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- Rejuvenescer células “velhas”, é possível?Publication . Bragança, José; Santos, JoãoThe possibility of reprogramming differentiated adult cells which are limited in their functions, into cells capable of generating all cell types of the adult organism has opened new horizons in regenerative medicine. However, for a safe use of reprogrammed cells in new clinical applications, the reprogramming process remains to be improved. We have showed that the use of the protein Cited2 in during the reprogramming procedure increases the efficiency and homogenizes the process.
- Bioelectrical signal detection using conducting polymer electrodes and the displacement current methodPublication . Inácio, Pedro; Mestre, Ana L G; Medeiros, C.R.; Asgarifar, Sanaz; ELAMINE, Youssef; Canudo, Joana; Santos, João; Bragança, José; Morgado, Jorge; Biscarini, Fabio; Gomes, Henrique L.Conducting polymer electrodes based on poly (3, 4 ethylenedioxythiophene): polystyrene sulfonate were used to record electrophysiological signals from autonomous cardiac contractile cells present in embryoid bodies. Signal detection was carried out by measuring the displacement current across the polymer/electrolyte double-layer capacitance, and compared with voltage detection. While for relatively low capacitance electrodes, the voltage amplification provides higher signal quality, and for high capacitive electrodes, the displacement current method exhibits a higher signal-to-noise ratio. It is proposed that the displacement current method combined with high capacitive polymer-based electrodes is adequate to measure clusters of cells and whole organs. Our approach has a great potential in fundamental studies of drug discovery and safety pharmacology.
- Exogenous WNT5A and WNT11 proteins rescue CITED2 dysfunction in mouse embryonic stem cells and zebrafish morphantsPublication . Santos, João; Mendes-Silva, Leonardo; Afonso,Vanessa; Martins, Gil; Machado, Rui; Lopes, Joao; Cancela, M. Leonor; Futschik, Matthias; Sachinidis, Agapios; Gavaia, Paulo; Bragança, JoséMutations and inadequate methylation profiles of CITED2 are associated with human congenital heart disease (CHD). In mouse, Cited2 is necessary for embryogenesis, particularly for heart development, and its depletion in embryonic stem cells (ESC) impairs cardiac differentiation. We have now determined that Cited2 depletion in ESC affects the expression of transcription factors and cardiopoietic genes involved in early mesoderm and cardiac specification. Interestingly, the supplementation of the secretome prepared from ESC overexpressing CITED2, during the onset of differentiation, rescued the cardiogenic defects of Cited2-depleted ESC. In addition, we demonstrate that the proteins WNT5A and WNT11 held the potential for rescue. We also validated the zebrafish as a model to investigate cited2 function during development. Indeed, the microinjection of morpholinos targeting cited2 transcripts caused developmental defects recapitulating those of mice knockout models, including the increased propensity for cardiac defects and severe death rate. Importantly, the co-injection of anti-cited2 morpholinos with either CITED2 or WNT5A and WNT11 recombinant proteins corrected the developmental defects of Cited2-morphants. This study argues that defects caused by the dysfunction of Cited2 at early stages of development, including heart anomalies, may be remediable by supplementation of exogenous molecules, offering the opportunity to develop novel therapeutic strategies aiming to prevent CHD.
- Induced pluripotent stem cells, a giant leap for mankind therapeutic applicationsPublication . Bragança, José; Lopes, João A. P.; Silva, Leonardo; Santos, JoãoInduced pluripotent stem cells (iPSC) technology has propelled the field of stem cells biology, providing new cells to explore the molecular mechanisms of pluripotency, cancer biology and aging. A major advantage of human iPSC, compared to the pluripotent embryonic stem cells, is that they can be generated from virtually any embryonic or adult somatic cell type without destruction of human blastocysts. In addition, iPSC can be generated from somatic cells harvested from normal individuals or patients, and used as a cellular tool to unravel mechanisms of human development and to model diseases in a manner not possible before. Besides these fundamental aspects of human biology and physiology that are revealed using iPSC or iPSC-derived cells, these cells hold an immense potential for cell-based therapies, and for the discovery of new or personalized pharmacological treatments for many disorders. Here, we review some of the current challenges and concerns about iPSC technology. We introduce the potential held by iPSC for research and development of novel health-related applications. We briefly present the efforts made by the scientific and clinical communities to create the necessary guidelines and regulations to achieve the highest quality standards in the procedures for iPSC generation, characterization and long-term preservation. Finally, we present some of the audacious and pioneer clinical trials in progress with iPSC-derived cells.
- CITED2 cooperates with ISL1 and promotes cardiac differentiation of mouse embryonic stem cellsPublication . Pacheco-Leyva, Ivette; Matias, Ana Catarina; Oliveira, Daniel V.; Santos, João; Nascimento, Rita; Guerreiro, Eduarda; Michell, Anna C.; van De Vrugt, Annebel M.; Machado-Oliveira, Gisela; Ferreira, Guilherme; Domian, Ibrahim; Bragança, JoséThe transcriptional regulator CITED2 is essential for heart development. Here, we investigated the role of CITED2 in the specification of cardiac cell fate from mouse embryonic stem cells (ESC). The overexpression of CITED2 in undifferentiated ESC was sufficient to promote cardiac cell emergence upon differentiation. Conversely, the depletion of Cited2 at the onset of differentiation resulted in a decline of ESC ability to generate cardiac cells. Moreover, loss of Cited2 expression impairs the expression of early mesoderm markers and cardiogenic transcription factors (Isl1, Gata4, Tbx5). The cardiogenic defects in Cited2-depleted cells were rescued by treatment with recombinant CITED2 protein. We showed that Cited2 expression is enriched in cardiac progenitors either derived from ESC or mouse embryonic hearts. Finally, we demonstrated that CITED2 and ISL1 proteins interact physically and cooperate to promote ESC differentiation toward cardiomyocytes. Collectively, our results show that Cited2 plays a pivotal role in cardiac commitment of ESC.
- Ectopic expression of CITED2 prior to reprogramming, promotes and homogenises the conversion of somatic cells into induced pluripotent stem cellsPublication . Charneca, João; Matias, Ana Catarina; Escapa, Ana Luisa; Fernandes, Catarina; Alves, Andre; Santos, João; Nascimento, Rita; Bragança, JoséCited2 plays crucial roles in mouse embryonic stem cells self-renewal, the initiation of the somatic reprogramming process into induced pluripotent stem cells (iPSC) and the suppression of cell senescence. Here, we investigated the potential of CITED2 expression in combination with the Oct4, Sox2, Klf4 and c-Myc factors for reprogramming of primary mouse embryonic fibroblasts (MEF) at passage 2 and 4. The ectopic CITED2 expression in primary MEF prior to the onset of the reprogramming process, generated iPSC with less variability in the expression of endogenous pluripotency-related genes. In contrast, part of the MEF reprogrammed without ectopic expression of CITED2 at passage 4 originated partially reprogrammed iPSC or pre-iPSC. However, the overexpression of CITED2 in the pre-iPSC was insufficient to complete the reprogramming process into iPSC. These results indicated that ectopic CITED2 expression at the onset of the reprogramming process in combination with the reprogramming factors promotes a complete and homogeneous conversion of somatic cells into iPSC.