém, os mecanismos moleculares que direcionam a indução da pluripotência ainda não estão muito bem esclarecidos. Alguns estudos revelaram que componentes chaves da via NFB estão envolvidos na regulação da pluripotência, bem como na diferenciação e destino celular das células-tronco. Neste estudo, analisamos a participação de componentes da via canônica (RelA e NFB1) e não-canônica (RelB e NFB2) de NFB nos processos de diferenciação e destino celular ou manutenção da pluripotência. Para isto usamos técnicas de PCR quantitativa em Tempo Real (qPCR) e Imunoprecipitação de Cromatina (ChIP) investigando os papéis das vias canônica e não-canônica de NFB na manutenção da pluripotência e diferenciação de CPs, em um modelo de indução de diferenciação celular mediado por ácido trans-retinóico (atRA) em células de carcinoma embrionário NTera-2. Foram avaliadas as ligações dos fatores de transcrição RelA e RelB nas regiões promotoras dos genes OCT4, SOX2, MYC, KLF4 e GFAP e a regulação transcricional associada. Nossos resultados identificaram que as células não tratadas com atRA apresentaram níveis baixos na expressão dos componentes da via canônica de NFB, RelA e NFB1, e GFAP e quando induzidas à diferenciação por atRA durante 4 dias esses níveis se elevaram. Uma situação oposta foi vista nos componentes da via não-canônica de NFB, RelB e NFB2, e na expressão dos fatores de pluripotência OCT4, NANOG, SOX2 e KLF4, que apresentaram níveis de expressão elevados nas células não tratadas com atRA e sofreram redução com a indução da diferenciação celular. O ensaio de ChIP revelou que RelA liga-se nas regiões de regulação dos genes OCT4, SOX2, KLF4, MYC e GFAP apenas quando a célula está em processo de diferenciação, enquanto RelB se apresentou ligado às mesmas regiões tanto nas células indiferenciadas quanto naquelas induzidas à diferenciação por 4 dias. Com estes dados sugerimos que a via canônica de NFB pode estar relacionada com o processo de diferenciação e destino celular através da regulação negativa executada por RelA e NFB1 nos genes responsáveis pela identidade pluripotente das células aqui estudadas enquanto a via não-canônica de NFB, representada pela ativação de RelB e NFKB2, pode participar na manutenção da pluripotência através da regulação positiva destes mesmos fatores. / Human pluripotent stem cells (hPSCs) are able to give rise to all the 200 cell types of the adult organism. In nature, there are three types of hPSCs: embryonic stem cells, germ line stem cells and embryonal carcinoma cells. hPSCs characteristics have allowed a major advance in basic research, and are thought to have great applicability in regenerative medicine. In the nucleus of hPSCs there are transcription factors responsible for the maintenance of their pluripotent identity. OCT4, NANOG, SOX2, KLF4 and MYC are considered the core pluripotency factors in hPSCs. A great deal of knowledge about the mechanisms that promote and maintain pluripotency has been generated. Based on these studies it was possible to generate induced pluripotent stem cells (iPSCs). However, the molecular mechanisms that drive the induction of pluripotency are not fully understood. Some studies have recently indicated that key components of the NFkB may be involved in regulating pluripotency as well as cell differentiation and cell fate. In this study we analyzed the involvement of components of the canonical (RelA and NFB1) and the non-canonical NFB pathways (RelB and NFB2) in the maintenance of pluripotency, differentiation and cell fate processes. The techniques of quantitative real-time PCR (qPCR) and chromatin immunoprecipitation (ChIP) were used to interrogate the roles of the canonical and non-canonical NFB pathways in maintenance of pluripotency and differentiation in a model of cell differentiation induced by all trans-retinoic acid (atRA) on embryonal carcinoma cells NTera-2. The transcription factors RelA and RelB occupancy in the promoter regions of OCT4, SOX2, KLF4, MYC and GFAP, and the transcriptional regulation associated were evaluated. Our results showed that undifferentiated cells exhibited low expression levels of canonical NFB pathway components, RelA and NFB1, while cells induced to differentiate for 4 days exhibited downregulated expression of these factors. In the other hand, the non-canonical NFB pathway components, RelB and NFB2, and the pluripotency factors OCT4, NANOG, SOX2 and KLF4 were expressed in higher levels in undifferentiated cells, and were downregulated upon the differentiation process. ChIP assay revealed that RelA binds to the regulatory regions of OCT4, SOX2, KLF4, MYC, and GFAP only when cells are induced to differentiate, while RelB was found bound to the same regions in both undifferentiated and differentiated cells. This data suggests that the canonical NFB pathway may be associated to differentiation and cell fate processes by downregulation of genes responsible for the pluripotent identity, and that the non-canonical NFB pathway may act in the maintenance of pluripotency through the upregulation of the same factors.