Biology subjects

Morsut, L.

Publications and source records attributed to Morsut, L..

8 recordsLinked to original sources

bioRxiv2026

A novel reaction-diffusion architecture for engineering self-organized patterns in mammalian cells

Reaction-diffusion circuits generate self-organized spatial patterns through local activation and long-range inhibition, but synthetic implementations in mammalian cells have been limited by the differential-diffusion requirement. Here, we introduce a novel architecture, juxtacrine activation with paracrine inhibition (JAPI), where the activator propagates through cell-cell contacts rather than diffusion. We demonstrate mathematically and numerically that JAPI accesses the same patterning regimes as classical diffusion-based circuits with one fewer free parameter. We then engineer compact synNotch-based JAPI circuits in mammalian fibroblasts and demonstrate their sufficiency for self-organized patterning through tunable, size-limited signal propagation. Functionalized to spatially control morphogen secretion, these circuits perturb feather bud formation on adjacent embryonic chicken epidermis. Finally, we develop a library-based approach to explore coupled, dual-JAPI circuits with tunable cross-inhibition, enabling programmable interactions between patterns and access to a broad morphospace of spatial states. Together, JAPI provides a compact, modular platform for programming self-organized multicellular patterning. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/727552v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@fae8c9org.highwire.dtl.DTLVardef@c8eddborg.highwire.dtl.DTLVardef@177d08corg.highwire.dtl.DTLVardef@11cc952_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Decoding human B cell ontogeny in prenatal and adult bone marrow and in vitro models via single-cell multiomics

In humans, the bone marrow becomes the primary site for B lymphopoiesis during the second trimester of pregnancy and continues throughout life. Prenatal and adult B cell progenitors play distinct roles in the aetiology and pathology of paediatric and adult hematopoietic malignancies, though the molecular drivers of these differences remain unclear. Here, we created a comprehensive multiomics atlas of over 500k cells covering immune and stromal compartment from prenatal and adult bone marrow, enabling high-resolution analysis of the cell-intrinsic and cell-extrinsic processes that modulate prenatal and adult B lymphopoiesis. Even though B cells follow broadly similar developmental trajectories, we identify a novel postnatal lateCLP subset equivalent to prenatal preProB cells, and uncover prenatal cells carry several signatures characteristic of leukemias, including enhanced proliferation, higher RAG1/RAG2 activity, extrinsic B cell signals such as IL7, and lower retention signals in the bone marrow. We also developed and characterised, at both cellular and molecular levels, a new experimental framework for generating B cell precursors from human induced pluripotent stem cells (hiPSCs), and show that it faithfully recapitulates key stages of B cell differentiation. Together, our single-cell multiomics atlas of B lymphopoiesis in vivo and in vitro offers detailed insights into the unique molecular features of prenatal and adult B cell lymphopoiesis, and serves as a powerful resource for investigating the early events that contribute to haematological disorders.

immunology↗

bioRxiv2025

Derivation of cardiomyocyte-propelled motile aggregates from stem cells

Robotics draws inspiration from biology, particularly animal locomotion based on muscle-driven contractions. While traditional engineering assembles components sequentially, locomotive animals are built via self-organized developmental programs. Stem cells, under the right conditions, can mimic these processes in vitro, offering a pathway to develop muscle-propelled biobots in a self-organized building process. Here, we demonstrate that existent cardiogenic gastruloid protocols can produce motile aggregates from mouse embryonic stem cells, although with very limited efficiency. We then identify a novel protocol that yields contractile aggregates with higher frequency and larger contractile areas. In this novel protocol, mesendoderm induction using TGF-beta ligands is followed by cardiogenic induction with FGFs and VEGF. Synthetic organizers further control contraction localization. Aggregates developed via this protocol show enhanced motility, marking a step forward towards building motile cardiobots from self-organized biological material. This strategy opens new possibilities for designing autonomous biobots and studying the evolution of muscle-powered movement of multicellular organisms and cardiovascular development.

synthetic biology↗

bioRxiv2024

Programming the elongation of mammalian cell aggregates with synthetic gene circuits

A key goal of synthetic morphogenesis is the identification and implementation of methods to control morphogenesis. One line of research is the use of synthetic genetic circuits guiding the self-organization of cell ensembles. This approach has led to several recent successes, including control of cellular rearrangements in 3D via control of cell-cell adhesion by user-designed artificial genetic circuits. However, the methods employed to reach such achievements can still be optimized along three lines: identification of circuits happens by hand, 3D structures are spherical, and effectors are limited to cell-cell adhesion. Here we show the identification, in a computational framework, of genetic circuits for volumetric axial elongation via control of proliferation, tissue fluidity, and cell-cell signaling. We then seek to implement this design in mammalian cell aggregates in vitro. We start by identifying effectors to control tissue growth and fluidity in vitro. We then combine these new modules to construct complete circuits that control cell behaviors of interest in space and time, resulting in measurable tissue deformation along an axis that depends on the engineered signaling modules. Finally, we contextualize in vitro and in silico implementations within a unified morphospace to suggest further elaboration of this initial family of circuits towards more robust programmed axial elongation. These results and integrated in vitro/in silico pipeline demonstrate a promising method for designing, screening, and implementing synthetic genetic circuits of morphogenesis, opening the way to the programming of various user-defined tissue shapes.

synthetic biology↗

bioRxiv2024

Defining and Controlling Axial Nephron Patterning in Human Kidney Organoids with Synthetic Wnt-Secreting Organizers

Current human pluripotent stem cell-derived kidney organoids contain nephron-like structures that lack organotypic patterning. It is thought that during human development, nephrons form their proximal-distal axial polarity in response to collecting duct-derived signals that are absent in kidney organoids. To delineate how nephron polarities establish, we profiled human kidney development by spatial transcriptomic approaches. Our analyses describe a new axial polarity in the nephron and demonstrate that the nephron proximal-distal polarity develops adjacent to a transcriptional boundary in the collecting duct where non-canonical WNT11 is downregulated and canonical WNT9B ligand is upregulated. The nephron region closest to this boundary in turn activates a series of canonical WNT target genes inferring positional nephron identities. To establish whether a canonical WNT source can improve organoid patterning to an in vivo-like state, we bioengineered self-organizing WNT-secreting synthetic organizers. Organizer-coupled kidney organoids respond to WNT ligands by forming expression gradients and developing distal cell identities. Tuning the WNT dose produced nephrons with continuous patterning along the proximal-distal axis. Strikingly, polarized iPSC-derived nephrons directed their distal tubules towards the WNT-source, indicating axial patterning and morphogenetic programs are tuned by WNTs from the synthetic organizers. Our data present a strategy to control organ patterning, build an artificial kidney, and highlights the power of synthetic organizer systems for advancing organoid models.

synthetic biology↗

bioRxiv2023

Engineering Programmable Material-To-Cell Pathways Via Synthetic Notch Receptors To Spatially Control Cellular Phenotypes In Multi-Cellular Constructs

Synthetic Notch (synNotch) receptors are modular synthetic components that are genetically engineered into mammalian cells to detect signals presented by neighboring cells and respond by activating prescribed transcriptional programs. To date, synNotch has been used to program therapeutic cells and pattern morphogenesis in multicellular systems. However, cell-presented ligands have limited versatility for applications that require spatial precision, such as tissue engineering. To address this, we developed a suite of materials to activate synNotch receptors and serve as generalizable platforms for generating user-defined material-to-cell signaling pathways. First, we demonstrate that synNotch ligands, such as GFP, can be conjugated to cell- generated ECM proteins via genetic engineering of fibronectin produced by fibroblasts. We then used enzymatic or click chemistry to covalently link synNotch ligands to gelatin polymers to activate synNotch receptors in cells grown on or within a hydrogel. To achieve microscale control over synNotch activation in cell monolayers, we microcontact printed synNotch ligands onto a surface. We also patterned tissues comprising cells with up to three distinct phenotypes by engineering cells with two distinct synthetic pathways and culturing them on surfaces microfluidically patterned with two synNotch ligands. We showcase this technology by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined spatial patterns towards the engineering of muscle tissue with prescribed vascular networks. Collectively, this suite of approaches extends the synNotch toolkit and provides novel avenues for spatially controlling cellular phenotypes in mammalian multicellular systems, with many broad applications in developmental biology, synthetic morphogenesis, human tissue modeling, and regenerative medicine.

synthetic biology↗

bioRxiv2022

Control of spatio-temporal patterning via cell density in a multicellular synthetic gene circuit

A major goal in synthetic development is to design and construct gene regulatory circuits that control the patterning and morphogenesis of synthetic multicellular structures. In natural development, an interplay between mechanical and chemical communication shapes the dynamics of gene regulatory circuits that underlie patterning and morphogenesis. However, for synthetic gene circuits, how the non-genetic properties of the growth environment impact circuit behavior remains poorly understood. Here, we describe an occurrence of mechano-chemical coupling in synthetic contact-dependent synNotch patterning circuits demonstrating that cell density modulates the transduction of signal between a sender and receiver cell. By exploiting density-dependent signaling, we construct multicellular signal propagation circuits with synNotch and control the patterning outcome both temporally and spatially via cell density gradients established in vitro via plating or small-molecule mediated modulation of proliferation. Our work demonstrates that synthetic gene circuits can be critically impacted by their context, providing an alternate means for programming multi-cellular circuit patterning outcomes.

synthetic biology↗

bioRxiv2019

A Modular Computational Framework for the Rational Design and Exploration of Synthetic Development

Synthetic development is a nascent field of research that uses the tools of synthetic biology to design genetic programs directing cellular patterning and morphogenesis in higher eukaryotic cells, such as mammalian cells. One specific example of such synthetic genetic programs was based on cell-cell contact-dependent signaling using synthetic Notch pathways, and was shown to drive formation of multilayered spheroids by modulating cell-cell adhesion via differential expression of cadherin-family proteins. The design method for these genetic programs relied on trial and error, which limited the number of possible circuits and parameter ranges that could be explored. Here we build a parametrized computational framework that, given a cellcell communication network driving changes in cell adhesion and initial conditions as inputs, predicts developmental trajectories. We first built a general computational framework where contact-dependent cell-cell signaling networks and changes in cell-cell adhesion could be designed in a modular fashion. We then use a set of available in vitro results (that we call the "training set" in analogy to similar pipelines in the machine learning field) to parametrize the computational model with values for adhesion and signaling. We then show that this parametrized model can qualitatively predict experimental results from a "testing set" of available in vitro data that varied the genetic network in terms of adhesion combinations, initial number of cells and even changes to the network architecture. Finally, this parametrized model is used to recommend novel network implementation for the formation of a 4-layered structure that has not been reported previously. The framework that we develop here could function as a testing ground to identify the reachable space of morphologies that can be obtained by controlling contact-dependent cell-cell communications and adhesion. Additionally, we discuss how the model could be expanded to include other forms of communication or effectors for the computational design of the next generation of synthetic developmental trajectories.

synthetic biology↗