Study of the molecular mechanisms involved in cell differentiation of embryonic stem cells

Potential heart progenitors from postnatal and adult heart have been identified in rodents and humans, and were shown to differentiate in vitro into mature cardiomyocytes. However, in some studies only immature cardiac cells were obtained, leading to concerns about whether the inefficient cell electrical maturation and electrical coupling of derivative cells will cause arrhythmias upon cellular therapies of injured hearts. Notwithstanding these concerns, recent clinical assays have indicated an improvement of heart function and life quality of patients treated with autologous cardiac progenitor cells. With the discovery of the capacity of embryonic stem cells (ESC) to differentiate into all lineages, including the cardiovascular lineage, many researchers have been focusing on developing strategies to efficiently direct ESC differentiation for the production of unlimited numbers of cardiomyocytes. Yet, the low efficiency of cardiac differentiation, its teratogenic nature and the lack of reliable protocols to purify the desired cells from ESC cultures are holding back their use for clinical application. Hence, understanding the molecular mechanisms that regulate commitment and differentiation of diverse muscle and non-­‐muscle cell lineages of the heart is essential to improve current therapies and to design novel heart stem cell-­‐ based therapeutic strategies for cardiac regenerative medicine. Many transcription factors, including Nkx2.5, Gata4 and Isl1, have been described as master regulators of cardiac morphogenesis. Indeed, they are required for the commitment and differentiation of cardiac progenitors in early embryonic development, as well as for the differentiation of ESC into cardiovascular lineages. Furthermore, mutations in these genes have been associated with congenital heart defects in humans. This finding underscores the clinical relevance of studying the role of cardiac transcription factors during heart development. Cited2 is a transcriptional regulator essential for mouse embryonic development. Amongst a wide spectrum of developmental defects presented by Cited2 knockout mouse embryos, the cardiovascular and left-­‐right patterning defects are the most prominent. Importantly, heterozygous mutations of human CITED2 in association with congenital heart disease have been reported by several research groups. Moreover, it has been suggested that Cited2 is important for ESC-­‐derived cardiovascular development and essential in the epiblast or its derivatives for normal cardiac left-­‐right patterning. Although Cited2 was shown to be required for the maintenance of pluripotency and self-­‐renewal of ESC, its role in early embryonic development and its mechanism of action are still not well explored. In the present study, we investigated the role of Cited2 in mouse ESC differentiation towards cardiac cells. By performing increasing and absence of Cited2 expression in ESC, we have established that Cited2 is essential for cell commitment and differentiation towards the mesoderm, ectoderm and endoderm lineages. Indeed, Cited2 depletion impaired ESC differentiation to several cell lineages. More importantly, Cited2 was required for the normal emergence of mesoderm cells from early ESC specification to cardiac lineages. Our results suggest that this response is primarily associated with the direct or indirect modulation of Nodal and Brachyury gene expression during mesodermal cell commitment, thus enabling a signaling cascade promoting cardiogenesis. Additionally, our results indicated that loss of Cited2 generated comparatively less cardiomyocytes in differentiated ESC cultures, whereas gain of Cited2 led to higher numbers of cardiomyocytes. These findings suggest Cited2 is an important protein for cardiac function. Furthermore, our data suggest that Cited2 can modulate the gene expression (Islet1, Gata4, Nkx2.5 and Tbx5) in cardiac progenitors, thus playing an important role during cardiac cell commitment. We have also found that Cited2 plays a role in cardiomyocyte proliferation, as indicated by the downregulation of Tbx5 and its targets genes upon Cited2 depletion. Furthermore, by overexpressing human CITED2 and Gata4 in murine ESC, we found that Cited2 could not prevent endoderm commitment induced by Gata4 overexpression. The same overexpression approach was used to test the role of CITED2-­‐ISLET1 interaction in cardiac differentiation, and the results indicate that the cardiogenic response of Cited2 might be additive in association with that induced by Islet1 (Isl1) overexpression. We have also found Cited2 expression in multipotent cardiac progenitor population, specifically in secondary heart field (SHF) progenitors, which are marked by the expression of Isl1 protein. Our data generated by increasing and absence of Cited2 Cited2 expression in SHF progenitor, suggest that finely regulated Cited2 gene expression levels could allow or disrupt the SHF-­‐differentiation program. Moreover, by performing GST-­‐pull down and by bi-­‐molecular fluorescence complementation assays, we gathered evidence indicating that CITED2 physically interacts with ISL1. By performing chromatin immunoprecipitation assays we have also found that CITED2 binds the Isl1 promoter region. Taken together, our results indicate that Cited2 is important for proper cardiovascular differentiation by acting at different levels during cardiac stem cell commitment: firstly, by inducing ESC towards mesoderm at early events during ESC commitment; and secondly, by inducing and maintaining the expression of cardiac transcription factors important for cardiogenesis. Although many Cited2 functions remain uncovered, our data indicate that Cited2 is an important cardiac differentiation regulator that can participate in heart regeneration, and therefore be useful for the development of alternative approaches to treat or prevent heart failure,Potenciais progenitores cardíacos de coração pós-­‐natal e adulto foram identificados em roedores e seres humanos, e verificou-­‐se serem capazes de se diferenciar in vitro em cardiomiócitos maduros. No entanto, em alguns estudos foram obtidos apenas células cardíacas imaturas, assim suscitando questões sobre se ineficiente maturação elétrica celular e acoplamento eléctrico das células derivadas não causará arritmias no contexto da aplicação de terapias celulares em lesões cardíacas. Apesar destas questões, ensaios clínicos recentes indicaram uma melhoria da função cardíaca e da qualidade de vida dos pacientes tratados com células progenitoras cardíacas autólogas. Com a descoberta da capacidade de células estaminais embrionárias (ESC) se diferenciarem em todas as linhagens, incluindo a linhagem cardiovascular, muitos investigadores se têm concentrado no desenvolvimento de estratégias para dirigir eficientemente a diferenciação de ESC para a produção de um número ilimitado de cardiomiócitos. No entanto, a baixa eficiência da diferenciação cardíaca, a sua natureza teratogénica e a falta de protocolos bem validados para purificar as células desejadas das culturas de ESC estão a atrasar o seu uso para aplicações clínicas. Assim, a compreensão dos mecanismos moleculares que regulam o comprometimento e diferenciação de diversas linhagens de células musculares e não-­‐ musculares do coração é essencial para melhorar as terapias atuais e para conceber novas estratégias terapêuticas baseadas em células estaminais para a medicina regenerativa cardíaca. Muitos factores de transcrição, incluindo Nkx2.5, Gata4 e ISL1, têm sido descritos como reguladores importantes da morfogénese cardíaca. Com efeito, eles são necessários para o comprometimento e diferenciação de células progenitoras cardíacas no desenvolvimento embrionário inicial, assim como para a diferenciação das ESC em linhagens cardiovasculares. Além disso, mutações nestes genes foram associadas a doenças cardíacas congénitas em pacientes humanos. Esta observação reforça a relevância clínica do estudo do papel dos fatores de transcrição cardíacos no desenvolvimento do coração. O Cited2 é um regulador de transcrição essencial para o desenvolvimento embrionário murino. Entre um amplo espetro de defeitos de desenvolvimento apresentados por embriões de ratinho Cited2 knockout, defeitos cardiovasculares e de padronização esquerda-­‐direita são os mais proeminentes. É importante ressaltar que mutações heterozigóticas de CITED2 humano em associação com cardiopatias congénitas têm sido relatados por vários grupos de investigação. Além disso, tem sido sugerido que Cited2 é importante para o desenvolvimento cardiovascular derivado de ESC e é essencial no epiblasto ou seus derivados para a padronização esquerda-­‐direita cardíaca. Embora o Cited2 seja necessário para a manutenção da pluripotência e auto-­‐ renovação de ESC, o seu papel no desenvolvimento embrionário precoce e o seu mecanismo de ação ainda não foram bem explorados. No presente estudo, investigamos o papel de Cited2 em diferenciação de ESC de ratinho em células cardíacas. Através da realização de experiências de ganho e de perda de função de Cited2 em ESC, nós verificámos que Cited2 é essencial para o comprometimento e diferenciação em células de linhagens de mesoderme, ectoderme e endoderme. De facto, a depleção de Cited2 perturbou a diferenciação de ESC para diversas linhagens de células. Mais importante ainda, Cited2 foi necessário para o aparecimento normal de células da mesoderme a partir da especificação precoce de ESC para linhagens cardíacas. Os nossos resultados sugerem que esta resposta está principalmente associada com a modulação direta ou indireta da expressão dos genes Nodal e Brachyury durante o comprometimento em célula mesodermal, permitindo assim uma cascata de sinalização que promove a cardiogénese. Além disso, os nossos resultados indicam que a perda de Cited2 levou à geração de menos cardiomiócitos em culturas de ESC diferenciadas, enquanto que o ganho de Cited2 levou a um maior número de cardiomiócitos. Estes resultados sugerem portanto que Cited2 é uma proteína importante para a função cardíaca. Além disso, nossos dados sugerem que Cited2 pode modular a expressão génica (Islet1, Gata4, Nkx2.5 e Tbx5) em progenitores cardíacos, desempenhando assim um papel importante durante o comprometimento de células cardíacas. Nós também descobrimos que Cited2 desempenha um papel na proliferação de cardiomiócitos, como indicado pela regulação negativa de Tbx5 e seus genes-­‐alvo na situação de depleção de Cited2. Além disso, através de sobreexpressão de CITED2 humano e Gata4 em ESC, descobrimos que Cited2 não impede o comprometimento em endoderme induzido pela sobreexpressão de Gata4. A mesma abordagem de sobre-­‐expressão foi usada para testar o papel da interação CITED2-­‐ ISLET1 na diferenciação cardíaca, e os resultados indicam que a resposta cardiogénica de Cited2 será aditiva em associação com aquela induzida pela sobreexpressão de Islet1 (ISL1). Também detetámos expressão de Cited2 em populações de progenitores cardíacos multipotentes, especificamente em progenitores do campo cardíaco secundário (SHF), que são marcados pela expressão da proteína ISL1. Os nossos dados obtidos em experiências de ganho e perda da função de Cited2 em progenitores SHF sugerem que níveis de expressão do gene Cited2 finamente regulados podem permitir ou interromper o programa de diferenciação SHF. Além disso, através da realização de ensaios de GST-­‐pull down e ensaios de fluorescência de complementação bi-­‐molecular, obtivemos dados indicando que CITED2 interage fisicamente com ISL1. Através da realização de ensaios de imunoprecipitação de cromatina também descobrimos que CITED2 se liga à região do promotor de ISL1. Em conjunto, os nossos resultados indicam que Cited2 é importante para uma diferenciação cardiovascular adequada, agindo em diferentes níveis durante o comprometimento de células estaminais cardíacas: em primeiro lugar através da indução de ESC para mesoderme em estadios precoces durante o comprometimento de ESC; e em segundo lugar através da indução e manutenção da expressão de fatores de transcrição cardíacos importantes para a cardiogénese. Embora muitas funções de Cited2 permaneçam por descobrir, os nossos dados indicam que Cited2 é um importante regulador da diferenciação cardíaca, que pode participar na regeneração do coração e, portanto, ser útil para o desenvolvimento de abordagens alternativas para o tratamento ou prevenção da insuficiência cardíaca.Fundação para a Ciência e Tecnologia - bolsa de doutoramento FCT (SFRH/BD/62054/2009)Universidade do Algarve, Departamento de Ciências Biomédicas e MedicinaMinistério Ciência, Tecnologia e Ensino Superior - Programa de Investigação em Medicina Regenerativa da Universidade do Algarv

Cdkn2a inactivation promotes malignant transformation of mouse immature thymocytes before the β-selection checkpoint

CDKN2A deletion is the most frequent genetic alteration in T-cell acute lymphoblastic leukemia (T-ALL), occurring across all molecular and immunophenotypic subtypes. CDKN2A encodes two functionally unrelated tumor suppressor proteins, ARF and INK4a, which are critical regulators of cell cycle and proliferation. Arf has been reported to suppress T-ALL development in post−b-selection thymocytes, but whether CDKN2A acts as a tumor suppressor gene in immature, pre−b-selection thymocytes remains to be elucidated. Resorting to a Rag2-deficient model of T-ALL, driven by the ETV6:: JAK2 fusion, we report that Cdkn2a haploinsufficiency at early stages of T-cell development facilitates leukemia developmentPPBI-POCI-01-0145-FEDER-022122; POCI-01-0145-FEDER-007274; NORTE01-0145-FEDER-000029info:eu-repo/semantics/publishedVersio

StemChecker: a web-based tool to discover and explore stemness signatures in gene sets

Stem cells present unique regenerative abilities, offering great potential for treatment of prevalent pathologies such as diabetes, neurodegenerative and heart diseases. Various research groups dedicated significant effort to identify sets of genes-so-called stemness signatures-considered essential to define stem cells. However, their usage has been hindered by the lack of comprehensive resources and easy-to-use tools. For this we developed StemChecker, a novel stemness analysis tool, based on the curation of nearly fifty published stemness signatures defined by gene expression, RNAi screens, Transcription Factor (TF) binding sites, literature reviews and computational approaches. StemChecker allows researchers to explore the presence of stemness signatures in user-defined gene sets, without carrying-out lengthy literature curation or data processing. To assist in exploring underlying regulatory mechanisms, we collected over 80 target gene sets of TFs associated with pluri- or multipotency. StemChecker presents an intuitive graphical display, as well as detailed statistical results in table format, which helps revealing transcriptionally regulatory programs, indicating the putative involvement of stemness-associated processes in diseases like cancer. Overall, StemChecker substantially expands the available repertoire of online tools, designed to assist the stem cell biology, developmental biology, regenerative medicine and human disease research community. StemChecker is freely accessible at http://stemchecker.sysbiolab.eu.Portuguese Fundacao para a Ciencia e Tecnologia (FCT) [RH/BPD/96890/2013, SFRH/BPD/70718/2010, PTDC/BIA/GEN/116519/2010, IF/00881/2013, PTDC/BIA-GEN/116519/2010]; Programa Doutoral ProRegeM-Mecanismos de Doenca e Medicina Regenerativa [PD/00117/2012]info:eu-repo/semantics/publishedVersio

Acute Loss of Cited2 Impairs Nanog Expression and Decreases Self-Renewal of Mouse Embryonic Stem Cells

Identifying novel players of the pluripotency gene regulatory network centered on Oct4, Sox2, and Nanog as well as delineating the interactions within the complex network is key to understanding self-renewal and early cell fate commitment of embryonic stem cells (ESC). While overexpression of the transcriptional regulator Cited2 sustains ESC pluripotency, its role in ESC functions remains unclear. Here, we show that Cited2 is important for proliferation, survival, and self-renewal of mouse ESC. We position Cited2 within the pluripotency gene regulatory network by defining Nanog, Tbx3, and Klf4 as its direct targets. We also demonstrate that the defects caused by Cited2 depletion are, at least in part, rescued by Nanog constitutive expression. Finally, we demonstrate that Cited2 is required for and enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells.National Portuguese through FCT-Fundacao para a Ciencia e a Tecnologia [PEst-OE/EQB/LA0023/2013, PTDC/SAU-ENB/111702/2009]; Camara Municipal de Oeiras; Merck Sharp & Dhome Foundation-Portugal; CRUK; Leukaemia & Lymphoma Research; Kay Kendall Leukaemia Fund; Wellcome Trust; Medical Research Council; Cancer Research UK [12796, 14633]; Great Ormond Street Hospital Childrens Charity [W1062]; Medical Research Council [MC_U137973817, G1000801g, MC_qA137913

Alginate Biosynthesis in Azotobacter vinelandii

The Gram-negative bacterium Azotobacter vinelandii can synthetize the biopolymer alginate that has material properties appropriate for plenty of applications in industry as well as in medicine. In order to settle the foundation for improving alginate production without compromising its quality, a better understanding of the polymer biosynthesis and the mechanism of regulation during fermentation processes is necessary. This knowledge is crucial for the development of novel production strategies. Here, we highlight the key aspects of alginate biosynthesis that can lead to producing an alginate with specific material properties with particular focus on the role of oxygen availability linked with the molecular mechanisms involved in the alginate production

NF-kappa B-dependent RANKL expression in a mouse model of immature T-cell leukemia

Activation of the receptor activator of nuclear factor-kappa B (RANK) by its ligand (RANKL) is involved in both solid and hematological malignancies, including multiple myeloma, acute myeloid leukemia and B-cell leukemia. Although RANKL expression has been described in normal T cells, a potential role in T-cell leukemia remains undefined. Here, we used a model of immature T-cell leukemia/lymphoma, the TEL-JAK2 transgenic mice, to assess RANKL expression in leukemic cells and its regulatory mechanisms. We found that Rankl mRNA was significantly overexpressed in leukemic T cells when compared to wild-type thymocytes, their nonmalignant counterparts. Moreover, Rankl mRNA and RANKL surface expression in leukemic cells was induced by T-cell receptor (TCR) signaling activation, dependently on the NFKB signaling pathway. These results indicate that TCR-activated leukemic T cells express high levels of RANKL and are potential inducers of RANK signaling in microenvironmental cells. (C) 2019 Elsevier Inc. All rights reserved.Fundacao para a Ciencia e a Tecnologia (Portugal)Portuguese Foundation for Science and Technology [PTDC/SAU-OBD/103336/2008, UID/BIM/04773/2013, POCI-01-0145-FEDER-007274, SFRH/BD/75137/2010, IF/00056/2012]European Regional Development Fund through COMPETE 2020 programEuropean Union (EU) [NORTE-01-0145-FEDER-000029