SyNPL: Synthetic Notch pluripotent cell lines to monitor and manipulate cell interactions in vitro and in vivo

Abstract

Cell-cell interactions govern differentiation and cell competition in pluripotent cells during early development, but the investigation of such processes is hindered by a lack of efficient analysis tools. Here we introduce SyNPL: clonal pluripotent stem cell lines which employ optimised Synthetic Notch (SynNotch) technology to report cell-cell interactions between engineered sender and receiver cells in cultured pluripotent cells and chimaeric mouse embryos. A modular design makes it straightforward to adapt the system for programming differentiation decisions non-cell-autonomously in receiver cells in response to direct contact with sender cells. We demonstrate the utility of this system by enforcing neuronal differentiation at the boundary between two cell populations. In summary, we provide a new tool which could be used to identify cell interactions and to profile changes in gene or protein expression that result from direct cell-cell contact with defined cell populations in culture and in early embryos, and which can be adapted to generate synthetic patterning of cell fate decisions.

Use of CRISPRoff and synthetic Notch to modulate and relay endogenous gene expression programs in engineered cells

“…An attractive feature of synthetic Notch is to provide flexibility in cellular responses with customized response behaviors to achieve combinatorial integration of user-defined environmental cues (Roybal et al, 2016;Toda et al, 2020). One of the most intriguing applications of synNotch is to trace and control stem cell fate decisions (Malaguti et al, 2022;Zhang et al, 2022;Lee et al, 2023). Here, we show that PAX6-driven expression of GFP ligand can induce the neural differentiation of H9 human embryonic stem cells through synNotch activation and its subsequently controlled Ngn2 transgene expression.…”

“…Thus, when synNotch receptors bind an immobilized ligand (i.e., presented on neighboring cells or anchored to the extracellular environment), the resultant mechanical force exposes protease intramembrane cleavage sites in the Notch core, potentiating release of the intracellular transcription factor. This allows for subsequent expression of user-specified target transgenes (Malaguti et al, 2022;Lee et al, 2023). In this way, synNotch activation can tune defined cellular responses to selected inputs.…”

Synthetic Morphogenesis: Introducing IEEE Journal Readers to Programming Living Mammalian Cells to Make Structures

“…The use of membrane-tethered molecules works in a similar way, to solve the problem of loss by diffusion, when only neighbours need to receive a signal. A very recent preprint shoes the value of this approach to 2D pattern elaboration of the type described in 3D in Fig 11 [48] It should be noted that a numbers of patterning systems have also been constructed in bacteria. The short generation times of these organisms means that they are frequently used as the first test-beds for ideas that later appear in mammalian systems, but they lie beyond the scope of this article.…”