Stress Inducible Protein 1 (STIP1) é uma co-chaperonina crucial no desenvolvimento murino. Nesse contexto, estudamos as funções reguladas por STIP1 usando células-tronco embrionárias murinas (CTEm). Nosso estudo mostrou um papel regulador para STIP1 na via JAK/STAT3, incluindo os fatores de transcrição NANOG, OCT4 e SOX2, caracterizando STIP1 como agente regulador na auto-renovação e pluripotência em CTEm. Adicionalmente, STIP1 modula a diferenciação em CTEm, uma vez sua expressão é requerida na formação de corpos embrioides (EBs) normais. Adicionalmente, ensaios de formação de teratoma mostraram inibição na formação do tumor e defeitos na diferenciação já que a formação de tecidos do mesoderma foi favorecida. Além disso, foi revelada a importância de STIP1 na proliferação celular já que sua ausência afetou a função, a qual foi parcialmente resgatada com tratamento de STIP1 exógena. Desse modo, nosso trabalho revela um papel crucial para STIP1 nas CTEm, caracterizando novas funções na compreensão do papel da co-chaperonina no desenvolvimento inicial em mamíferos. / Stress Inducible Phosphoprotein 1 (STIP1) is a crucial co-chaperonin in mice development. In this context, we studied the functions regulated by STIP1 using murine embryonic stem cells (CTEm). Our study shows a regulatory role for STIP1 in JAK/STAT3 pathway, including the transcription factors NANOG, OCT4 and SOX2, characterizing STIP1 as a regulatory agent in self-renewal and pluripotency in CTEm. In addition, an essential role of STIP1 in differentiation was demonstrated since its expression is required in embryoid bodies (EBs) formation with appropriate size and morphology. Moreover, teratoma formation assays showed inhibited tumor formation and defects in differentiation when formation of mesoderm was favored. Furthermore, were revealed the importance of STIP1 in cell proliferation, since its absence affects the function which was partially rescued after treatment with exogenous STIP1. Thus, our work reveals a central role for STIP1 in CTEm, characterizing new functions to understand the biological role of the co-chaperonin in early mammalian development.

Made available in DSpace on 2015-03-05T17:19:10Z (GMT). No. of bitstreams: 0 Previous issue date: 22 / Nenhuma / Na sociedade contemporânea, o homem moderno, em sua sede de conhecer e dominar, por meio da ciência e utilizando a razão, empreende métodos que lhe permitem os mais surpreendentes avanços científicos e tecnológicos, como é o caso das pesquisas e terapias com células-tronco embrionárias. A utilização da clonagem terapêutica e de embriões excedentes produzidos através de técnicas de fertilização in vitro faz emergir a problemática do começo da vida humana e as contradições acerca dos riscos e dos benefícios das terapias. Nesse contexto de riscos e incertezas diante dos novos direitos, a bioética se configura num sistema baseado na reflexão crítica e na busca por caminhos eticamente sustentáveis. Na tentativa de reduzir os riscos do uso de células-tronco embrionárias em terapias, a responsabilidade ética e o princípio da dignidade humana constituem fundamentos do pensamento bioético e ponto de partida para a formulação de limites jurídicos que, por sua vez, permeiam as decisões presentes e as possibilidades futu / In the society contemporary, the human being, with all the will to know and control, using the science and the reason, develop methods that permitt amazings scientific and biotecnology advances, as the cases of research and terapies with embryonic stem cells. The using of therapeutical cloning and of excesses embryos produced through tecniques of fertilization in vitro makes the problematic of the human life beginning shows up, as well as contradictions about the risks and benefits of the therapy. Among risks and insures ahead of the new rights, the bioethics configures itself on a system based on the critical reflection and on the search for ways ethicly sustainable. Trying to reduce risks using embryonic stem cells on therapies, the ethic responsability and the human dignity principles build the bedding of the bioethics thoughts and, starting point to formulate the legal limits that, on its turn, permeate the present decisions and the future possibilities

The mammalian epigenome is globally reprogrammed at two stages of development; this involves the erasure and re-establishment of DNA methylation by both passive and active mechanisms, including DNA repair pathways, and occurs concurrently with an increase in developmental potency. In addition to Uhrf1 and the Tet enzymes, the interplay between activation induced cytidine deaminase (AID) and the DNA repair machinery has been implicated in epigenetic reprogramming of various in vivo and in vitro systems including mouse primordial germ cells, zygotes and induced pluripotent stem cells. AID deaminates cytosine to uracil and can also deaminate methylcytosine, whereas the primary role of UNG is to maintain the integrity of the genome through erasure of uracil. In this thesis, I have aimed to investigate the role of DNA repair in demethylation. To do this I have focused on the specific role of AID and UNG in the demethylation of a static system – primed serum ESCs and a dynamic system – serum to 2i (naïve) to epiblast-like ES cells. As the role of both AID and UNG involves genomic uracil, the central theme of my thesis is the impact of accumulation of uracil on DNA methylation levels in the genome. Therefore, my first aim was to develop a quantitative method to detect low levels of genomic uracil in DNA firstly, by mass spectrometry and secondly, by whole genome sequencing. In Chapter Three, I show that the impact of deamination during DNA preparation can be minimised, such that the level of genomic ESC uracil can be accurately determined as around 12,000 uracil per genome and that, as anticipated, Ung null ESCs have almost twice the genomic uracil content of wildtype ESCs. Secondly, I address the main question which is the impact of uracil accumulation on methylation levels. In order to do this, I generate two cell lines: Ung knockout and Aid over expressing, both of which should result in an increase in genomic uracil. I demonstrate that while over expression of Aid stimulates demethylation in static system and in a dynamic demethylating system, the impact of Ung knockout is less clear. In (static) serum ESCs, loss of Ung results in hypomethylation however, in order to transition to 2i (naïve) ESCs, a process which involves demethylation of the genome, it appears the Ung is required as loss of this gene inhibits proper demethylation. As such, I conclude that UNG-mediated DNA repair functions alongside passive demethylation, by reduction of UHRF1 levels, to demethylate 2i ESCs. To probe the mechanism by which accumulation of uracil in the genome alters methylation levels, I investigate the impact of Ung KO and Aid OE on global levels of DNA damage. I show that both cell lines have a greater incidence of double strand breaks compared to a wild type cell line, and accordingly, upregulate their DNA damage response pathway and the expression of certain repair genes. I suggest that increasing genomic levels of uracil causes genomic instability and that DNA demethylation occurs as a consequence of the repair of extensive DNA damage. More broadly, I suggest that ESCs are uniquely poised, due to their heightened DNA damage response, to use uracil as an intermediate of DNA demethylation. Interestingly, I also note that the biological impact on serum ESCs of loss of Ung appears to be an increase in pluripotency.

O transplante de células-tronco hematopoéticas (CTHs) é o tipo mais bem-sucedido de terapia celular realizado até os dias atuais. No entanto, apesar do sucesso e da relevância clínica das CTHs isoladas a partir de fontes adultas, o uso destas células tem algumas limitações em relação à sua disponibilidade, compatibilidade imunológica e risco de contaminação. Desse modo, busca-se o desenvolvimento de soluções para as dificuldades apontadas para suprir a demanda de transplantes. Uma abordagem emergente para superar este problema é baseada na cultura e diferenciação de células-tronco embrionárias humanas (CTEhs). Estas são célulastronco pluripotentes e indiferenciadas com elevada capacidade de auto-renovação e diferenciação em todas as células derivadas dos três folhetos germinativos. No entanto, os métodos de diferenciação utilizados para a produção de CTHs a partir de células pluripotentes ainda não são eficientes. Os protocolos descritos até o momento têm gerado números variados e populações de células heterogêneas, e produz apenas CTHs muito primitivas e imaturas com baixa capacidade funcional in vivo. Parte desta dificuldade pode decorrer da ineficiência do microambiente de cultura para a diferenciação. Neste trabalho, nós demonstramos um eficiente protocolo de diferenciação hematopoética baseado em cocultivo de CTEhs com fibroblastos embrionários murinos com alto rendimento na geração de célulastronco/progenitoras hematopoéticas (CTPHs) que expressam os antígenos CD45, CD43, CD31 e CD34, e apresentam potencial clonogênico in vitro equivalente ao de células mononucleares isoladas de sangue de cordão umbilical. Nós fomos capazes de produzir todas as células das linhagens eritróide e mielóide em diferentes estágios de maturação, como também células positivas para marcadores linfóides. Demonstramos ainda que as células hematopoéticas surgem no sistema de cultura a partir de um endotélio-hemogênico constituído por células CD34+CD31+. No entanto, apesar das características maduras das CTPHs obtidas por tal método, os ensaios de reconstituição hematopoiética mostraram que estas células ainda possuem limitada capacidade funcional de enxertamento em camundongos imunocomprometidos quando transplantadas por via retro-orbital. / Hematopoietic stem cells (HSC) transplant is the most successful type of cell therapy carried out to date. However, despite the success and the clinical relevance of HSC isolated from adult sources, these cells have some limitations regarding its availability, immunological compatibility and risk of contamination. Thus, we seek to develop solutions to overcome these difficulties to supply the demand for transplants. An emerging approach to overcome this problem is based on human embryonic stem cells (hESCs) culture and differentiation. These are pluripotent and undifferentiated stem cells with high capacity for self-renewal and differentiation in all cells derived from the three embryonic germ layers. However, differentiation methods used for HSC production from pluripotent cells are not efficient yet. Protocols described so far have generated varying numbers and heterogeneous cell populations, and produce only very primitive and immature HSC with low in vivo functional capacity. Part of this difficulty may result from the inefficiency of the microenvironment of culture for differentiation. Here, we demonstrate an efficient protocol based on co-culture of hESCs with mouse embryonic fibroblasts for hematopoietic differentiation with high performance to generate in vitro hematopoietic stem/progenitor cells (HSPCs) that express CD45, CD43, CD31 and CD34 antigens with high purity of positive cells. We were able to produce all cells of erythroid and myeloid lineages at different stages of maturation. Lymphoid potential of hematopoietic cells was also evidenced. We demonstrated the primitive origin of hematopoietic cells through capillary-like structures constituted by hemogenic CD34+CD31+ cells. However, despite mature features of HSPCs obtained by our protocol, hematopoietic reconstitution assays showed that these cells have yet limited functional capacity for grafting into immunocompromised mice when exogenously transplanted by retro-orbital route.

Macrophages belong to the mononuclear phagocyte system and they perform fundamental roles to maintain homeostasis in the organism. Macrophage development, survival, proliferation and functionality depend upon the colony stimulating factor 1 (CSF1) and interleukin-34 (IL-34), which signal through the CSF1 receptor (CSF1R). CSF1R is a type III tyrosine kinase receptor that is present in the plasma membrane of monocytes and macrophages. Mutations in Csf1r in mice produce the loss of many tissue macrophage populations and multiple developmental abnormalities. In humans, abnormal enhancement of CSF1R expression has been correlated to adverse prognosis in a subset of carcinomas; and mutations in the human CSF1R are associated with an autosomal-dominant neurodegenerative disease. CSF1R is encoded by the c-fms proto-oncogene and its expression is partially controlled by the fms-intronic regulatory element (FIRE). The FIRE sequence is highly conserved across species and contains binding motifs for multiple transcription factors, which are relevant for haematopoiesis. Previous results from murine Csf1r transgenes showed that FIRE is essential for driving Csf1r expression, and that interactions between FIRE and multiple myeloid transcription factors contribute to maximal regulatory activity. This project aimed to study the role of FIRE in its normal chromatin context, in vivo. A FIRE knockout (FIRE-/-) mouse model was generated using the CRISPR/Cas9 technology in mouse embryonic stem cells (ESCs) and in mice. In ESCs, the deletion severely compromised the differentiation of macrophages from embryoid bodies generated in vitro. In mice, the frequency of the FIRE- /- genotype in the progeny does not follow a Mendelian distribution and about 5% of the offspring developed hydrocephalus. Unlike Csf1r -/-mice, which die before weaning, most surviving FIRE-/- mice grew normally and were fertile. The impact of the mutation on macrophage populations is selective. FIRE-/- mice are not monocyte deficient (identified as F4/80+ Csf1r+ cells in peripheral blood), although these cells have reduced levels of Csf1r mRNA and do not bind porcine CSF1 Fc fusion protein. The development of peritoneal macrophages and Iba-1+ microglia was abolished, but Adgre1+ (F4/80+) macrophage populations in liver and spleen were unaffected. Csf1r was greatly reduced in bone marrow progenitors, but about 30% of these cells were able to differentiate into macrophages in vitro, upon exposure to recombinant human CSF1 (rhCSF1). This study shows that FIRE is essential for the development of a subset of tissue-resident macrophage populations. In FIRE-/- mice, potential compensation from additional regulatory elements within Csf1r might underlie the development of unaffected tissue-resident macrophages.

The process of generating the vast diversity of immunoglobulin receptors and secreted antibodies begins with the recombination of the joining (JH), diversity (DH) and variable (VH) genes in the immunoglobulin heavy chain locus. The ability to produce antibodies is restricted to the B cell lineage and is tightly regulated, starting with the temporal separation of the recombination process, in which DH-JH precedes VH-DHJH recombination. Successful recombination of both heavy and light chain loci results in the expression of an antigen receptor on the cell surface. Subsequent selection stages remove non‑functional and autoreactivity receptors from the final pool of antigen responding B cells that ultimately give rise to antibody secreting plasma cells. Understanding the complexity of the recombination processes and the diversity of the resulting antibody repertoire has been a major focus of academic and industrial research alike. Therapeutic monoclonal antibodies have seen many successful applications within the clinic and they constitute a billion-dollar industry. However, limitations therein have resulted in the emergence of antibody engineering approaches and the use of natural sources of alternative heavy chain only antibodies (HCAbs/nanobodies). The biotechnology company Crescendo Biologics has taken the highly desired characteristics of HCAbs a step further with the creation of a mouse platform capable of producing fully humanized HCAbs. The Crescendo platform presents a unique opportunity to expand our understanding of how mouse B cell development functions by exploiting the features of heavy chain only antibody production. Furthermore, the platform enables the expansion of our limited knowledge of the epigenetic mechanisms involved in the recombination of the human immunoglobulin heavy chain locus. Using flow cytometry, with dimensionality reduction analysis approaches, I investigated B cell development in the context of HCAbs. These studies revealed a previously uncharacterised developmentally intermediate B cell population. Due to ethical and availability limitations to studies of human bone marrow, the primary pre-selection human B cell repertoire has not been studied in detail. The isolation of several B cell developmental stages and the use of our novel DNA-based high-throughput unbiased repertoire quantification technique, VDJ-seq, allowed me to study recombination of the human IGH locus sequence and observe HCAb repertoire selection within the mouse environment. The adaptation of next generation sequencing techniques to antigen receptor repertoire quantification has provided an unprecedented insight into repertoire diversity and the alterations it undergoes during infection or ageing. Our VDJ-seq assay is unique in its ability to interrogate DNA recombinants. To expand its capabilities, I investigated several limitations of the technique, including mispriming and PCR/sequencing errors, and implemented experimental and bioinformatics solutions to overcome them, which included the creation of a comprehensive analysis workflow. Finally, I have developed and applied a novel network visualisation method for genome-wide promoter interaction data generated by promoter capture Hi-C. The availability of high quality human pluripotent stem cell datasets allowed me to utilise the new techniques to further our understanding of the dynamics of genome organisation during early human embryonic development. This visualisation approach will be directly applicable to understanding B cell development.

SCRATCH2 é um fator de transcrição envolvido no desenvolvimento neural expresso em células pós-mitóticas. Identificamos que a retenção nuclear de SCRATCH2 é dada pelo domínio zinc-finger. A atividade repressora é modulada pelo domínio SCRATCH e não depende do domínio SNAG. O alinhamento de ortólogos de SCRATCH2 identificou uma sequência conservada na região N-terminal contendo os resíduos de fosforiláveis Y77 e S78. Mutação em Y77 ou S78 reduz a capacidade repressora de SCRATCH2, enquanto mutações em ambos resíduos resgatam sua função. Nossos dados sugerem que o domínio zinc-finger é responsável pela localização nuclear enquanto a atividade repressora é mediada pelo domínio SCRATCH. Já a identidade de Y77 e S78 é importante para a conformação correta proteína. O nocaute de SCRATCH2 aumenta a expressão de marcadores de progenitores intermediários (IP) e reduz o a expressão de marcadores de neurônios pós-mitóticos durante a neurodiferenciação de células-tronco. Esses dados sugerem que SCRATCH2 atua na manutenção de IP, e participa do início da diferenciação neural. / SCRATCH2 is a transcription factor involved in neural development expressed in postmitotic neural cells. Here, we identify that the nuclear retention of SCRATCH2 is controlled by the zinc-finger domain. The repressor activity is modulated by the SCRATCH domain and is independent of the SNAG domain. An analysis of SCRATCH2 through homology comparison identified a N-terminal conserved sequence containing two phosphorylatable residues, Y77 and S78. Single mutation in Y77 or S78 reduces SCRATCH2 repression ability while concomitant mutation in both rescue SCRATCH2 function. Our data suggest that the zinc-finger domain is responsible for nuclear retention of SCRATCH2 while residues Y77 and S78 are relevant for the protein correct conformation. In mouse embryonic stem cells neural induced towards corticogenesis, SCRATCH2 KO increases the levels of intermediate progenitors (IP) markers and reduces the level of early born neurons markers. This data suggests that SCRATCH2 plays a role in the maintenance of IP pool, thereby regulating the onset of neural differentiation.

Since the first successful derivation of human embryonic stem cells (hESC), rapid progress has been attained in the development of strategies in differentiation of these cells into various neural lineages, with the fundamental objective of using these cells for replacement and repair of damaged neuronal circuits in the central nervous system (CNS). Of particular interest are midbrain dopaminergic (mDA) neurons, which play a central role in regulation of voluntary movement. Degeneration or loss of function of mDA neurons in the nigrostriatal pathway is associated with Parkinson disease (PD). Stromal-Derived Inducing Activity (SDIA) is recognized as one of the most efficient methods in restricting ESC differentiation to a dopaminergic lineage, and refers to the property of mouse stromal cell lines such as PA6 or MS5 to cause ESC to differentiate to DA neurons. Although this strategy has been extensively used to generate mDA neurons from hESC, the biochemical nature of SDIA is yet unknown.  In the present study mDA neurons were generated from the BG01V2 hESC line by SDIA. To examine whether SDIA exerts its effect directly on hESC and is responsible for early dopaminergic induction, neural progenitor cells (NPC) were enyzmatically isolated from the co-cultures and allowed to differentiate in feeder-free conditions. The isolated cells were committed to a mesencephalic neural lineage, and were capable of maintaining their phenotype and developing into postmitotic mDA neurons in feeder-free conditions. The mDA neurons showed neuronal excitability and dopamine transporter function. The in vitro proliferation and differentiation of the NPC was also investigated by a BrDU incorporation assay. Next, the maintenance of cellular memory and capacity for proliferation of the mesencephalic NPC was assessed. The NPC could be expanded in vitro by five-fold as neurospheres for up to two weeks while retaining their DA differentiation potential, but did not retain a stable phenotype over extended periods of time. Preliminary transplantation experiments of neurospheres in striatal lesioned animals indicated, however, that these cells could survive and conserve their phenotype in vivo. To gain additional insight into the biochemical role of SDIA in early dopaminergic induction of hESC, the separate contributions of cell surface activity and secreted factors were examined. The data revealed that the PA6 cell surface activity promoted cell survival and was mainly responsible for enhanced neurogenesis of hESC, whereas secreted factors provided DA lineage-specific instructions. In order to identify the soluble factors responsible for the DA phenotype-inducing component of SDIA, the gene expression profile of PA6 cells was compared to that of cell lines lacking the DA-inducing property. A number of soluble factors known to be associated with CNS development that were highly expressed in PA6 cells were identified as potential DA differentiation-inducing candidates. These differentially-expressed genes included stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these factors, termed SPIE, were applied to the hESC, they induced dopaminergic neuronal differentiation of hESC line, BG01V2 and other karyotypically normal hESC lines in vitro. Thus, it appears that SPIE comprises the DA phenotype-inducing property of SDIA. This may provide a simple and direct means of differentiating hESC to form DA neurons in a single step, without a requirement for co-culture, animal cell lines, or animal products. / QC 20100916

The derivation of embryonic stem cells (ESCs) has created an invaluable resource for scientific study and discovery. Further improvement in differentiation protocols is necessary to generate the large number of cells needed for clinical relevance. The goal of this work was to develop a method to incorporate biomaterial microparticles (MPs) within stem cell aggregates and to evaluate their use for local control of the cellular microenvironment for directed differentiation. The effects of unloaded MPs on ESC differentiation were first determined by controlled incorporation of poly(lactic-co-glycolic acid) (PLGA), agarose and gelatin MPs. Embryoid body (EB) formation, cell viability, and gross morphology were not affected by the presence of the MPs. Further analysis of gene expression and patterns of phenotypic marker expression revealed alterations in the differentiation profile in response to material incorporation. The ability of MPs to direct ESC differentiation was investigated by incorporation of growth factor loaded MPs within EBs. MPs were loaded with bone morphogenetic protein-4 (BMP-4). BMP-4 loaded MPs incorporated within EBs induced mesoderm gene expression while inhibiting expression of an ectoderm marker compared to untreated EBs. Finally, magnetic MPs (magMPs) were incorporated within EBs to induce magnetic sensitivity. The responsiveness of EBs to applied magnetic fields was controlled by the number of magMPs incorporated within the aggregates. Magnetic guidance was then used to control the precise location of single EBs or populations of EBs for bioreactor culture and for construction of heterogeneous cell constructs. Overall, the results indicated that PSC differentiation within spheroids is sensitive to various types of biomaterials. Incorporation of MPs within EBs can be used to direct ESC differentiation by control of the cellular environment from microscale interactions, by delivery of soluble factors, to macroscale interactions, by control of EB position in static and suspension cultures.

Lentivirus efficiently transduce stem cells, however are notably silenced in embryonic stem cells (ESC). Provirus can be silent, expressing, or variegated when clonal single copy ESCs spawn daughters that revert expression despite containing identical integration sites (IS) indicating epigenetic regulation. In the silent state, variegated provirus are bound by H1 and MeCP2, where H1 compensates for MeCP2 binding in DNA methylation null ESCs, consistent with a model of heterochromatin formation dependent on concentrations of its constituent components. ESC Variegation was hypothesized to result from spreading of nearby heterochromatin. Global IS analysis indicates Variegated IS favour gene deserts, repeat clusters, and LINEs while Expressers prefer gene density with stable modest expression and SINEs. Chromatin data does not support a role for the spread of heterochromatin possibly a consequence of the dynamic/dispersed nature of ESC heterochromatin. Variegation thus may depend on stochastic chromatin regulation by pluripotency factors at proximal genome organizing repeats.