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Biophysical modulation of cell fate through chromatin remodelling
Publication . Lima, Ana Francisca Silva de; Neves, Ricardo; Enver, Tariq; Ponte, Manuel
The use of non-lethal stimuli as cell modulators is a growing research area. The use of such strategies enables the control of cell behaviour and phenotype using stimuli that occur within the normal cellular environment. By using these stimuli, is possible to take advantage of their controlled application in benefit of a targeted cell phenotype modification and interfere with fate determination. Osmoregulatory mechanisms are constituted by diversified signalling mechanisms, which share the objective of maintenance of cell integrity and function when an extracellular osmolarity disturbance occurs. Cellular osmoregulation comprises the regulation of cell volume and osmolyte transport and also protein structure and turnover, and genomic integrity. These mechanisms are observed under distinct physiological and pathophysiological scenarios. This work aims to contribute to a better understanding of the relevance of the environmental osmotic modulation on cell physiology and its potential relevance to cell behaviour and phenotype. The hyposmotic modulation described in this work induced differences in the transcriptional process by an increased RNA polymerase II binding to the chromatin. This has been mediated by the intervention of specific transcription factors such as zinc finger proteins to attain a specific RNA polymerase II binding profile. The hyposmotic modulation has the ability to interfere with cell fate determination processes such as reprogramming and transdifferentiation. The potential of interfering with such complex processes has to do with the broad effect of the hyposmotic modulation on cellular signalling molecules (like calcium and adenosine triphosphate), mitochondrial activity and morphology, chromatin structure, epigenetic landscape, alternative splicing events and transcriptional pattern. The area of cellular therapies is growing exponentially and the basic knowledge on cellular behaviour and cellular response to osmotic changes as well as the development of new tools responsive to osmotic changes can have a crucial role for the development of future therapeutic applications.
Osmotic modulation of chromatin impacts on efficiency and kinetics of cell fate modulation
Publication . Lima, A. F.; May, G.; Colunga, J.; Pedreiro, S.; Paiva, A.; Ferreira, L.; Enver, T.; Iborra, F. J.; Pires Das Neves, R.; Faculdade de Ciências e Tecnologia (FCT); Nature Publishing Group
Chromatin structure is a major regulator of transcription and gene expression. Herein we explore the use of osmotic modulation to modify the chromatin structure and reprogram gene expression. In this study we use the extracellular osmotic pressure as a chromatin structure and transcriptional modulator. Hyposmotic modulation promotes chromatin loosening and induces changes in RNA polymerase II (Pol II) activity. The chromatin decondensation opens space for higher amounts of DNA engaged RNA Pol II. Hyposmotic modulation constitutes an alternative route to manipulate cell fate decisions. This technology was tested in model protocols of induced pluripotency and transdifferentiation in cells growing in suspension and adherent to substrates, CD34 + umbilical-cord-blood (UCB), fibroblasts and B-cells. The efficiency and kinetics of these cell fate modulation processes were improved by transient hyposmotic modulation of the cell environment.
Soft culture substrates favor stem-like cellular phenotype and facilitate reprogramming of human mesenchymal stem/stromal cells (hMSCs) through mechanotransduction
Publication . Gerardo, Heloísa; Lima, Ana; Ramos, João R. D.; Couceiro, Sofia; Carvalho, João C. L.; Travasso, Rui D. M.; Pires das Neves, Ricardo; Grãos, Mário; Faculdade de Ciências e Tecnologia (FCT); Nature Publishing Group
Biophysical cues influence many aspects of cell behavior. Stiffness of the extracellular matrix is probed by cells and transduced into biochemical signals through mechanotransduction protein networks, strongly influencing stem cell behavior. Cellular stemness is intimately related with mechanical properties of the cell, like intracellular contractility and stiffness, which in turn are influenced by the microenvironment. Pluripotency is associated with soft and low-contractility cells. Hence, we postulated that soft cell culture substrates, presumably inducing low cellular contractility and stiffness, increase the reprogramming efficiency of mesenchymal stem/stromal cells (MSCs) into induced pluripotent stem cells (iPSCs). We demonstrate that soft substrates (1.5 or 15 kPa polydimethylsiloxane – PDMS) caused modulation of several cellular features of MSCs into a phenotype closer to pluripotent stem cells (PSCs). MSCs cultured on soft substrates presented more relaxed nuclei, lower maturation of focal adhesions and F-actin assembling, more euchromatic and less heterochromatic nuclear DNA regions, and increased expression of pluripotency-related genes. These changes correlate with the reprogramming of MSCs, with a positive impact on the kinetics, robustness of colony formation and reprogramming efficiency. Additionally, substrate stiffness influences several phenotypic features of iPS cells and colonies, and data indicates that soft substrates favor full iPSC reprogramming.
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Fundação para a Ciência e a Tecnologia
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SFRH
Número da atribuição
SFRH/BD/51942/2012