Biology subjects

Pedone, E.

Publications and source records attributed to Pedone, E..

9 recordsLinked to original sources

bioRxiv2025

Harnessing Dynamic Cell Responses to Optimise Pluripotency Protocols

Pluripotent stem cells have a high potential for research and development applications in biomedicine. They are undifferentiated cells capable of unlimited self-renewal and maintenance of pluripotency, yet can be induced to differentiate into specific cell types through defined cues. Various protocols have been proposed to maintain mouse embryonic stem cells (mESCs) in the pluripotent state; state of the art cell culture protocols are based on serum-free media, and continuous provision of drugs inhibiting pathways associated to differentiation. We explore here the possibility to avoid continuous drug exposure. First, we employ an external feedback control microfluidics/microscopy platform to steer the expression of a pluripotency reporter. Control experiments show a significant delay in cell response to drugs; these dynamics are confirmed by flow-cytometry open-loop experiments. We then harness such delays to design novel culture protocols with limited cell exposure to small-molecule inhibitors; in so doing, we reduce cell exposure to drug, but we maintain pluripotency. Our results suggest new approaches to design cell culture protocols based on the quantitative analysis and control of cellular dynamics.

synthetic biology↗

bioRxiv2022

Development of a single layer microfluidic device for dynamic stimulation, culture and imaging of mammalian cells

The possibility to tightly control the cellular microenvironment within microfluidic devices represents an important step forward precision analysis of cellular phenotypes in vitro. In particular, microfluidic platforms that allow both long-term mammalian cell culture and dynamic modulation of the culture environment can support quantitative studies of cells responses to drugs. Here, we report the design and testing of a novel microfluidic device of easy production (single Polydimethylsiloxane layer), which integrates a micromixer with vacuum-assisted cell loading for long-term mammalian cell culture and dynamic mixing of 4 different culture media. Finite element modelling was used to predict the flow rates and device dimensions to achieve diffusion-based fluid mixing. The device showed efficient mixing and dynamic exchange of media in the cell trapping chambers. This work represents the first attempt to integrate single layer microfluidic mixing devices with vacuum-assisted cell loading systems for mammalian cell culture and dynamic stimulation.

bioengineering↗

bioRxiv2020

ChipSeg: an automatic tool to segment bacteria and mammalian cells cultured in microfluidic devices

Extracting quantitative measurements from time-lapse images is necessary in external feedback control applications, where segmentation results are used to inform control algorithms. While such image segmentation applications have been previously reported, there is in the literature a lack of open-source and documented code for the community. We describe ChipSeg, a computational tool to segment bacterial and mammalian cells cultured in microfluidic devices and imaged by time-lapse microscopy. The method is based on thresholding and uses the same core functions for both cell types. It allows to segment individual cells in high cell-density microfluidic devices, to quantify fluorescence protein expression over a time-lapse experiment and to track individual cells. ChipSeg enables robust segmentation in external feedback control experiments and can be easily customised for other experimental settings and research aims.

synthetic biology↗

bioRxiv2020

Cheetah: a computational toolkit for cybergenetic control

Advances in microscopy, microfluidics and optogenetics enable single-cell monitoring and environmental regulation and offer the means to control cellular phenotypes. The development of such systems is challenging and often results in bespoke setups that hinder reproducibility. To address this, we introduce Cheetah - a flexible computational toolkit that simplifies the integration of real-time microscopy analysis with algorithms for cellular control. Central to the platform is an image segmentation system based on the versatile U-Net convolutional neural network. This is supplemented with functionality to robustly count, characterise and control cells over time. We demonstrate Cheetahs core capabilities by analysing long-term bacterial and mammalian cell growth and by dynamically controlling protein expression in mammalian cells. In all cases, Cheetahs segmentation accuracy exceeds that of a commonly used thresholding-based method, allowing for more accurate control signals to be generated. Availability of this easy-to-use platform will make control engineering techniques more accessible and offer new ways to probe and manipulate living cells.

synthetic biology↗

bioRxiv2020

β-catenin perturbations control differentiation programs in mouse embryonic stem cells

The Wnt/{beta}-catenin pathway is involved in development, cancer and embryonic stem cell (ESC) maintenance; its dual role in stem cell self-renewal and differentiation is still controversial. Here, by applying an in vitro system enabling inducible gene expression control, we report that moderate induction of transcriptionally active exogenous {beta}- catenin in {beta}-catenin null mouse ESCs promotes epiblast-like cell (EpiLC) derivation in vitro. Instead, in wild type cells moderate chemical pre-activation of the Wnt/{beta}-catenin pathway promotes EpiLC in vitro derivation. Finally, we suggest that moderate {beta}- catenin levels in {beta}-catenin null mouse ESCs favour early stem cell commitment towards mesoderm if the exogenous protein is induced only in the ground state of pluripotency condition, or endoderm if the induction is maintained during the differentiation. Overall, our results confirm previous findings about the role of {beta}-catenin in pluripotency and differentiation, while indicating a role for its doses in promoting specific differentiation programs.

cell biology↗

bioRxiv2019

Senolytic treatment targets aberrant p21-expression to restore liver regeneration in adult mice

Young mammals possess a limited regenerative capacity in tissues such as the liver, heart and limbs, but which is quickly lost upon maturation or transition to adulthood. Chronic cellular senescence is a known mediator of decreased tissue function in aging and disease. Here we investigated whether senescence plays a role in the progressive loss of liver regenerative capacity that develops in young adult mice. We find that following partial hepatectomy, the senescence markers p21, p16Ink4a and p19Arf become dynamically expressed at an age when regenerative capacity decreases. In addition, we demonstrate that treatment with a senescence-inhibiting drug improves regenerative capacity, through targeting of aberrant p21 expression. Surprisingly, we also find that the senescence marker p16Ink4a is expressed in a different cell-population to p21, and is unaffected by senescence targeting. This work suggests that senescence may initially develop as a heterogeneous cellular response, and that treatment with senolytic drugs may aid in promoting organ regeneration.

cell biology↗

bioRxiv2019

Towards automated control of embryonic stem cell pluripotency

Mouse embryonic stem cells (mESCs) have been shown to exist in three distinct pluripotent states (ground, naive and primed pluripotent states), depending on culture conditions. External feedback control strategies have been, so far, mainly used to automatically regulate gene expression in bacteria and yeast. Here, we exploit a microfluidics/microscopy platform and segmentation and external feedback control algorithms for the automatic regulation of pluripotency phenotypes in mESCs. We show feasibility of automatically controlling, in living mESCs, levels of an endogenous pluripotency gene, Rex1, through a fluorescent reporter, used as control output, and drugs commonly used to modulate pluripotency (i.e. MEK kinase and Gsk3{beta} inhibitors) as control inputs. Our results will ultimately aid in the derivation of superior protocols for pluripotency maintenance and differentiation of mouse and human stem cells.

developmental biology↗

bioRxiv2019

Canonical Wnt pathway controls mESCs self-renewal through inhibition of spontaneous differentiation via β-catenin/TCF/LEF functions

The Wnt/{beta}-catenin signalling pathway is a key regulator of embryonic stem cell self-renewal and differentiation. Constitutive activation of this pathway has been shown to significantly increase mouse embryonic stem cell (mESC) self-renewal and pluripotency marker expression. In this study, we generated a novel {beta}-catenin knock-out model in mESCs by using CRISPR/Cas9 technology to delete putatively functional N-terminally truncated isoforms observed in previous knock-out models. While we showed that aberrant N-terminally truncated isoforms are not functional in mESCS, we observed that canonical Wnt signalling is not active in mESCs, as {beta}-catenin ablation does not alter mESC transcriptional profile in LIF-enriched culture conditions; on the other hand, Wnt signalling activation represses mESC spontaneous differentiation. We also showed that transcriptionally silent {beta}-catenin ({Delta}C) isoforms can rescue {beta}-catenin knock-out self-renewal defects in mESCs, cooperating with TCF1 and LEF1 in the inhibition of mESC spontaneous differentiation in a Gsk3 dependent manner.

cell biology↗

bioRxiv2018

A tunable dual-input system for on-demand dynamic geneexpression regulation.

Cellular systems have evolved numerous mechanisms to finely control signalling pathway activation and properly respond to changing environmental stimuli. This is underpinned by dynamic spatiotemporal patterns of gene expression. Indeed, in addition to gene transcription and translation regulation, modulation of protein levels, dynamics and localization are also essential checkpoints that govern cell functions. The introduction of tetracycline-inducible promoters has allowed gene expression control using orthogonal small molecules, facilitating rapid and reversible manipulation to study gene function in biological systems. However, differing protein stabilities means this solely transcriptional regulation is insufficient to allow precise ON-OFF dynamics, thus hindering generation of temporal profiles of protein levels seen in vivo. We developed an improved Tet-On based system augmented with conditional destabilising elements at the post-translational level that permits simultaneous control of gene expression and protein stability. Integrating these properties to control expression of a fluorescent protein in mouse Embryonic Stem Cells (mESCs), we found that adding protein stability control allows faster response times to changes in small molecules, fully tunable and enhanced dynamic range, and vastly improved microfluidic-based in-silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to finely modulate levels of signalling pathway components in stem cells.

synthetic biology↗