Ana Lima

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.

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.

Noise in gene expression is a main determinant of phenotypic variability. Increasing experimental evidence suggests that genome-wide cellular constraints largely contribute to the heterogeneity observed in gene products. It is still unclear, however, which global factors affect gene expression noise and to what extent. Since eukaryotic gene expression is an energy demanding process, differences in the energy budget of each cell could determine gene expression differences. Here, we quantify the contribution of mitochondrial variability (a natural source of ATP variation) to global variability in gene expression. We find that changes in mitochondrial content can account for ∼50% of the variability observed in protein levels. This is the combined result of the effect of mitochondria dosage on transcription and translation apparatus content and activities. Moreover, we find that mitochondrial levels have a large impact on alternative splicing, thus modulating both the abundance and type of mRNAs. A simple mathematical model in which mitochondrial content simultaneously affects transcription rate and splicing site choice can explain the alternative splicing data. The results of this study show that mitochondrial content (and/or probably function) influences mRNA abundance, translation, and alternative splicing, which ultimately affects cellular phenotype.

Herein, we report the use of biodegradable nanoparticles (NPs) containing perfluoro-1,5-crown ether (PFCE), a fluorine-based compound (NP170-PFCE) with the capacity to track cells in vivo by magnetic ressonance imaging (MRI) and efficiently release miRNA. NP170-PFCE complexed with miRNAs accumulate whitin the cell’s endolysosomal compartment and interact with higher frequency with argonaute2 (Ago2) and GW182 proteins, which are involved in the biological action of miRNAs, than commercial complexes formed by commercial reagents and miRNA, which in turn accumulate in the cell cytoplasm. The release of miRNA132 (miR132) from the NPs increased 3-fold the survival of endothelial cells (ECs) transplanted in vivo and 3.5-fold the blood perfusion in ischemic limbs relatively to control.