Elise Cachat

2- and 3-dimensional synthetic large-scale de novo patterning by mammalian cells through phase separation.

Synthetic biology provides an opportunity for the construction and exploration of alternative solutions to biological problems - solutions different from those chosen by natural life. To this end, synthetic biologists have built new sensory systems, cellular memories, and alternative genetic codes. There is a growing interest in applying synthetic approaches to multicellular systems, especially in relation to multicellular self-organization. Here we describe a synthetic biological system that confers large-scale de novo patterning activity on 2-D and 3-D populations of mammalian cells. Instead of using the reaction-diffusion mechanisms common in real embryos, our system uses cadherin-mediated phase separation, inspired by the known phenomenon of cadherin-based sorting. An engineered self-organizing, large-scale patterning system requiring no prior spatial cue may be a significant step towards the construction of self-assembling synthetic tissues.

A library of mammalian effector modules for synthetic morphology

BackgroundIn mammalian development, the formation of most tissues is achieved by a relatively small repertoire of basic morphogenetic events (e.g. cell adhesion, locomotion, apoptosis, etc.), permutated in various sequences to form different tissues. Together with cell differentiation, these mechanisms allow populations of cells to organize themselves into defined geometries and structures, as simple embryos develop into complex organisms. The control of tissue morphogenesis by populations of engineered cells is a potentially very powerful but neglected aspect of synthetic biology.ResultsWe have assembled a modular library of synthetic morphogenetic driver genes to control (separately) mammalian cell adhesion, locomotion, fusion, proliferation and elective cell death. Here we describe this library and demonstrate its use in the T-REx-293 human cell line to induce each of these desired morphological behaviours on command.ConclusionsBuilding on from the simple test systems described here, we want to extend engineered control of morphogenetic cell behaviour to more complex 3D structures that can inform embryologists and may, in the future, be used in surgery and regenerative medicine, making synthetic morphology a powerful tool for developmental biology and tissue engineering.

Application of synthetic biology to regenerative medicine

Synthetic biology uses interchangeable and standardized “bio-parts” to construct complex genetic networks that include sensing, information processing and effector modules: these allow robust and tunable transgene expression in response to a change in signal input. The rise of this field has coincided closely with the emergence of regenerative medicine as a distinct discipline. Unlike synthetic biology, regenerative medicine uses the natural abilities of cells to make trophic factors and to produce new tissues as they would in normal development and tissue maintenance. In this article, we argue that bringing these young fields together, so that synthetic biology techniques are applied to the problem of regeneration, has the potential significantly to enhance our ability to help those in clinical need. We first review the synthetic tool kit available for engineered mammalian networks, then examine the main areas in which synthetic biology techniques might be applied to promote regeneration: (i) biosynthesis and controlled release of therapeutic molecules, (ii) synthesis of scaffold material, (iii) regulation of stem cells, and (iv) programming cells to organize themselves into novel tissues. We finally consider the long-term potential of synthetic biology for regenerative medicine, and the risks and challenges ahead.

Synthetic biology meets tissue engineering

Classical tissue engineering is aimed mainly at producing anatomically and physiologically realistic replacements for normal human tissues. It is done either by encouraging cellular colonization of manufactured matrices or cellular recolonization of decellularized natural extracellular matrices from donor organs, or by allowing cells to self-organize into organs as they do during fetal life. For repair of normal bodies, this will be adequate but there are reasons for making unusual, non-evolved tissues (repair of unusual bodies, interface to electromechanical prostheses, incorporating living cells into life-support machines). Synthetic biology is aimed mainly at engineering cells so that they can perform custom functions: applying synthetic biological approaches to tissue engineering may be one way of engineering custom structures. In this article, we outline the ‘embryological cycle’ of patterning, differentiation and morphogenesis and review progress that has been made in constructing synthetic biological systems to reproduce these processes in new ways. The state-of-the-art remains a long way from making truly synthetic tissues, but there are now at least foundations for future work.

Dual-controlled optogenetic system for the rapid down-regulation of protein levels in mammalian cells

Optogenetic switches are emerging molecular tools for studying cellular processes as they offer higher spatiotemporal and quantitative precision than classical, chemical-based switches. Light-controllable gene expression systems designed to upregulate protein expression levels meanwhile show performances superior to their chemical-based counterparts. However, systems to reduce protein levels with similar efficiency are lagging behind. Here, we present a novel two-component, blue light-responsive optogenetic OFF switch (‘Blue-OFF’), which enables a rapid and quantitative down-regulation of a protein upon illumination. Blue-OFF combines the first light responsive repressor KRAB-EL222 with the protein degradation module B-LID (blue light-inducible degradation domain) to simultaneously control gene expression and protein stability with a single wavelength. Blue-OFF thus outperforms current optogenetic systems for controlling protein levels. The system is described by a mathematical model which aids in the choice of experimental conditions such as light intensity and illumination regime to obtain the desired outcome. This approach represents an advancement of dual-controlled optogenetic systems in which multiple photosensory modules operate synergistically. As exemplified here for the control of apoptosis in mammalian cell culture, the approach opens up novel perspectives in fundamental research and applications such as tissue engineering.

Organoid And Tissue Patterning Through Phase Separation: Use Of A Vertex Model To Relate Dynamics Of Patterning To Underlying Biophysical Parameters

Exactly a century ago, D’Arcy Thompson set an agenda for understanding tissue development in terms of underlying biophysical, mathematically-tractable mechanisms. One such mechanism, discovered by Steinberg in the 1960s, is adhesion-mediated sorting of cell mixtures into homotypic groups. Interest in this phase separation mechanism has recently surged, partly because of its use to create synthetic biological patterning mechanisms and partly because it has been found to drive events critical to the formation of organoids from stem cells, making the process relevant to biotechnology as well as to basic development. Here, we construct quantitative model of patterning by phase separation, informed by laboratory data, and use it to explore the relationship between degree of adhesive difference and speed, type and extent of resultant patterning. Our results can be used three ways; to predict the outcome of mix-ing cells with known properties, to estimate the properties required to make some designed organoid system, or to estimate underlying cellular properties from observed behaviour.